Mycologia, 85(3), 1993, pp. 415-427.
? 1993, by The New York Botanical Garden, Bronx, NY 10458-5126
RIBOSOMAL
DNA
INTERNAL
DIVERGENCE
SEQUENCE
TRANSCRIBED
SPACER
WITHIN
1 OF
THE
SCLEROTINIACEAE
IGNAZIO CARBONE AND LlNDA M. KOHN
Department of Botany, University of Toronto, Erindale College,
Mississauga, Ontario L5L 1C6, Canada
ABSTRACT
Based on morphological and immunological studies, we hypothesize that there are two lineages within
the Sclerotiniaceae, a family of plant-infecting ascomycetes in the order Helotiales: 1) genera producing
sclerotia, which are tuberlike, melanized masses of hyphae, and 2) genera producing substratal stromata,
which are mats of compact hyphae that incorporate plant tissues. We sequenced the Internal Transcribed
Spacer (ITS 1), defined by primers ITS 1 and 2, in 43 isolates: 29 sclerotial isolates (19 species in 9
genera), 11 substratal isolates (8 species in 4 genera), and 3 outgroup isolates in the Leotiaceae (3 species
in 3 genera). Direct, double-stranded sequencing yielded ca 170 bases for sclerotial isolates and ca 200
bases for substratal and outgroup isolates. MACVECTOR and MULTALIN were used for global
alignment, and multiple alignment with hierarchical clustering, respectively. The Internal Transcribed
Spacer showed close similarity among most of the sclerotial taxa (76 to 100% similarity to Sclerotinia
sclerotiorum). This supports our hypothesis that a sclerotial lineage exists and suggests that this lineage
has evolved relatively recently. Isolates of the asexual (mitotic) species Sclerotium cepivorum showed
98% similarity to those of the genus Sclerotinia. Sequence divergence was greater (45 to 65% similarity
to S. sclerotiorum) amongst the substratal taxa and our outgroups. Parsimony analysis produced one
statistically strongly supported tree for a group of species in the genus Rutstroemia, including Sclerotinia
homoeocarpa. Although such subclusters of species can be distinguished using parsimony analysis, we
conclude that a substratal lineage cannot be discerned based on sequence data from the ITS. Among
these more distantly related taxa, including some substratal ingroup taxa and the outgroup taxa, ITS
1 is saturated with changes and shows relatively equal dissimilarity. The variation observed in the ITS
does not resolve among more distantly related taxa.
Key Words: ascomycetes, discomycetes, internal transcribed spacer, taxonomy
All members of the Sclerotiniaceae
produce
one of two basic types of stromata, the determinate tuberlike sclerotium or the indeterminate
platelike substratal stroma (Whetzel, 1945; Kohn
and Grenville, 1989a, b). The determinate sclerotial genera include those genera which are necrotrophic plant pathogens, such as Sclerotinia
and Botryotinia. The indeterminate
substratal
stromatal genera include those genera which are
presumed to be plant saprobes, such as Rutstroemia.
In pure culture many of these fungi produce
only mycelium and stroma. Production of the
apothecial teleomorph may require further preparation and incubation of stromata, in addition
of stromata in heterothallic
to spermatization
species. In nature, some ubiquitous plant infecting sclerotiniaceous
species rarely or never pro?
duce apothecia. One good example is Sclerotinia
homoeocarpa, causal agent of dollar spot of turf,
a common disease in North America, Europe,
and Australasia, whose teleomorph has not been
415
reported and examined since the
conclusively
species was described. Several studies have suggested that S. homoeocarpa is not a member of
the genus Sclerotinia and that it should be reclassified among the substratal stromatal Sclerotiniaceae (Kohn, 1979; Kohn and Grenville,
1989a, b; Novak and Kohn, 1991). Examination
of apothecia putatively thought to belong to this
species suggested placement in either Lanzia or
Moellerodiscus (Korf, pers. comm.), but unfortunately none of these apothecia were retained.
Rutstroemia (= Poculum), Lanzia and Moeller?
odiscus are delimited primarily on the basis of
their apothecial microanatomy, with ectal excipula composed of filamentous cells in gel, filamentous cells lacking gel, and globose cells, respectively
(Dumont,
1976). White's (1941)
broader definition of Rutstroemia, still accepted
by some workers, includes Lanzia and some el?
ements of Moellerodiscus (Dumont, 1976). No
in particular,
description of the microanatomy,
the ectal excipular structure of S. homoeocarpa
416
Mycologia
was furnished in the original description, no type
or authentic herbarium material remains, and
attempts to produce apothecia in vitro have been
unsuccessful. Using the taxonomic methods necessary to compare the morphological characters
that define Rutstroemia, Lanzia, and Moellerodiscus, it is impossible to reclassify S. homoeocarpa without having the teleomorph in hand.
There have been no comparative molecular stud?
ies of any of these taxa, which have not been
extensively or copiously collected and, with the
exception of S. homoeocarpa, have rarely been
accessioned in culture collections. Another example is Sclerotium cepivorum, causal agent of
soft rot of onions, which does not produce a tel?
eomorph, only mycelia and determinate sclerotia
and, consequently, under conventional interpretations of Article 59 of the International Code
of Botanical Nomenclature (Greuter, 1988), can?
not be classified among the holomorphic
scle?
rotial taxa in the Sclerotiniaceae with which it
shares many features (Kohn and Grenville, 1989a,
b; Novak and Kohn, 1991).
Depending on the relative character weighting
for stromatal anamorphs (sclerotia versus sub?
stratal stromata, morphological
type), conidial
anamorphs (presence or absence, morphological
type), and apothecial microanatomy (gross mor?
phology, ectal excipular type), holomorphic gen?
era have been defined very broadly or very narrowly. Rutstroemia sensu White versus sensu
Dumont is one example. In another example,
based on a broad interpretation stressing apo?
thecial morphology and the production of scle?
rotia, Dennis (1978) and, to an even greater extreme, von Arx (1981) recognized virtually all
sclerotial taxa as one genus Sclerotinia. These
workers perhaps recognized a clade or generic
lineage rather than a single genus. Other authors
(Korf, 1973; Kohn, 1979; Schumacher and Kobn,
1985) have considered more equally characteristics of apothecial morphology along with those
of anamorph morphology (conidia and stromata)
and ecology, recognizing and segregating many
genera. However, these workers have not resolved relationships among genera. While teleo?
morph and anamorph morphology have largely
defined genera in the Sclerotiniaceae, the type of
stroma produced and the relationship of the fun?
gus to the host have afforded key characters for
grouping genera within the family and for rec?
ognizing relationships between the pleomorphic,
sexual (meiotic) species in the family and mainly
or exclusively asexual (mitotic) species, such as
Sclerotium cepivorum and Sclerotinia homoeo?
carpa, both of which lack conidial anamorphs.
A recent study in our laboratory (Novak and
Kohn, 1991) has shown that developmental stro?
matal storage proteins occur in both the sclerotial
and substratal stromatal taxa and that it is possible to distinguish between these groups of taxa
on the basis ofthe ability of these stromatal pro?
teins to cross-react with the major 36-kDa pro?
tein of Sclerotinia sclerotiorum. Using western
hybridizations,
only sclerotial proteins were
shown to cross-react when treated with antibody
raised to the 36-kDa protein of S. sclerotiorum.
No cross-reactivity was observed with substratal
stromatal proteins. Based on these immunological studies and on morphological studies (Kohn
and Grenville, 1989a, b), we hypothesized that
there are two lineages within the Sclerotiniaceae:
the sclerotial lineage and the substratal stromatal
lineage.
In the present study, sequence divergence in
the nuclear ribosomal
Internal Transcribed
Spacer (ITS 1), bounded by the 18S and 5.8S
genes of the nuclear ribosomal DNA, was explored as a potential source of characters for
studies at the generic level in the Sclerotiniaceae.
Although the noncoding ITS regions are generally more variable than the coding regions ofthe
adjacent nuclear ribosomal RNA genes, given
our previous observations
of no intraspecific
variation within Sclerotinia sclerotiorum (Kohn
and Anderson, unpubl.), we postulated three
things: 1) that the sclerotial taxa might show se?
quence similarity to each other and sequence di?
vergence from the substratal stromatal taxa, 2)
that Sclerotium cepivorum would show a high
level of sequence similarity to the sclerotial taxa,
and 3) that Sclerotinia homoeocarpa would show
sequence similarity to the substratal stromatal
taxa and a lack of relatedness to Sclerotinia. The
primary objective of this study was to sequence
the ITS 1 in a larger sample within the Sclero?
tiniaceae, including closely related species and
genera within the two proposed lineages, as well
as outgroup isolates representing the morphologically and ecologically most closely related
family, the Leotiaceae. Outgroup isolates from
the Leotiaceae were chosen based on the alternative hypotheses that either substratal stro?
matal taxa show closer relatedness to the Leo?
tiaceae than to the sclerotial lineage or that
substratal stromatal taxa show equal relatedness
Carbone
and
Kohn:
Molecular
to the Leotiaceae and to the sclerotial lineage.
Neurospora crassa Shear & Dodge was included
in the sample to facilitate alignment of the coding
ends of the region amplified with these primers,
although no close relationship to the Scleroti?
niaceae was anticipated. In order to determine
where, and what type of variation was present
in the ITS 1, sequences were first aligned and
grouped using programs for global alignment and
with visual adjustment.
multiple alignment
Aligned sequences were then compared and
grouped by hierarchical clustering and parsi?
mony analysis. The results of this study were
data from a precompared with immunological
vious study (Novak and Kohn, 1991) for a subset
of our isolates.
materials
and methods
Cultures and DNA extraction.?Isolates used in this
study are listed in Table I. Cultures were grown on
potato-dextrose agar (PDA) in the dark and at room
temperature (20-22 C). Mycelium was grown on liquid
complete yeast medium (CYM), harvested after 2-4
days and freeze-dried for DNA extraction (Kohn et al.,
1991; Kohli et al., 1992). DNA extraction was by the
small scale (mini-prep) method of Zolan and Pukkila
(1986). DNA was stored at -20 C until required for
amplification.
Amplification of genomic DNA. ?The ITS 1 region de?
fined by primers ITS 1 and ITS 2 (White et al., 1990)
was amplified via the polymerase chain reaction (PCR).
DNA was diluted 200-fold and 50 iA was placed in a
0.5 ml polypropylene tube to which 50 ^1of a 2 x mix
of all other reaction components was added as described by Kohn et al. (1991). Double-stranded DNA
amplification products were generated by using equimolar (50 pM) amounts of each primer. A DNA-free
control tube was included with each run as a safeguard
against contamination. The thermal program followed
was as described in Kohn et al. (1991). PCR products
were observed by electrophoresing 10 yXfrom each tube
on 1.5%agarose gels in Tris-acetate-EDTA (TAE) buff?
er. The presence of a single bright band in each lane
was a check for a successful amplification, while the
absence of a band in the control lane was a check
against contamination. The remaining PCR reaction
product (90 iA) was purified using GENECLEAN
(BioCan Scientific). All purified DNA samples were
stored at -20 C until required for sequencing.
was sequenced directly
Sequencingprocedure.?UNA
using the dideoxy chain termination method (Sanger
et al., 1977). The ITS 1 region was sequenced for a
sample of 43 isolates (Table I), consisting of 29 sclero?
tial isolates (19 species in 9 genera), 11 substratal stro?
matal isolates (8 species in 4 genera) and 3 outgroup
isolates (3 species in 3 genera). For each isolate both
complementary ITS 1 strands were sequenced follow?
ing the protocol of Winship (1989) as modified by An?
derson and Stasovski (1992).
Systematics
of Sclerotiniaceae
417
Analysis of DNA sequences. ?Sequence data were
checked between complementary strands and compared with other sequence data with the assistance of
MACVECTOR (Lipman and Pearson, 1985; Pearson
and Lipman, 1988) and MULTALIN (Corpet, 1988).
The first step in sequence analysis was the comparison
of complementary strands to check for reading errors
and resolve ambiguities if possible. Complete sequenc?
es were then globally aligned with a computerized
alignment algorithm (MACVECTOR). This algorithm
compares all the residues in one sequence to all positions in a specified reference sequence only, in this case
Sclerotinia sclerotiorum. To every pair of residues
compared, a positive weight is assigned for a match
and a negative weight for a mismatch, deletion or a
gap. The generated value is the score ofthe alignment
and represents the best (optimal) alignment ofthe nonreference sequence to the reference sequence for the
weights used. The final alignment of all sequences to
S. sclerotiorum was analyzed visually and the deletion/
gap weights were adjusted to obtain the best visual
alignment of all sequences. The order ofthe sequences
and weight parameters in the final MACVECTOR
alignment were the order and weight parameters used
for MULTALIN. MULTALIN performs all possible
pairwise comparisons using an algorithm similar to
MACVECTOR, with S. sclerotiorum as the query se?
quence. Initially a hierarchical clustering of the se?
quences is done using these scores (Corpet, 1988). This
is followed by pairwise comparisons of clusters of
aligned sequences to obtain the complete multiple
alignment. MULTALIN then builds a hierarchical
clustering ofthe sequences using the scores of all pair?
wise comparisons in the multiple alignment as a measure of the similarity between sequences. Similarity
scores were represented on a fractional (or percentage)
similarity scale where a value of 1.0 (or 100%) corresponds to no sequence divergence (i.e., identical se?
quences). In general, as sequence divergence increases,
the fractional similarity (S) between two sequences decreases (S < 1.0) and the distance from the reference
sequence, defined as 1-S increases (Swofford and Olsen,
1990). The final multiple alignment was visually ad?
justed and parsimony analysis of the multiple align?
ment, both to compare branching topologies and branch
lengths with the hierarchical clustering, was performed
using PAUP 3.0n (Swofford, 1990).
RESULTS
DNA sequencing
Direct, double-stranded
yielded ca 170 bases for sclerotial isolates and ca
200 bases for the substratal stromatal and out?
group isolates. In general, DNA sequences could
be determined with accuracy by reading only the
antisense strand but as a further check for reading
errors and for resolution of ambiguous bases, it
was necessary to align both strands in all isolates.
A preliminary MACVECTOR
global align?
ment of all the sequences with S. sclerotiorum
(LMK 2) as the reference sequence revealed very
little sequence divergence within the sclerotial
418
Mycologia
Table I
Isolates representing
the Sclerotiniaceae
and selected
outgroups
Carbone
and
Kohn:
Systematics
Molecular
of Sclerotiniaceae
419
Table I
Continued
a Collection no., in parentheses, designates accession number in the collection of L. M. Kohn.
b Localities are abbreviated as follows: N.W.T., Ellesmere Island, North West Territories; N.S.W., New South
Wales, Australia.
c Sources of cultures are abbreviated as follows: ATCC, American Type Culture Collection; CUP, Cornell
University Plant Pathology Herbarium; ATRI, Australian Turfgrass Research Institute.
d Isolate represents an asexual (mitotic) species known only from anamorphs.
e Isolate represents an anamorph of a pleomorphic species with a known teleomorph in the Sclerotiniaceae.
Unless designated otherwise, isolates in this table represent pleomorphic species with known teleomorphs in
the Sclerotiniaceae.
isolates and a high level of divergence within the
substratal stromatal and outgroup isolates. Be?
cause of this, visual sequence alignment within
the sclerotial isolates was easily accomplished,
but alignment between sclerotial and substratal
stromatal and outgroup isolates was difncult, with
dubious results. The best visual alignment that
we could achieve of all of the sequences with
MACVECTOR was obtained with a match score
of 4 and a mismatch score of 2, a deletion penalty
of 20 (rather than the default of 12), and a gap
penalty of 4. The order ofthe sequences did not
have any effect on the final MACVECTOR align?
ment but was found to have an effect on the final
multiple alignment (data not shown). Therefore,
the order of the sequences given by MACVEC?
TOR was the order used for MULTALIN.
Multiple alignment of all the sequences (Fig.
1) revealed that there was very little divergence
close to the ends of the sequences, and no di?
vergence when the ends extended into the coding
regions ofthe 18S and 5.8S rRNA genes. Little
intraspecific sequence variation was observed.
Among the sclerotial isolates most of the variability was located in the middle of the ITS 1
region, and was either in the form of single base
substitutions only [Sclerotinia spp., Sclerotium
cepivorum, and Cristulariella moricola (teleo?
morph = Grovesinia pyramidalis M. Cline, J. L.
Crane, & S. Cline)] or base substitutions accommotif (Bopanied by a 2 bp insertion/deletion
trytis spp., Moniliniafructicola,
Myriosclerotinia
spp., and Ciborinia ciborium). Among the sub?
stratal and outgroup isolates single or multiple
base substitutions and insertion/deletion
events
were observed throughout the ITS 1 region.
Hierarchical clustering based on the multiple
alignment by MULTALIN, with S. sclerotiorum
(LMK 2) as the query sequence, is shown in Fig.
characters from a previous
2; immunological
study of stromatal proteins (Novak and Kohn,
1991) and morphological types of the macroconidial anamorphs are also shown in Fig. 2. The
purpose of the cluster analysis was mainly to
order the data set and to compartmentalize
like
and unlike isolates, not to estimate true evolu?
tionary distance. The final sequence alignment
within those clusters with >70?/o similarity was
found to be relatively insensitive to the choice
of weights and a reasonable alignment could also
be attained by eye. Sequences that were outside
these clusters had ambiguous alignments and gaps
were inserted as necessary by the alignment algorithm to maximize the total similarity score
when summed over all positions. The choice of
query sequence did not affect the clustering; the
same clustering was obtained when isolates of
each of four other taxa in the sample [Sclerotinia
Mycologia
420
30
40
10
20
50
60
70
<-5.8S
ACT-- AC^CAGACGACATTAATAAAAAGAGITrrGAT-AT
TCCGTIUITGAMGTITrAACTATTATAT2
44
57
3
115
118
36
47
55
1
71
126
161
18
20
21
521
523
12
0-7
E76
0-2
431
501
476
448
415
540
537
410
52
402
403
404
394
395
8
10
185
5
400
102
541
Ncr
.
.
.
.
.
.
.
.
.
.
.
.
.
.g_
.g_
.g_
.
.g_
.aat_
.g.t.
.gat.
.t.gat.
.g.g.g.t....
.t.
.aa.t.g.t
.c.-ag.t.
.g.g.ag.g.
.c.cat.c.ttc.
.a.
. .a
. .a
. .g
.t. .
.a.c
.
. . .g.t. .
at.t.c.
.g.gg.ag.g.
.a.g.gt.
.c_t.tc.
.na.t.
.t.tc.
.a.at.tagt.c.
.a.at.tagt.c.
.g.c.c.g.c.ttcagag.
.g.c.c.g.c.
.g.c.c.g.c.
.g.
.e.g.c.
.c...
.ct.
.g.
. . ta. t. . aa. . . tt.gagaca.
.g. . .gg. .ggg.
. t.gagt.
.gg.g.cc.
. . .g.c. .
c. . . . gagt.. gattcacc
.g_att.tt.t.tn.
.g. . . .a.t.tt.g_att.tt. . .t.tt.
. .c. .ttcagag.
. . .t.tt.
. .c. .ttcagag.
.gtgatt.
.ttcagag.
.gtgatt.
.ttcagag.
.gtgatt.
. .a.g.at.g.tg.
agag. . . agggg.
c. ttc.gggtc
. a. . atc. . gagt. ggg. cctc.
. . a. t. tca. . ag. . t. gtt.
Fig. 1. Multiple alignment of ITS 1 sequences against that of S. sclerotiorum (LMK 2). The 5.8S and 18S
ribosomal RNA genes start at positions 17 and 212, respectively. Reference sequence at the top corresponds to
the antisense strand. Isolate accession numbers are indicated at left. In the aligned sequences, a dot = match, a
lower-case letter = mismatch, a dash = gap, and Ncr = Neurospora crassa. Sequence data for Neurospora crassa
is from Chambers et al. (1986). Asterisks denote sequences that extend beyond position 216: LMK 400,
*gccgactggagcattttttgagtttttaatgand LMK 541, *cctgctgctacagtaggagactcctcctttgtaatg.
Carbone
and
80
Kohn:
Molecular
90
100
TCTCTGGCGAGCATACA2
44
57
3
115
118
36
47
55
1
71
126
161
18
20
21
521
523
12
0-7
E76
0-2
431
501
476
448
415
540
537
410
52
402
403
404
394
395
8
10
185
5
400
102
541
Ncr
Systematics
of Sclerotiniaceae
110
120
-AGGCCCCGA-
a
a
a
?g
?g
?g
?g
gc
ct
c
c
a
cgag.g.
421
130
140
-AGAQCAGCTCGCC
g
g
g
g
ag
ag
ag
ag
ag
ag
ag
ag
ag
ag
ag
a.a
at
tc
gt.cg
gag
tga.gcattttaga
.cg
?g?.
ag
.g. .cctgt
?g
cc.g..ga.gcggtggccc
ctgaca.gcgcgggc
.cgg.
gccgaagccagcggt
ccgg
ccaggg..g.
.gg.g..
.c..gaggc
...ggc.
?c.tggc
g. . gng. an.
-.ct.c.gcgaacg.
tctcaggctcgaaagcttgaggctctggg-caattaagccc..g
-.et.c.gcgaacg.
tctcaggctcgaaagcctggggctctggg-caattaagccc..g
-.ctcc.gcg.gcg.
tctcaggctcgaaagcttgaggccttggg.cagttaaggcc.ag
ct
-.ctcc.gc..gcg.
tcccaggcccg..aaggcgctgg...tgtcccccgagggtgc-.
ct
-.ctcc.gc..gcg.
tcccaggcccg..aaggcgctgg...tgtcccccgagggtgc-.
ct
..ctcc.gc..gcg.
tctccggccccggagggcgctg....tgtccccggaagggtc..
ct
..ctcc.gc..gcg.
tctccggccccggagggcgctg....tgtccccggaagggtc..
ct
..ctcc.gc..gcg.
tctccggccccggagggcgctg....tgtccccggaagggtc..
c.
-.ctct.gcg.gcg.
tcttcggcttgtgaaagctaa.t.aacggtttagggct..<
c...c...ac..gggg-.
gct.tg.-.
g.gcgt
g.gggca.tca.cag.gg
-.cccggtggccgat.
g..c..
cgg. act. cg. . ggg.. gccgcgagcgggagacccgaggat..
gg.. ggcccgaaggcctttccggac
Fig. 1. See p. 420 for explanation.
homoeocarpa (LMK 8), Botrytis cinerea (LMK
18), Rutstroemia petiolorum (LMK 402), and
Monilinia megalospora (LMK 415)] were used
as query sequences (data not shown). Most ofthe
sclerotial isolates, including the three isolates of
Sclerotium cepivorum, were tightly clustered together showing close similarity to Sclerotinia
sclerotiorum (S = 0.76-1.0). The remaining iso-
422
Mycologia
150
160
170
180
190
200
210
18S->
AAAGC
AACAAAGTAATAATACACAAGGGTGGGAGGTCTACCCTTT-CGGGCATG-AACICTGTAATG
2
44
57
3
115
118
36
47
55
1
71
126
161
18
20
21
521
523
12
0-7
E76
0-2
431
501
476
448
415
540
537
410
52
402
403
404
394
395
8
10
185
-J
400
102
541
Ncr
. . .ta.g.a.
.t.
. gt. . . . agg. t. tgcc. ttttt.
a. . . g. a. .
g.
.cg.a.
.a.
.gtt.
gtta. . ataca.gcg.a.
. . . a. . gt. . ga. atcta. . ccga. . . . a.
.tt
at.
. . g.gt.t.
.a.
.tggg...g...a..gt....agg.t.tgcccctgt......g.-..
..
.. g.cg....
. n.g..
c.gc.
.g...g..
.ta.g.a.
.ta.-.g.a.
. .t. . . tag.g.g.a.
.cg., .ta.g.g.a.
?tg., .ta.g.g.a.
?g.
.tg., ?gag.t.g.g.a-g.
?g.
.tg., ?gag.t.g.g.a-g.
?g.
?gag.t.g.g.a-g.
.tg.
. . .a.
.g.tcg.ga.agcc.t.ta.g.g.act.
.
. . .c. . . .tc.ttc.aa.g.ggt.ggcacttcaatc.cgctgacgg.
.gc.g.gc*
g.g.g.
. cg. g-.
. . g.t....t..c.a.tg.
actataa.
a. gcga. *
acc. t. gggagggggcc.
tggtcgtcgagcag.
.gt...g.c.g..c....t..g...
.
cc...gc.g.g.c..ccg...gggtaa.attcgc.atggtttg.gggagtttt..?
Fig. 1. See p. 420 for explanation.
lates, representing all of the substratal stromatal
taxa, the outgroups, and three sclerotial taxa,
Monilinia megalospora, M. oxycocci, and Ciborinia erythronii, showed less similarity to S.
sclerotiorum (S = 0.45-0.65) and formed several
clusters. Within the sclerotial cluster were two
subclusters of very similar taxa, the Sclerotinia
cluster (S > 0.98) and the Botrytis cluster (S >
Carbone
and
Kohn:
Molecular
Systematics
of Sclerotiniaceae
423
0.96). Among the predominantly substratal stro?
matal and outgroup isolates there was one notable large cluster including a subcluster with
and Sclerotinia hoRutstroemia henningsiana
=
and
a
subcluster with R.
moeocarpa (S
0.81),
> 0.87).
petiolorum, R. sydowianaandR.firma(S
branch to all Rutstroemia
species, including
Sclerotinia homoeocarpa, supported in 100 out
of 100 bootstrap replications.
Although this cluster of Rutstroemia species in?
cluding S. homoeocarpa could be distinguished,
a substratal stromatal lineage could not be discerned. As shown in Fig. 2, similarity to the
query sequence among two of the three outgroup
isolates from the Leotiaceae (LMK 540, Bisporella citrina and LMK 537, Ionomidotis sp.), a
closely related family in the same order (Helotiales), was within the same range (S = 0.45-0.65)
Results of multiple alignment and hierarchical
clustering in this study show that most of the
sclerotial taxa, including Sclerotium cepivorum,
cluster together, showing close similarity to S.
sclerotiorum. This supports our hypothesis that
a sclerotial lineage exists and suggests that this
lineage has evol ved relatively recently. Although
a cluster of species within Rutstroemia can be
distinguished, we conclude that a substratal stro?
matal lineage cannot be discerned based on this
genomic region. Among these taxa, the ITS 1
sequences are saturated with changes. The vari?
ation in ITS 1 does not resolve among the more
distantly related taxa. The positions among deeply
rooted branches in Fig. 2 are therefore not significant. As a result, it is not surprising that the
three outgroup taxa representing the Leotiaceae
are found among the substratal stromatal ingroup in Fig. 2. With the exception ofthe rela?
tionships within the Rutstroemia cluster, essentially all of the substratal stromatal taxa and the
three outgroup taxa are equally unrelated with
respect to ITS 1. Our alternative hypotheses concerning the relatedness of the substratal stro?
matal taxa to taxa in the sclerotial lineage and
the Leotiaceae cannot be tested with sequence
data from the ITS. Within the Rutstroemia clus?
ter, however, the position of Sclerotinia homoeo?
carpa is significant among the other Rutstroemia
as among the ingroup substratal stromatal iso?
lates and three sclerotial isolates from the Sclero?
tiniaceae. Neurospora crassa rooted most deeply
in the clustering (S = 0.30, with an expected
random match of S = 0.25); although the align?
ment of the internal portion of ITS 1 of N. crassa
to those of the other isolates in our sample was
very poor, the ends extending into the coding
regions aligned well.
Even with powerful computing,
parsimony
analysis of the data set was problematic because
of the site saturation and the larger sequence size
of ITS 1 in the substratal stromatal and outgroup
isolates, especially when compared to the rela?
tively conserved sequences (yielding few char?
acters) of most of the sclerotial isolates. The
branch-and-bound
search function with PAUP
failed to produce a tree, even when taxa were
reordered and one isolate was used to represent
each taxon. A heuristic search with PAUP with
only one isolate representing each taxon pro?
duced a tree with similar topology to that pro?
duced by hierarchical
but with
clustering,
branches supported no more than 46-78%. Subclusters defined by hierarchical clustering with
MULTALIN and branch-and-bound
searches of
certain subsets of taxa with PAUP were similar.
Trees produced by these branch-and-bound
searches are shown in Figs. 3-8. In trees shown
in Figs. 3 and 5, no branch is supported in >95
of 100 bootstrap replications. Sequences of iso?
lates in the Botrytis cluster produced 57 most
parsimonious
trees, with little support for
branching by bootstrap resampling (Fig. 5) or
50% majority rules (Fig. 6), and with little resolution by strict consensus (Fig. 7). Parsimony
analysis of the Rutstroemia cluster resulted in
one most parsimonious
tree (Fig. 8) with the
DISCUSSION
species.
The results of this study are in agreement with
immunological data from a previous study (Novak and Kohn, 1991), particularly the western
blot data as indicated in Fig. 2 of this paper, and
enthe discriminant
analysis of competitive
immunosorbent
zyme-linked
assays (ELISAs;
Fig. 6; Novak and Kohn, 1991). In both the
discriminant analysis ofthe competitive ELISAs
and our hierarchical clustering and parsimony
analyses, the sclerotial isolates including Scle?
rotium cepivorum were clustered together, show?
ing close similarity to S. sclerotiorum, while the
substratal stromatal isolates showed less simi?
and
larity and did not cluster. Immunological
sequence similarity data also show that Sclero?
tinia homoeocarpa is more closely related to the
substratal stromatal genera than to other Sclero-
424
Mycologia
WBMS
?
0.4
0.6
Similarity
0.8
Sclerotinia sclerotiorum(2)
S. sclerotiorum(44)
S. sclerotiorum(57)
' S. minor(3)
S. minor(115)
S. minor(118)
r S. trifoliorum(36)
i S. trifoliorum(47)
'
S. trifoliorum(55)
cepivorum(1)
S. cepivorum(71)
HSclerotium
S. cepivorum(126)
? Cristulariellamoricola (161)
clnerea (18)
B. clnerea (20)
HBotrytis
B. cinerea (21)
sp. (521)
KBotrytis
B. calthae (523)
Moniliniafructicola (12)
i? Myriosclerotiniadennisii (0-7)
Ti- Ciboriniaciborium(E76)
*- Myriosclerotiniascirpicola (0-2)
?
Moniliniafructigena (431)
?
Ciboriacaucus (501)
?
C. acerina (476)
?
Verpatiniacalthicola (448)
?
Moniliniamegalospora (415)
?
Bisporella citrina (540)
?
lonomidotissp. (537)
?
Moniliniaoxycocci (410)
^? Ciboriniaerythronii(52)
?
Rutstroemiapetiolorum (402)
?
R. sydowiana (403)
?
R. firma (404)
r R. henningsiana(394)
i- R. henningsiana(395)
Sclerotiniahomoeocarpa (8)
S. homoeocarpa(10)
S. homoeocarpa(185)
Lambertellasubrenispora (5)
L. langei (400)
Piceomphale bulgarioides (102)
Ascocoryne cylichnium(541)
1.0
scale
Fig. 2. Hierarchical clustering based on multiple alignment of ITS 1 sequence data and comparison with
other characters. Pairwise similarity scores are represented on a fractional similarity scale where a value of 1.0
(or 100%) corresponds to identical sequences. Isolate accession numbers in the collection of L. M. Kohn are
shown in brackets. Column WB shows results of western blot analysis from Novak and Kohn (1991); +, crossreaction; ?, no cross-reaction; n, no data available. Column MS designates macroconidial state; B, Botrytis; C,
Cristulariella; MJ, Monilia lacking disjunctors; MD, Monilia with disjunctors; o, no macroconidia; *, have
distinctive macroconidial states that are morphologically different from those of the Sclerotiniaceae.
Carbone
and
Kohn:
Molecular
B. cinerea (18)
C. moricola (161)
S. cepivorum(1)
?
S. trifoliorum(36)
l?j. S. minor(3)
S. sclerotiorum(2)
Systematics
of Sclerotiniaceae
- S. sclerotiorum(2)
J-
B. cinerea (18)
B. calthae (523)
M. dennisii (0-7)
,?
C. ciborium(E76)
?
M. scirpicola (0-2)
??
Fig. 3. Unrooted phylogenetic tree based on the
multiple alignment of the ITS 1 of five species in the
Sclerotinia cluster with Botrytis cinerea (LMK 18) as
the outgroup. One of two most parsimonious trees is
shown. Horizontal distance is proportional to branch
length; vertical distance does not contribute to branch
length. The scale bar equals two character state changes.
The consistency index of both trees was 1.000. No
branch was supported in >95 of 100 bootstrap repli?
cations.
tinia spp., suggesting that it should be reclassified. Parsimony analysis ofthe Rutstroemia subcluster (Fig. 8) with bootstrapping suggests that
S. homoeocarpa may be accommodated
in Rut?
stroemia, but a comparison with other species in
closely related genera, such as Lanzia and Moel?
lerodiscus, is needed.
With the exception of a single base substitution
in one isolate of S. minor (LMK 118), and vari?
ation in a single base in the two isolates of Rut?
stroemia henningsiana (LMK 394, 395), the ab?
sence of intraspecific variation in ITS 1 within
our sample of sclerotiniaceous
isolates was notable. In contrast, O'Donnell (1992) recently re?
ported multiple ITS types within Fusarium sambucinum Fckl. (teleomorph = Gibberella pulicaris
(Fr.) Sacc). Different rates of ITS evolution, with
examples in the literature of resolution at intra?
specific, species, genus, and suprageneric levels
among different groups of fungi, could be due
Al fructigena (431)
M. fructicola (12)
Botrytis sp. (521)
Fig. 5. Unrooted phylogenetic tree based on the
multiple alingment ofthe ITS 1 of seven species in the
Botrytis cluster with S. sclerotiorum (LMK 2) and M.
fructigena (LMK 431) as outgroups. One of 57 most
parsimonious trees is shown. Horizontal distance is
proportional to branch length; vertical distance does
not contribute to branch length. The scale bar of branch
lengths equals two character state changes. The consistency index ofthe tree shown was 0.880. No branch
was supported in >95 of 100 bootstrap replications.
either to a molecular clock with variable rates or
to the as yet unexplained effects of life cycle strategies on rates of speciation (Bruns et al., 1991).
There are several interesting taxonomic implications in our study. First, clustering based on
similarity does not suggest that all sclerotial taxa
should be accommodated under one genus Scle?
rotinia. Sequences of Sclerotium cepivorum and
Cristulariella moricola show 98% similarity to
that of Sclerotinia sclerotiorum, suggesting a close
relationship that is also supported by other biochemical and morphological
characters. Taxo?
nomic placement ofthe apparently "mitotic speS. sclerotiorum(2)
B. cinerea (18)
- B. cinerea (18)
B. calthae (523)
M. fructicola (12)
. C. moricola (161)
M. dennisii (0-7)
?S. cepivorum(1)
?S. trifoliorum(36)
M. scirpicola (0-2)
S. minor(3)
L? S. sclerotiorum(2)
Botrytis sp. (521)
Fig. 4. Strict consensus of the two most parsimo?
nious trees in which only the branching topology is
significant. Branch lengths are not drawn to scale.
425
C. ciborium(E76)
M. fructigena (431)
Fig. 6. Unrooted phylogenetic tree based on 50%
majority rule of the 57 trees. Numbers indicate the
percentage of trees that support the branch.
426
Mycologia
S. sclerotiorum(2)
. S. sclerotiorum
(2)
?
B. clnerea (18)
-
?
Botrytis sp. (521)
?
B. calthae (523)
?
M. fructicola (12)
?
M. dennisii (0-7)
?
C. ciborium(E76)
?
M. scirpicola (0-2)
?
M. fructigena (431)
?
Fig. 7. Strict consensus of the 57 most parsimo?
nious trees in which only the branching topology is
significant. Branch lengths are not drawn to scale.
cies" (see Reynolds and Taylor, 1992) within a
genus in the Sclerotiniaceae would require both
more molecular characters, yielding a statistically well-supported
placement
phylogenetic
within a genus, and revision of Article 59 of the
International Code of Botanical Nomenclature.
Another intriguing question is whether the unusual Cristulariella conidial anamorph is derived
from sclerotia or appressoria rather than a modification of the Botrytis conidial form.
Isolates of all Botrytis species examined, in?
cluding LMK 521, a new species on Rubus from
and Schumacher,
in
Norway (Holst-Jensen
manuscript), show >96% similarity. Although a
well-supported lineage cannot be discerned from
this data, the Botrytis cluster (Figs. 2, 5-7) shares
a unique deletion/substitution
motif between po?
sitions 110 and 140 (Fig. 1).
The >97% similarity of Ciborinia ciborium to
Myriosclerotinia isolates is cause to question the
recent transfer of this Carex- and Eriophoruma genus
infecting species from Myriosclerotinia,
of fungi infecting members of the Cyperaceae and
Juncaceae, to Ciborinia (Schumacher and Kohn,
1985). The divergence in ITS 1 between C. ci?
borium and C. erythronii is notable; we think that
Ciborinia as presently circumscribed is a heterogeneous grouping in need of monographic revi?
sion.
While Monilinia fructicola and M. fructigena
in the Junctoriae group of the genus Monilinia
(Batra, 1991) fall within the exclusively sclerotial
cluster (both with >76% similarity to S. scleroti?
orum), M. megalospora and M. oxycocci, in the
Disjunctoriae group, are outside of the sclerotial
cluster (with 65% and 56% similarity to S. scle-
P. bulgarioides
(102)
r R. petiolorum
(402)
I? R. sydowiana(403)
R. firma(404)
R. henningsiana
(394)
S. homoeocarpa
(8)
?L subrenispora
(5)
8
Fig. 8. Unrooted phylogenetic tree based on the
multiple alignment of the ITS 1 of six species in the
Rutstroemia cluster (including Lambertella subrenis?
pora) with S. sclerotiorum (LMK 2) and P. bulgarioides
(LMK 102) as outgroups. Horizontal distance is proportional to branch length; vertical distance does not
contribute to branch length. The scale bar equals ten
character state changes. The consistency index of the
tree shown was 0.902. Asterisks indicate those branch?
es that are supported in >95 of 100 bootstrap repli?
cations.
rotiorum, respectively). In an independent study,
utilizing some ofthe same isolates as in our sam?
ple, Holst-Jensen (1992) analysed morphologi?
cal, ecological, and restriction fragment length
polymorphism (RFLP) characters using UPGMA
and PAUP, deriving a strongly supported branch
(>95 out of 100 bootstrap replications) separating the Junctoriae (lacking disjunctors between
macroconidia and infecting rosaceous hosts with
fleshy fruits) from the Disjunctoriae (possessing
disjunctors and infecting ericaceous or rosaceous
hosts with relatively dry fruits), in agreement with
our ITS 1 data. Interestingly, the branching topologies of both his dendrograms and cladograms support the separation of M. megalospora
and M. oxycocci that we observed; he hypothesizes that this reflects the earlier divergence time
of the former species.
Lastly, the rejection of Honey's lectotypification of Rutstroemia
(Kohn and Schumacher,
in
Rabenh.
1984) with Peziza bulgarioides
Kalchbr. (= Piceomphale bulgarioides) is sup?
ported by the lack of similarity in ITS 1 of our
isolate of this species to the Rutstroemia cluster
(Fig. 8) especially in comparison to our isolate
of the species that was ultimately conserved as
the type species R. firma.
In addition to the taxonomic implications, ITS
variability is being exploited for disease diagnosis and identification of other phytopathogenic
fungi (Xue et al., in press; Nazar et al., 1991) and
could be used to identify mycelia and sclerotia
Carbone
and
Kohn:
Molecular
of the sclerotial Sclerotinaceae infecting or infesting plants and seeds. Data from this study
could be used in the development of diagnostic
probes for the identification of
oligonucleotide
taxa or groups of taxa.
acknowledgments
This research was supported by a Summer Bursary
for IC and an Operating Grant to LMK from the Natural Sciences and Engineering Research Council of
Canada. We thank all those who furnished cultures.
Thanks also to Jim Anderson and Jerry Brunner.
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Accepted for publication February 11, 1993