Mycol. Res. 108 (4): 393–402 (April 2004). f The British Mycological Society
393
DOI: 10.1017/S0953756204009372 Printed in the United Kingdom.
Polymorphisms in nuclear rDNA and mtDNA reveal the
polyphyletic nature of isolates of Phomopsis pathogenic
to sunflower and a tight monophyletic clade of defined
geographic origin
Djaouida REKAB1, Giovanni DEL SORBO2, Carmen REGGIO2, Astolfo ZOINA2 and Giuseppe FIRRAO1*
1
Dipartimento di Biologia Applicata alla Difesa delle Piante, Università di Udine, via Scienze 208, Udine 33100, Italy.
Dipartimento di Arboricoltura, Botanica e Patologia vegetale, Sez. di Patologia vegetale, Università di Napoli ‘ Federico II ’,
via Università 100, I-80055 Protici (Napoli), Italy.
E-mail : firrao@pldef.uniud.it
2
Received 3 June 2003; accepted 29 December 2003.
The molecular diversity of Diaporthe helianthi (anamorph Phomopsis helianthi), the causal agent of sunflower stem
canker, was studied in 16 isolates of different geographic origin using nuclear and mitochondrial markers. PCR products
corresponding to the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA gene, and to the
mitochondrial atp6 gene were sequenced. The ITS1 and ITS2 sequences were compared with those of Phomopsis spp. and
Diaporthe spp. obtained from databases. The diversity in the region surrounding the atp6 gene was also studied by
restriction analysis using four enzymes. The analyses revealed a marked diversity within the sunflower-isolated strains,
which appear to belong to phylogenetically unrelated groups. Noticeably, all the isolates collected in France and in the
former Yugoslavia, where severe epiphytotics of sunflower stem canker are frequently reported, showed high similarity to
each other forming a clade which clearly differentiated from all other ones within the genus Phomopsis. Conversely, all
the isolates collected in Italy, where, despite favourable environmental conditions, the incidence of the disease is low,
were only distantly related to the former group and showed sequence similarity with other previously established
phylogenetic clades within the Phomopsis/Diaporthe complex.
INTRODUCTION
Diaporthe helianthi (anamorph Phomopsis helianthi) is
the causal agent of sunflower stem canker, a disease
reported to cause serious economic damage in several
countries (Mihaljevic, Petrov & Cvetkovic 1980,
Pentericci 1988, Delos & Moinard 1995). The fungus
was first described in the late 1970s in the then
Yugoslavia (Mihaljevic et al. 1980), and has been reported in Romania (Iliescu et al. 1985), Hungary
(Varos, Leranth & Vajnal 1983), France (Lamarque &
Perny 1985) and the USA (Herr, Lipps & Walters
1983). In Italy, the anamorph of the fungus was found
on sunflower in 1987 (Zazzerini, Tosi & Losavio 1988).
Heavy epiphytotics of stem canker have been reported
in France and the former Yugoslavia, whereas in Italy,
despite the climatic conditions which are favourable for
infection, the disease only occurs sporadically and does
not cause significant yield losses.
The taxonomic relationships within the Diaporthe/
Phomopsis complex have been historically difficult to
establish. Most often, these fungi have been classified
* Corresponding author.
on the basis of the diseases they cause on plants and a
strict association between host plant and pathogen
species has been suggested (Uecker 1988). A similar
situation applies also to the Phomopsis teleomorph,
Diaporthe, resulting in a proliferation of species names.
In the revision of the genus Diaporthe by Wehmeyer
(1933), the number of accepted species was reduced
from 650 to 70, assuming that some species of
Diaporthe may occur on more than one host. Similarly,
evidence that host association is not a basis for delimiting species in Phomopsis was provided by Brayford
(1990), who showed that Phomopsis occurring in twigs
and bark of Ulmus species form two genetically and
morphologically discrete groups, and that both groups
could also be found in Acer pseudoplatanus, Fagus
sylvatica and Fraxinus excelsior. Rehner & Uecker
(1994) presented results of a phylogenetic analysis
based on ITS region sequence of 51 isolates showing
that in most cases there was no evidence of coevolution
between Phomopsis and its hosts at any hierarchical
level revealed within the ITS phylogeny.
Attempts have been made to determine relationships
between Phomopsis isolates on the basis of morphological characters, but were practically limited by
Polymorphisms in nuclear rDNA and mtDNA
character plasticity, as variation within a single isolate
may be large (Wehmeyer 1933, Nitimagi 1935, Parmeter 1958, Morgan-Jones 1985). Phomopsis anamorphs produce two kinds of conidia : alpha-conidia,
which are viable and infective, and beta-conidia, which
are thought to be unable to germinate and do not have
any apparent role in epidemiology. Early work on D.
helianthi has demonstrated significant morphological
variability among isolates. In the former Yugoslavia,
two groups were described (Maric, Masirevic & Li
1982) : one mainly producing alpha-conidia, and the
other a prevalence of beta-conidia. In the USA three
groups were described, two corresponding to the
Yugoslavian ones, and a third one producing both
types of conidia in equal proportions (Herr, Lipps &
Walters 1983).
Beside morphological criteria, biochemical and molecular methods have been used to study the variability
among phytopathogenic Phomopsis isolates (MorganJones 1985, Meijer, Megnegneau & Linders 1994,
Muntanola-Cvetkovic, Vukojevic & Mihaljcevic 1996,
Vannacci et al. 1996). In particular, the sequence
analysis of the ITS region has been the subject of several recent studies (Rehner & Uecker 1994, Uddin et al.
1998, Zhang et al. 1998, Farr, Castlebury & PardoSchultheiss 1999, Guo, Hyde & Liew 2000, Harrington
et al. 2000, Kanematsu et al. 2000, Mostert et al. 2001).
More than 200 entries can now be found in public
databanks reporting sequences of either ITS1 or ITS2
or both of isolates affiliated to Phomopsis or Diaporthe.
Despite this noteworthy sequencing effort, a comprehensive picture of the taxonomy of this group has not
yet been provided, because the degree of divergence
among sequences is high, resulting in low support for
some clades obtained by parsimony analysis. Farr et al.
(1999) found relatively low bootstrap support in their
phylogenetic analysis, and suggested that the ITS
regions sequences alone may be not appropriate for
determining the relationships among those fungi.
Although a significant number of sequences of
Diaporthe spp. have been determined, only a small number of isolates of D. helianthi have been included in the
cited papers. Recently, Says-Lesage et al. (2002) carried
out a more comprehensive study on the diversity of
D. helianthi strains isolated in France, as deduced from
AFLP analysis ; those authors also determined the
rDNA ITS1 and ITS2 sequences of seven French
D. helianthi isolates, and showed that sunflower isolates
clustered separately from soybean reference isolates.
The study of Says-Lesage et al. (2002) was, however,
limited to French isolates.
All molecular studies concerning the phylogenetic
relationships within the Diaporthe/Phomopsis complex
have been based on nuclear rDNA sequences. The relevance of the analysis of independent loci to support
taxonomically sound clades of microorganisms has
been repeatedly highlighted (Waalwijk et al. 1996,
O’Donnell, Cigelnik & Nirenberg 1998, Young 2001).
Mitochondrial DNA has been shown to be highly
394
Table 1. List of Phomopsis/Diaporthe isolates used in the study,
with host and geographical origin.
Lane
(Fig. 2)
Isolate
Received as
Origin
Host
1
N1
D. helianthi
Sunflower
2
CBS 592/81 D. helianthi
3
IMI 313865
D. helianthi
4
5
6
7
8
9
10
11
12
13
14
15
16
17
IMI 313866
IMI 313861
F2
F1
A3
A2
A1
R1
CBS 187/87
I4
I3
I2
I1
227-98
18
21-99
19
10-99
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. helianthi
D. phaseolorum
var. sojae
D. phaseolorum
var. caulivora
D. phaseolorum
var. meridionalis
Former
Yugoslavia
Former
Yugoslavia
Former
Yugoslavia
France
France
France
France
Argentina
Argentina
Argentina
Romania
Italy
Italy
Italy
Italy
Italy
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Sunflower
Soybean
Soybean
Soybean
valuable in the detection of polymorphisms and in
tracing phylogenetic relationships among phytopathogenic fungi (Forster et al. 1986, Bates, Buck & Brasier
1993, Fukuda et al. 1994, Guillamòn et al. 1994, Varga
et al. 1994). The sequences of a 402 bp fragment of the
coding region of the gene encoding cytochrome b, were
used for classification of several species of the section
Fumigati in the genus Aspergillus (Wang et al. 2000).
In Candida glabrata the differentiation of isolates originating from Brazil from those originating in the USA
was possible on the basis of polymorphisms in the
COX2 gene, encoding the subunit 2 of cytochrome c
oxidase (Sanson & Briones 2000).
In the present paper, mitochondrial DNA polymorphisms occurring in the sequence of the atp6 gene
coding region and RFLPs in a region surrounding this
gene have been used in combination with sequence
polymorphisms in the nuclear rDNA ITS regions to
study variability occurring in isolates of Diaporthe
pathogenic to sunflower to reveal association of DNA
polymorphisms with geographic origin of isolates.
MATERIALS AND METHODS
Fungal isolates and nucleic acid extraction
The Diaporthe isolates used in this work are listed in
Table 1. They include all the available strains isolated
in Italy (available on request) and all strains available
from the major international culture collections. All
isolates had been previously identified as D. helianthi
D. Rekab and others
based upon morphological criteria and were routinely
grown on potato dextrose agar medium (PDA ; Difco
Laboratories, Detroit, MI) in Petri dishes at 25 xC in
the dark for 7 d. For long-term storage, spores were
put on silica gel and kept at x20 x. Mini-preparation
of total genomic DNA were obtained from 1-week-old
mycelia grown on PDA plates covered by Bio-Rad
membrane (Bio-Rad Laboratories, Hercules, CA) at
25 x in the dark for 7 d. Total nucleic acids were extracted from 350 mg of fresh mycelium as described in
Lecelier & Silar (1994), resuspended in 50 ml of sterile
distilled water and stored at x20 x. When larger
amount of nucleic acids were needed (e.g. for Southern
blot analysis), 100 ml shaken cultures in PDB were
inoculated with small (1 mm3) agar plugs cut from
the edge of growing colonies. Fungal mycelia (1 wk old)
were collected on sterile filter paper by vacuum filtration, extensively washed with sterile water, immediately frozen under liquid nitrogen and freeze dried.
DNA was extracted from 1 g freeze dried mycelium
according to Garber & Yoder (1984) and stored in TE
at 4 x.
Nuclear rDNA sequence analysis
The ITS region of the nuclear rDNA was PCR amplified using primers ITS4 and ITS5 (White et al. 1990)
and 2 ml of the fungal nucleic acid extract obtained as
described above. Amplification was carried out in 50 ml
of PCR Buffer (Boehringer Mannheim, Mannheim) in
the presence of 150 ng of each primer, 200 mM of each
dNTP (Boehringer Mannheim) and 2 U of Taq polymerase (Boehringer Mannheim). Reactions were cycled
35 times with the following parameters : 95 x (30 s),
56 x (75 s) and 72 x (90 s) on a model 9600 thermocycler
(PerkinElmer, Norwalk, NJ). Amplified DNA was
electrophoresed on 1 % (w/v) agarose gels run at 100 V
for 1 h in Tris-acetate buffer, stained with ethidium
bromide, and photographed under UV light. PCR
products were purified with Qiaquick PCR purification
Kit 50 (Qiagen, Hilden) according to the manufacturer
instructions. Purified DNA was quantified by measuring the absorbance at 260 nm wavelength. Cyclesequencing reactions with both primers ITS4 and ITS5
were carried out using the dye terminator procedure
with fluorescent dideoxynucleotides.
Sequence analysis of the mitochondrial atp6 gene
Degenerate primers ATP6-FOR1 (5k AGTCCWYTWGMYCAATTTGAA 3k) and ATP6-REV1
(5k CATGTGACCWSWTAAWATRTTWGC 3k) were
used for PCR amplification of a 558 bp fragment of
the atp6 gene. Both primers were designed on two
sequences (SPL(D/T)(Q/E)FE and ANILSGHM)
conserved at protein level, which were found in the
alignment of the coding part of the of Aspergillus
nidulans (GenBank accession no. J01390), Cochliobolus
heterostrophus (X13439), Podospora anserina (X15602),
395
Penicillium chrysogenum (L19866) and Trichophyton
rubrum (X88896). PCR reaction mixtures were prepared in a total volume of 50 ml PCR buffer (Promega)
containing 300 ng of genomic DNA as template,
200 mM dNPTs, 150 ng of each primer, 1.5 mM MgCl2,
2.5 U of Taq polymerase (Promega). Samples were denatured at 94 x for 2 min and then subjected to 30 cycles
of amplification under the following conditions : 30 s at
94 x, 45 s at 50 x and 2 min at 72 x. Extension of incomplete products was done with a final incubation at
72 x for 7 min. The 558 bp amplification products were
gel purified, eluted from agarose with the QIAEXII
Gel extraction kit (Qiaex), ligated in pGEM-T Easy
(Promega, Madison, WI) and transformed into
Escherichia coli JM109 according to standard molecular techniques. For each Diaporthe strain amplification
products obtained in three independent PCR reactions
were cloned and sequenced on both strands using the
dye terminator procedure with fluorescent dideoxynucleotides.
Southern blot analysis of mitochondrial DNA
Four digests (EcoRI, HaeIII, HindIII and TaqI; all
Promega) were performed on DNA of each of the
19 isolates of Diaporthe. Digestions (20 mg DNA) were
performed overnight using 20 units of enzyme in a
volume of 200 ml at 37 x (for EcoRI, HaeIII, HindIII) or
65 x (TaqI). After digestion, DNA was precipitated by
addition of 20 ml of 3 M sodium acetate pH 5.2 and
440 ml absolute ethanol, incubated at x20 x for 3 h and
recovered by a short (3 min) centrifugation at 14 000 g.
Each digest was redissolved in 20 ml of TE and electrophoresed at 50 V for 4 h. Southern blot analysis was
performed according to standard molecular procedures
(Sambrook et al. 1989) using a digoxigenin-labeled
558 bp fragment obtained by PCR amplification of the
atp6 gene on genomic DNA of isolate F2 with primers
ATP6-FOR1 and ATP6-REV1 (see above). Labeling
of the probe with digoxigenin and chemiluminescent
detection of hybridization bands were performed using
a DIG-labeling and detection kit (Roche Diagnostics,
IN) according to manufacturer instructions.
Sequence analysis
Pairwise alignments of the sequences were produced
using SeqPup (D. G. Gilbert, Indiana University, IN).
Alignments were adjusted manually and used to score
nucleotide differences between each pair of sequences.
In order to compare the sequences diversity of the
nuclear rDNA ITS and the mitochondrial atp6 gene, the
Shannon–Weaver diversity index (Shannon & Weaver
1949) was calculated considering each nucleotide position as an independent character and then normalized
according to Goodwin et al. (1993) to give the diversity
index Dk which ranges from 0 (all isolates are identical)
to 1 (maximum diversity among isolates).
For the phylogenetic analysis of the ITS region of
nuclear rDNA, the sequence data matrix (M1105)
Polymorphisms in nuclear rDNA and mtDNA
396
Table 2. Number of nucleotide differences scored in pairwise comparison between sequences of the fungal strains isolated from sunflower.
Upper triangle: mitochondrial atp6 sequences. Lower triangle: nuclear rDNA ITS.
N1
N1
CBS 592/81
IMI 318861
A3
IMI 313866
F1
IMI 318865
F2
I2
I3
I4
CBS 187/87
A2
A1
I1
R1
CBS
592/81
0
0
0
4
2
2
3
4
33
33
32
34
32
41
36
56
0
4
2
2
3
4
33
33
32
34
32
41
36
56
IMI
318861
0
0
4
2
2
3
4
33
33
32
34
32
41
36
56
A3
0
0
0
3
3
4
5
31
31
31
31
31
39
35
54
IMI
313866
0
0
0
0
0
1
2
32
32
33
33
32
40
35
54
F1
0
0
0
0
0
1
2
32
32
33
33
32
40
35
54
associated with the phylogenetic study (SN220) carried
out by Farr et al. (1999) was retrieved from TreeBase.
The sequences obtained in this study and that of
Says-Lesage et al. (2002) were then introduced in the
data set and aligned, with the exclusion of 21 positions,
to produce a new matrix of 52 taxa and 328 characters.
Details on the sequences included as reference are
reported in Farr et al. (1999). The resulting matrix
has been deposited in the TreeBase database with the
accession number SN1051-2914. Maximum parsimony
trees were inferred using the heuristic search (random
sequence addition, 10 replicates) and branch swapping
(tree bisection–reconnection) options of PAUP 4.0b2
(D. L. Swofford, Sinauer Associates, Sunderland,
MA). Relative support for the branches were estimated
with 1000 bootstrap replications.
Identification by sequence comparison
The sequences of the ITS region of the nuclear rDNA
obtained as described above were aligned to nearly all
sequences deposited as ribosomal ITS sequences of
Diaporthe spp. or Phomopsis spp. in public databases.
When ITS1 and ITS2 sequences for certain isolates
were deposited as two distinct accessions, sequences
were reconstructed and included only when data were
available to confirm source identity with certainty. The
nucleotide positions corresponding to 5.8S rRNA gene
were excluded from the alignment. The alignment
was carried out manually using the multiple sequence
editor SeqPup. About 35 nucleotide positions were
then excluded due to the difficulties in establishing a
consensus and therefore an unambiguous alignment.
The final matrix (TreeBase accession no. SN1051-2919)
consisted in 154 taxa and 317 nucleotide positions.
Distances between sequences were calculated using the
Jukes & Cantor (1969) coefficient by the program
DNAdist of the package PHYLIP (J. Felsenstein,
University of Washington, WA). As some positions
IMI
318865
0
0
0
0
0
0
3
31
31
31
31
32
39
35
54
F2
I2
I3
I4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
20
20
20
20
20
20
20
20
1
1
1
1
1
1
1
1
1
19
34
34
33
35
33
42
37
57
0
25
25
13
18
16
50
25
25
13
18
16
50
2
28
27
22
50
CBS
187/87
0
0
0
0
0
0
0
0
0
20
1
27
27
22
49
A2
A1
I1
R1
2
2
2
2
2
2
2
2
2
22
3
2
20
20
20
20
20
20
20
20
20
9
19
20
22
20
20
20
20
20
20
20
20
20
10
19
20
22
11
0
0
0
0
0
0
0
0
0
20
1
0
2
20
20
23
19
57
18
47
46
were excluded, some null distances resulted even between non-identical sequences.
RESULTS
DNA sequencing
Nuclear rDNA ITS1 and ITS2 sequences were obtained from 16 Diaporthe helianthi strains and three
reference strains of D. phaseolorum varieties by direct
sequencing of the PCR product. All sequences were
deposited in GenBank database (accession nos
AJ312348–AJ312366).
Primers ATP6-FOR1 and ATP6-REV1 allowed the
amplification of a single product of the size expected
for the atp6 gene (558 bp) from all isolates of Diaporthe. For each of the 19 isolates used in the present
study, sequencing of both strands of three independently obtained PCR products gave identical results.
All sequences were deposited in the GenBank database
(accession nos AY263688–AY263706).
A preliminary pairwise comparison between the
sequences (Table 2) revealed that all seven isolates of
D. helianthi from France and the former Yugoslavia
and isolate A3 from Argentina were very similar either
in rDNA ITS (>98.5 % identity between each pair of
sequences) and in the atp6 gene (all sequences identical). Because this group of isolates included all isolates
from the former Yugoslavia, where the fungus was first
detected and described, it is hereafter referred to as
D. helianthi s. str. The nuclear rDNA ITS sequences
from isolates I4 and CBS 187/87 were similar to each
other, as were those from isolates I2, I3 and A2. The
nuclear rDNA ITS sequences of the remaining isolates
were not similar to any other. In general, the sequences
showed a relatively high degree of variation from strain
to strain (diversity index Dk=0.138), thus confirming
the value of this region for the inference of taxonomic
relationships within the Phomopsis complex.
D. Rekab and others
Conversely, the sequence diversity detected in the
mitochondrial atp6 gene was much lower (diversity index Dk=0.018). The sequences of 12 of the 19 isolates
examined were invariant, the 12 isolates all having the
same sequence at the atp6 locus included all isolates
originating from France (IMI 313866, IMI 318861, F1,
F2) and the former Yugoslavia (N1, CBS 592/81, IMI
313865) as well as the Argentinian isolate A3, two (CBS
187/87 and I2) out of the four isolates from Italy,
the isolate R1 from Romania and isolate 227/98 of
D. phaseolorum var. meridionalis. Since the variations
within the chosen region of atp6 appeared not to be
adequate for consistent differentiation of taxa, the
investigations were extended to a larger part of the
mitochondrial genome by RFLP analysis (see below).
Phylogenetic analysis of nuclear rDNA ITS
The sequences obtained were aligned to those of
Says-Lesage et al. (2002) and to those of the isolates
selected from public databases by Farr et al. (1999) as
representatives of the Phomopsis/Diaporthe complex.
Phylogenetic analysis of this data set resulted in 16
most parsimonious trees. A comparison of these
results with those of Farr et al. (1999) indicates that
D. helianthi s. str. strains represented a novel, wellsupported clade (Fig. 1) (bootstrap=100%). Conversely, the phylogenetic position of the other strains
could not be precisely determined, as most internal
branches were supported by bootstrap values lower
than 50 %.
Cluster assignment by rDNA ITS sequence comparison
In order to obtain additional information about the
identity of the strains not assigned to the Diaporthe
helianthi s. str. clade, the sequences were aligned to 135
Phomopsis or Diaporthe orthologous sequences from
public databases. Distances (d) between each pair of
sequences were computed and stored in a matrix of
distances, which was used to identify the most closely
related sequence. Three strains of D. phaseolorum were
included to test the performance of this identification
matrix.
D. phaseolorum var. caulivora 21-99 and D. phaseolorum var. meridionalis 227-98 were similar to soybean
isolates grouped in clusters A and B by Zhang et al.
(1998), respectively. D. phaseolorum var. sojae 10-99
was similar to an isolate from Asparagus officinalis
examined by Rehner & Uecker (1994) and belonged to
their cluster C. These results are consistent with those
of Zhang et al. (1998), who could define phylogenetic
clades corresponding to D. phaseolorum var. caulivora
(cluster A) and D. phaseolorum var. meridionalis
(cluster B), but not for D. phaseolorum var. sojae.
The results from the distance matrix (Table 3) indicated that the Italian isolates I2 and I3 were similar
(d=0.0036) to a Phomopsis sp. isolate (FAU 638)
from melon and included in cluster C in the study of
397
Rehner & Uecker (1994). The sequence of one isolate
from Argentina (A2) was highly similar to that of a
Phomopsis sp. isolated from Chamaedorea sp., strain
FAU629, also belonging to cluster C in the study of
Rehner & Uecker (1994).
Isolates I4 and CBS 187/87 were related each other
but distinct from I2 and I3. They were moderately
similar (d<0.02) to a Diaporthe ambigua strain (STEU2657) isolated from Prunus sp. (Mostert et al. 2001).
Several sequences from strains belonging to the cluster
C in the study of Rehner & Uecker (1994) showed distances around 0.03.
Isolate I1 showed high similarity (d=0.0036) to two
Phomopsis sp. isolated from Stokesia sp., FAU 455 and
FAU 458, one from Actinidia chinensis (GJS 83-377)
and one from Capsicum sp. (FAU 484). FAU 455,
FAU 458 and FAU 484 all belonged to cluster C in the
study of Rehner & Uecker (1994).
Isolate R1, from Romania, showed high similarity
(d=0.0036–0.0072) to sequences obtained from isolates of Phomopsis oblonga, Diaporthe eres and D. perniciosa by Kanematsu et al. (2000). It was also similar
(d<0.02) to several Phomopsis sp. isolates examined by
Rehner & Uecker (1994) and belonging to their cluster
A1 (isolated from Epigea repens, Paulonia imperialis,
Kalmia latifolia, Sassafras albidum).
Finally, no isolate was found whose RDNA ITS
sequence was related (i.e. d<0.02) to that of isolate A1
from Argentina.
RFLP of mtDNA
The RFLP analysis of a larger part of the mitochondrial genome encompassing the coding sequence of
the atp6 gene was carried out using a 558 bp probe
obtained by PCR on genomic DNA of isolate F2 with
primers ATP6-FOR1 and ATP6-REV1. Digestions
were first performed with enzyme EcoRI. Hybridization profiles. (Fig. 2) revealed identical hybridizing
bands of all isolates defined above as Diaporthe
helianthi s. str. (lanes 1–8). A great variability was displayed in hybridizing bands of other isolates of the
collection, as each isolate showed a different haplotype.
Nevertheless, a certain degree of conservation in positions of restriction sites was found. For instance, isolates I3 and I2 displayed the same hybridizing pattern
(Fig. 2, lanes 14, 15). Substantially similar results were
obtained from the hybridizations of HindIII, HaeIII
and TaqI digests (not shown).
The results of the RFLP analysis corroborate those
from alignments of the sequences of the nuclear rDNA
ITS, indicating the occurrence of a cluster of eight isolates (corresponding to D. helianthi s. str., as defined
above), displaying the same coding sequence at the atp6
locus and the same restriction sites around it. Other
isolates (CBS 187/87, I2, R1 and 227/98), which share
the same sequence with isolates of the mentioned cluster at the atp6 locus, reveal great differences in their
RFLP patterns.
Polymorphisms in nuclear rDNA and mtDNA
398
Phomopsis sp FAU578
Phomopsis sp FAU500
Phomopsis sp FAU529
60
Diaporthe phaseolorum FAO125
Phomopsis sp FAU1053
Phomopsis sp FAU1068
Phomopsis sp FAU624
Diaporthe phaseolorum ST-3
95 Diaporthe phaseolorum ST-2
58
Diaporthe helianthi R1
Phomopsis sp FAU445
Phomopsis sp FAU522
Phomopsis sp FAU532
Diaporthe phaseolorum SW93-1
Phomopsis sp FAU446
Phomopsis sp FAU572
100
Phomopsis amygdali FAU1052
Phomopsis amygdali FAU1005
Diaporthe ambigua CMW2489
Phomopsis sp FAU537
70
Phomopsis sp FAU649
Diaporthe helianthi A3
Diaporthe helianthi F1
Diaporthe helianthi F2
Diaporthe helianthi IMI313866
Diaporthe helianthi IMI313865
Diaporthe helianthi Dh95045
Diaporthe helianthi Dh95099
Diaporthe helianthi Dh95004
100
57
60
Diaporthe helianthi N1
Diaporthe helianthi CBS592/81
Diaporthe helianthi IMI313861
63
Diaporthe helianthi Dh95049
Diaporthe helianthi Dh95048
Diaporthe helianthi Dh95016
Diaporthe helianthi Dh95057
Diaporthe helianthi I4
90 Diaporthe helianthi CBS187/87
97
Diaporthe helianthi A1
Diaporthe helianthi I1
Diaporthe sp Ds96002
Diaporthe helianthi A2
Diaporthe phaseolorum 973-B
51
57 Diaporthe helianthi I2
Diaporthe helianthi I3
Diaporthe sp FAU458
Phomopsis sp FAU453
Diaporthe sp 83-377-2
Diaporthe phaseolorum FAO126
Phomopsis sp FAU452
Phomopsis longicolla 3113
71 Phomopsis sp FAU600
1
Fig. 1. One of the 16 cladograms based on ITS sequences of selected isolates used in this work and sequences retrieved
from public databases selected as representative of the Phomopsis/Diaporthe complex by Farr et al. (1999). Bootstrap values
larger than 50 % are reported on branches. The sequence accessions corresponding to the strains included in the analysis are
either reported in Table 1 of this paper or in Farr et al. (1999) or Says-Lesage et al. (2002).
D. Rekab and others
399
Table 3. Distances between ITS sequences of fungal strains isolated from sunflower not belonging to the Diaporthe helianthi s. str. clade
and their closest relatives.
Straina
Distanceb
Strainc
Accession no.d
Speciese
Hostf
Cluster assignmentg
I1
0.004
0.004
0.004
0.004
0.007
0.007
0.011
0.011
0.011
0.014
0.015
FAU 484
FAU 455
FAU 458
GJS 83-377
10-99
FAU 499
FAU 456
FAU 457
FAU 459
FAU 438
STE-U2657
PS11334
PS11320
PS11323
AF102999
AJ312359
PS11337
PS11321
PS11322
PS11324
PS11314
AF230767
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
D. phas. soyae
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
D. ambigua
Capsicum annuum
Stokesia laevis
Stokesia laevis
Actinidia chinensis
Glycine max
Asparagus offic.
Stokesia laevis
Stokesia laevis
Stokesia laevis
Actinidia chinensis
Prunus sp.
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
N.A. – Farr et al. (1999)
This study
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
D. ambigua – Mostert et al. (2001)
I2
0.004
0.004
0.011
I3
FAU 638
FAU 452
AJ312363
PS11361
PS11318
D. helianthi
Phomopsis sp.
Phomopsis sp.
Helianthus annuus
Cucumis melo
Cucumis melo
This study
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
I3
0.004
0.007
0.014
I2
FAU 638
FAU 452
AJ312348
PS11361
PS11318
D. helianthi
Phomopsis sp.
Phomopsis sp.
Helianthus annuus
Cucumis melo
Cucumis melo
This study
C – Rehner & Uecker (1994)
C – Rehner and Uecker (1994)
I4
0.000
0.018
CBS 187/87
STE-U2657
AF230767
D. helianthi
D. ambigua
Helianthus annuus
Prunus sp.
This study
D. ambigua – Mostert et al. (2001)
0.000
0.018
I4
STE-U2657
AF230767
D. helianthi
D. ambigua
Helianthus annuus
Prunus sp.
This study
D. ambigua – Mostert et al. (2001)
A1
0.022
FAU 452
PS11318
Phomopsis sp.
Cucumis melo
C – Rehner & Uecker (1994)
A2
0.000
0.015
0.015
0.015
0.015
0.015
0.015
FAU 629
GJS 83-377
FAU 452
FAU 455
FAU 458
FAU 484
FAU 638
PS11359
AF102999
PS11318
PS11320
PS11323
PS11334
PS11361
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Chamaedorea sp.
Actinidia chinensis
Cucumis melo
Stokesia laevis
Stokesia laevis
Capsicum annuum
Cucumis melo
C – Rehner & Uecker (1994)
N.A. – Farr et al. (1999)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
C – Rehner & Uecker (1994)
R1
0.004
0.004
0.007
0.011
0.011
0.011
0.014
0.014
0.014
0.014
0.014
0.014
ATCC 42550-2
CBS 730.79-1
ATCC 38578-7
FAU 461
FAU 533
FAU 537
FAU 526
FAU 1073
FAU 506
SW-93-13
CBS 102.81
FAU 540
AB017729
AB017725
AB017724
PS11325
PS11348
PS11349
PS11344
AF103001
PS11339
AF001018
AB017727
PS11350
P. oblonga
D. eres
D. perniciosa
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
Phomopsis sp.
P. amygdali
Phomopsis sp.
D. phas. soyae
D. medusaea
Phomopsis sp.
Apple
Lunaria annua
Apple
Citrus limon
Epigaea repens
Kalmia latifolia
Sassafras albidum
Prunus sp.
Cornus florida
Glycine max
Juglans regia
Tsuga canadensis
B – Kanematsu et al. (2000)
B – Kanematsu et al. (2000)
B – Kanematsu et al. (2000)
N.A. – Rehner & Uecker (1994)
A1 – Rehner & Uecker (1994)
A1 – Rehner & Uecker (1994)
A1 – Rehner & Uecker (1994)
N.A. – Farr et al. (1999)
A1 – Rehner & Uecker (1994)
C1 – Zhang et al. (1998)
B – Kanematsu et al. (2000)
A1 – Rehner & Uecker (1994)
CBS 187/87
a
Strain from whose sequence the distance was calculated.
Distance calculated as described in the text.
c
Strain to whose sequence the distance was calculated.
d
Database accession of the sequence of the strain listed in c.
e
Species assignment of the strain listed c according to the database entry description.
f
Host for the strain listed c according to the database entry description.
g
Cluster assignment of the strain listed c according to the phylogenetic analysis where its ITS sequence was included, and reference.
N.A., not assigned to any cluster.
b
DISCUSSION
This study examined isolates of Diaporthe that have
been previously identified as D. helianthi, based upon
their occurrence on Helianthus annuus, their ability to
cause stem canker on this host, and morphological
characteristics. Phylogenetic analysis of nuclear rDNA
ITS sequences from these isolates revealed that a subgroup of them was closely related. This subgroup,
D. helianthi s. str., is composed of all isolates from
France and the former Yugoslavia, and also one from
Argentina, and constitutes a well-defined and supported clade. In contrast, all strains from Italy, one
from Romania and two from Argentina were not related to D. helianthi s. str. The taxonomic significance
of the D. helianthi s. str. was further supported by the
RFLP analysis of mtDNA, which is likely to be subject
to independent evolution from nuclear rDNA ITS
sequences. The isolates assigned to D. helianthi s. str.
share the same atp6 sequence and the same RFLP
haplotype with all restriction enzymes used. The
other isolates from sunflower showed different RFLP
Polymorphisms in nuclear rDNA and mtDNA
M
1
2
3
4
5
6
7
8
400
9
10
11
12
13
14
15
16
17
18
19
Fig. 2. DNA gel blot analysis of total DNA of Phomopsis/Diaporthe isolates digested with EcoRI. Lane numbers
correspond to isolate identifications reported in Table 1. A HindIII digest of l phage DNA, loaded in lane labeled M, was
used as marker.
patterns, although for some of them the sequence of the
atp6 was identical to that of D. helianthi s. str., probably due to the high conservation of the fragment
chosen for the sequence analysis. Therefore our combined results of nuclear rDNA ITS sequence analysis
and RFLP analysis of mtDNA suggest that eight out of
the 16 isolates examined were not related to D. helianthi
s. str. The ITS sequences themselves are the tool of
choice for the assessment of the phylogenetic position
of these strains, considering the high degree of ITS
sequence divergence and the very large set of ITS sequences already determined from Diaporthe and
Phomopsis species. An innovative approach was used
for this purpose, because recent attempts to classify
novel isolates by phylogenetic analyses on few selected
representative sequences taken from previous studies
only partially accomplished their scope, due to the reduction of the data set and the limited significance of
the branches (Farr et al. 1999, Mostert et al. 2001).
Another commonly used technique to assess the identity of environmental isolates is the comparison of their
ribosomal gene sequences to the entire database using
the BLAST or FASTA algorithms (e.g. Guo et al. 2000).
Those algorithms, optimized for database searches and
not for comparisons, may not be appropriate when
highly similar sequences have to be evaluated in order
to identify the most closely related on a taxonomic or
phylogenetic ground (e.g. the gap initiation and gap
extension penalties used by those algorithms may be
misleading). Hence, in order to address the identity of
isolates not related to D. helianthi s. str., we used the
complete set of data available in public databases to
build a large alignment of Diaporthe and Phomopsis
nuclear rDNA ITS sequences. By direct comparison of
the aligned sequences, we determined the most similar
sequences to those of our isolates, and evaluated them
in the context of previous studies. The isolates from
Italy did not belong to a single clade and appeared
to be related to different isolates within cluster C of
Rehner & Uecker (1994). This large group included
remarkably diverse isolates, and probably represented
more than one species, according to Rehner & Uecker
(1994). One isolate from Argentina and one from
Romania appeared even less related to any other
isolates.
In conclusion, the origin of the isolates infecting
sunflower is polyphyletic, spanning the entire range of
diversity within the genus Phomopsis. A high diversity
among sunflower isolates was also shown using polyacrylamide gel electrophoresis of mycelial isozymes and
other phenetic characters (Vannacci et al. 1996, Rekab
et al. 2002). These results are similar to those obtained
on Phomopsis spp. affecting peach (Farr et al. 1999),
and by grapevine (Mostert et al. 2001), soybean
D. Rekab and others
(Zhang et al. 1998), and Vaccinium spp. (Rehner &
Uecker 1994). In all cases, the investigated isolates were
reported to belong to at least two phylogenetically
unrelated groups. However, the diversity among Helianthus annuus isolates is interesting because the severity
of the disease in geographically distinct areas correlates
with the isolate groups determined by ITS sequence
analysis. All the isolates from France and the former
Yugoslavia, where losses due to sunflower canker up to
40 % (Carré 1993) and 50% (Pentericci 1988) have
been reported, represent a tight monophyletic clade
(D. helianthi s. str.). On the other hand, no significant
losses due to D. helianthi have been reported to date in
Italy and, significantly, none of the Italian isolates is
closely related to the tight group of D. helianthi s. str.
Accordingly, we speculate that D. helianthi s. str. is a
sunflower specialized pathogen, whilst the other strains
isolated from the same host in Italy, Romania and
Argentina may be polyphagous isolates of more limited
phytopathological concern. This hypothesis should be
tested by investigations to assess the in vivo pathogenicity of strains. Unfortunately, the in vitro production of D. helianthi ascospores, which are regarded
as the most epidemiologically relevant inoculum in
nature, is difficult. Therefore, the development of a
robust pathogenicity test has proved a challenging
task, although encouraging preliminary results have
been obtained (Pennisi et al. 2003, Tosi et al. 2003).
Nevertheless, the possibility that potentially dangerous isolates may not yet have been introduced into
Italy cannot be ruled out. The occurrence of diverse,
non-specialized Phomopsis isolates might be precursor.
to the spread of the host-adapted, phylogenetically
defined D. helianthi s. str. isolates. In early observations, Acimovic & Straser (1981) showed differences
among the former Yugoslavian isolates of P. helianthi
in their morphological development, biology and
epidemiology. Later, Muntanola-Cvetkovic et al.
(1996) reported ‘distinguishing characteristics with
respect to other cogeneric fungi ’ for their local isolates. At the same time, a larger degree of variability
was noticed among isolates collected in different
European countries (Vannacci et al. 1996, Vukojevic,
Franic Mihajlovic & Mihaljcevic 1996). On the basis of
these reports, it may be postulated that initially the
sunflower was infected by polyphagous Phomopsis
spp., until replacement by highly competitive and well
adapted D. helianthi s. str. isolates. It is therefore
relevant for the sunflower industry in Italy that strict
measures are taken in order to avoid the introduction
of potentially dangerous isolates from France and the
former Yugoslavia.
ACKNOWLEDGMENTS
We thank P. McCabe (University of California, Davis, CA) for
the critical reading of the manuscript. We are also grateful to Antonio
Zazzerini (University of Perugia), Caterina Cristani (University of
Pisa), Santa Olga Cacciola (University of Catania) for providing the
401
fungal strains, Matteo Lorito, Sheridan Woo and Francesco Vinale
(University of Naples) for helpful discussion and assistance and
Giovanni Vannacci (University of Pisa) for providing the scientific
context where this project could develop. This work was supported
by the Italian Ministry of the University and Scientific Research
(Programme PRIN 1999 ‘ Diaporthe helianthi-sunflower: study of a
pathosystem ’).
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Corresponding Editor: S. J. Assinder