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 ’). REFERENCES Acimovic, M. & Straser, N. (1981) Phomopsis sp. a new parasite in sunflower. Helia 4: 43–58. Bates, M. R., Buck, K. W. & Brasier, C. M. (1993) Molecular relationships of the mitochondrial DNA of Ophiostoma ulmi and the NAN and EAN races of O. novo-ulmi determined by restriction fragment length polymorphism. Mycological Research 97: 1093–1100. Brayford, D. (1990) Variation in Phomopsis isolates from Ulmus species in the British Isles and Italy. Mycological Research 94: 691–697. Carré, M. A. (1993) Maladies du tournesol: le choix variétal avant tout. Cultivar 332 : 46–51. Delos, M. & Moinard, J. (1995) Evolution du Phomopsis du tournesol en France. Un bref hystorique. Phytoma 473: 22–24. Farr, D. F., Castlebury, L. A. & Pardo-Schultheiss, R. A. (1999) Phomopsis amygdali causes peach shoot blight of cultivated peach trees in the southeastern United States. Mycologia 91: 1008–1015. Forster, H., Kinscherf, T. G., Leong, S. A. & Maxwell, D. P. (1986) Analysis of restriction fragment polymorphism of the mtDNA of the formae speciales of Phytophthora megasperma. Phytopathology 76: 1146–1151. Fukuda, M., Nakai, Y. F., Hibbett, D. S., Matsumoto, T. & Hayashi, Y. (1994) Mitochondrial DNA restriction length polymorphisms in natural populations of Lentinula edodes. Mycological Research 98: 169–175. Garber, R. C. & Yoder, O. C. (1984) Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus. Current Genetics 8 : 612–628. Guillamòn, J. M., Barrio, E., Huerta, T. & Querol, A. (1994) Rapid characterization of four species on the Saccharomyces sensu stricto complex according to mitochondrial DNA patterns. International Journal of Systematic Bacteriology 44: 708–714. Goodwin, S. B., Saghai-Maroof, M. A., Allard, R. W. & Webster, R. K. (1993) Isozyme variation within and among populations of Rhynchosporium secalis in Europe, Australia and the United States. Mycological Research 97: 49–58. Guo, L. D., Hyde, K. D. & Liew, E. C. Y. (2000) Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. New Phytologist 147: 617–630. Harrington, T. C., Steimel, J. P., Workneh, F. & Yang, X. B. (2000) Molecular identification of fungi associated with vascular discoloration of soybean in the North Central United States. Plant Disease 84: 83–89. Herr, L. J., Lipps, P. E. & Walters, B. H. (1983) Diaporthe stem canker of sunflower. Plant Disease 67: 911–913. Iliescu, H., Jinga, V., Ciurea, A. & Iionita, A. (1985) Investigations related to the prognosis of Sunflower stem canker (Diaporthe helianthi). Helia 8: 51–56. Jukes, H. & Cantor, C. R. (1969) Evolution of protein molecules. In Mammalian Protein Metabolism (H. N. Munro, ed.) 3 : 21–132. Academic Press, New York. Kanematsu, S., Minaka, N., Kobayashi, T., Kudo, A. & Ohtsu, Y. (2000) Molecular phylogenetic analysis of ribosomal DNA internal transcribed spacer regions and comparison of fertility in Phomopsis isolates from fruit trees. Journal of General Plant Pathology 66: 191–201. Lamarque, C. & Perny, R. A. (1985) Nouvelle maladie du tournesol: le phomopsis. Cultivar 179: 57–59. Polymorphisms in nuclear rDNA and mtDNA Lecelier, G. & Silar, P. (1994) Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Current Genetics 25 : 122–123. Maric, A., Masirevic, S. & Li, S. (1982) Prilog proucavanju Phomopsis spp. (Diaporthe sp.) prouzrokovaca sive pegavosti stabla suncokreta. Zastita Bilja 33: 403–419. Meijer, G., Megnegneau, B. & Linders, E. G. A. (1994) Variability for isozyme, vegetative compatibility and RAPD markers in natural populations of Phomopsis subordinaria. Mycological Research 98: 267–276. Mihaljevic, M., Petrov, M. & Cvetkovic, M. (1980) Phomopsis sp. novi parazit suncokreta u Jugoslaviji. Savremena Poljoprivreda 28: 531–539. Morgan-Jones, G. (1985) The Diaporthe/Phomopsis complex of soybean: morphology. In Proceedings of the Conference on Diaporthe/ Phomopsis Disease Complex of Soybean: 1–7. USDA, Beltsville, MD. Mostert, L., Crous, P. W., Kang, J. C. & Phillips, A. J. L. (2001) Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: morphological, cultural, molecular and pathological characterization. Mycologia 93: 146–167. Muntanola-Cvetkovic, M., Vukojevic, J. & Mihaljcevic, M. (1996) Cultural growth patterns and incompatibility reactions in Diaphorthe and Phomopsis populations. Journal of Phytopathology 144: 285–295. Nitimagi, N. M. (1935) Studies in the genera Cytosporina, Phomopsis and Diaporthe. VII. Chemical factors influencing spore characters. Annals of Botany (London) 49: 19–40. O’Donnell, K., Cigelnik, E. & Nirenberg, H. I. (1998) Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90: 465–493. Parmeter, J. R. (1958) An effect of substrate on spore form in Phomopsis. Phytopathology 48: 396–397. Pennisi, A. M., Abenavoli, M. R., Maimone, B., Cacciola, S. O. & Magnano di San Lio, G. (2003) Studio in vitro e in vivo delle interazioni ospite patogeno nel patosistema Diaporthe helianthigirasole. Notiziario di Protezione delle Piante 15: 413–414. Pentericci, S. (1988) La diffusione della Phomopsis nei paesi CEE e varietà resistenti. Sementi Elette 41: 38–39. Rehner, S. A. & Uecker, F. A. (1994) Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the Coelomycete Phomopsis. Canadian Journal of Botany 72 : 1666–1674. Rekab, D., Cacciola, S. O., Firrao, G. & Pappalardo, P. (2002) A taxonomic study on Diaporthe helianthi. In Proceeding of the 6th European Conference on Fungal Genetics (G. Vannacci & S. Sarrocco, eds): 407. Dipartimento di Coltivazione e Difesa delle Specie Legnose, Università di Pisa, Pisa. Sambrook, J. F., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sanson, G. F. O. & Briones, M. R. S. (2000) Typing of Candida glabrata in clinical isolates by comparative sequence analysis of the cytochrome c oxidase subunit 2 gene distinguishes two clusters of strains associated with geographical sequence polymorphisms. Journal of Clinical Microbiology 38: 227–235. Says-Lesage, V., Roeckel-Drevet, P., Viguié, A., Tourvieille, J., Nicolas, P. & Tourvieille de Labrouhe, D. (2002) Molecular 402 variability within Diaporthe/Phomopsis helianthi from France. Phytopathology 92: 308–313. Shannon, C. E. & Weaver, W. (1949) The Mathematical Theory of Communication. University of Illinois Press, Urbana, IL. Tosi, L., Della Torre, G., Quaglia, M. & Zazzerini, A. (2003) Diaporthe helianthi: epidemiologia e fattori di virulenza (fitotossine). Notiziario di Protezione delle Piante 15 : 411–412. Uddin, W., Stevenson, K. L., Pardo-Schultheiss, R. A. & Rehner, S. A. (1998) Pathogenic and molecular characterization of three Phomopsis isolates from peach, plum and Asian pear. Plant Disease 82: 732–737. Uecker, F. A. (1988) A world list of Phomopsis names with notes on nomenclature, morphology and biology. Mycological Memoir 13: 1–231. Vannacci, G., Pecchia, S., Cacciola, S. O. & Zazzerini, A. (1996) Studi sulla variabilità di isolati di Diaporthe helianthi. Petria 6: 264–265. Varga, J., Keveii, F., Vriesema, A., Debets, F., Kozakiewicz, Z. & Croft, H. (1994) Mitochondrial DNA restriction fragment length polymorphisms on field isolates of the Aspergillus niger aggregate. Canadian Journal of Microbiology 40: 612–621. Varos, J., Leranth, J. & Vajnal, L. (1983) Overwintering of Diaporthe helianthi a new destructive pathogen of sunflower in Hungary. Acta Phytopathologica Academiae Scientiarum Hungariacae 18: 303–305. Vukojevic, J., Franic Mihajlovic, D. & Mihaljcevic, M. (1996) The comparative investigation of Phomopsis helianthi isolates from different European localities. In Proceedings of 14th International Sunflower Conference 2 : 734–739. Beijing-Shenyang. Waalwijk, C., de Koning, J. R. A., Baayen, R. P. & Gams, W. (1996) Discordant groupings of Fusarium spp. from sections Elegans, Liseola and Dlaminia based on ribosomal ITS1 and ITS2 sequences. Mycologia 88: 361–368. Wang, L., Yokohama, K., Miyaji, M. & Nishimura, K. (2000) Mitochondrial cytochrome b gene of Aspergillus fumigatus and related species. Journal of Clinical Microbiology 38: 1352–1358. Wehmeyer, L. E. (1933) The genus Diaporthe Nitschke and its segregates. University of Michigam Studies, Science Series 9: 1–349. White, T. J., Brunus, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomial RNA genes for phytopathogenics. In PCR Protocols: a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 313–322. Academic Press, San Diego. Young, J. M. (2001) Implications of alternative classifications and horizontal gene transfer for bacterial taxonomy. International Journal of Systematic and Evolutionary Microbiology 51: 945–953. Zazzerini, A., Tosi, L. & Losavio, N. (1988) Rilievi fitopatologici su varietà di girasole a confronto nel 1987. L’Informatore Agrario 44(13): 85–88. Zhang, A. W., Riccioni, L., Pedersen, W. L., Kollipara, K. P. & Hartman, G. L. (1998) Molecular identification and phylogenetic grouping of Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean. Phytopathology 88 : 1306–1314. Corresponding Editor: S. J. Assinder