Colletotrichum clavatum sp. nov. identified as the causal agent of olive anthracnose in Italy

Headnote

Summary. Molecular analyses of a large population of isolates, previously identifi ed as group B or genetic group A4 of the Colletotrichum acutatum species complex, mainly of Italian origin from olive, but also from other hosts collected since 1992, confi rmed a well-resolved phylogenetic lineage with distinctive phenotypic characters which can be recognized as a separate species. Based on RAPD genomic fi ngerprinting, ITS and ß-tubulin DNA sequences, this species was clearly distinct from C. acutatum sensu stricto, C. fi oriniae and C. simmondsii as well as from the genetic groups A1, A6, A7 and A8, all previously referred to as C. acutatum sensu lato. Group A4 is widespread in Europe, being responsible for olive anthracnose epidemics in some Mediterranean countries, including Greece, Italy, Montenegro, Portugal and Spain; moreover, it causes anthracnose diseases on a wide range of other hosts including about 20 different genera of woody and herbaceous plants, ornamentals and fruit trees. This new anamorphic taxon is described as Colletotrichum clavatum sp. nov.

Key words: molecular phylogeny, RAPD-PCR, ß-tubulin, ITS region.

Introduction

Colletotrichum acutatum sensu lato (s. l.) is a species group as it includes variants with distinct morphological and molecular characters (Sreenivasaprasad and Talhinhas, 2005; Hyde et al., 2009). It comprises important fungal pathogens which are responsible for economically signifi cant diseases of temperate, subtropical and tropical crops, commonly recognized as anthracnoses (Peres et al., 2005; Sreenivasaprasad and Talhinhas, 2005; Johnston et al., 2005). Colletotrichum acutatum J.H. Simmonds ex J.H. Simmonds was fi rst reported as a distinct species on pawpaw (Carica papaya) in Queensland (Australia) by Simmonds (1965), who validated the species later with a broad concept and designated a holotype and six paratypes varying considerably in morphological and molecular characteristics (Simmonds, 1968). Subsequently, C. acutatum was referred to as causal agent of anthracnose diseases on a large number of crops and non-cultivated plant species (Shi et al., 1996; Zulfi qar et al., 1996; Johnston and Jones, 1997; Freeman et al., 1998). The teleomorph of C. acutatum, Glomerella acutata J.C. Guerber & J.C. Correll, was fi rst obtained in vitro by crossing different self-sterile monoconidial strains of C. acutatum (Guerber and Correll, 2001) and it was subsequently observed on naturally infected fruits of highbush blueberry in Norway (Talgø et al., 2007).

The main morphological characters adopted to differentiate C. acutatum from other species of Colletotrichum have been the shape of conidia, which are fusiform and pointed at both ends, and the slow growth rate in culture. However, as C. acutatum in a broad sense shows a high degree of phenotypic and genetic diversity, it has been diffi cult to discriminate from other species of Colletotrichum, including C. gloeosporioides which exhibits a few overlapping morphological traits and host range (Talhinhas et al., 2002).

Colletotrichum acutatum s. l. was fi rst introduced by Johnston and Jones (1997) to accommodate isolates that clustered with C. acutatum sensu Simmonds and others that showed a wide range of morphological and genetic diversity. Later, Lardner et al. (1999) used RAPD analyses and morphological and cultural features to split C. acutatum s. l. into seven distinct taxa, including fi ve morphologically and genetically uniform groups, designated as A, B, C, D, E, and two species, Glomerella miyabeana (Fukushi) Arx and C. acutatum f. sp. pineum Dingley & J.W. Gilmour. Subsequently, the analysis of the Internal Transcribed Spacer (ITS) regions of the ribosomal DNA (rDNA) and a fragment of the ß-tubulin-2 gene enabled the rearrangement of a global C. acutatum population into eight different molecular groups (from A1 to A8). These showed some degree of correlation with phenotypic characteristics, host association patterns and geographical distribution (Sreenivasaprasad and Talhinhas, 2005). The same genes have been more recently utilized to reassess three different genetic groups within C. acutatum s. l. and describe two new species, C. fi oriniae (Marcelino & S. Gouli) R.G. Shivas & Y.P. Tan and C. simmondsii R.G. Shivas & Y.P. Tan (Shivas and Tan, 2009). The new species corresponded to group C (also known as A3) and group D (also known as A2), respectively, whereas a third group was defi ned as C. acutatum (sensu Simmonds) and corresponded to group A (also known as A5).

Colletotrichum acutatum group B or genetic group A4, as identifi ed, respectively, by Lardner et al. (1999) and Sreenivasaprasad and Talhinhas (2005), is a cosmopolitan pathogen on a wide host range, including Olea europaea, Fragaria × ananassa, Lycopersicon esculentum, Malus domestica, Ficus carica, Eriobotrya japonica, Feijoa sellowiana, Hepatica acutiloba, Sambucus nigra, Prunus dulcis, Rhododendron spp., Rubus sp., Ceanothus sp., Vitis sp., Juglans sp., Primula sp., Camellia sp. and Bergenia sp. This group has been demonstrated to be responsible for epidemic outbreaks of fruit anthracnose of olive (O. europaea) in Greece, Italy, Andalucia (southern Spain) and Montenegro, while in Portugal, South Africa and Australia other C. acutatum groups are predominant as causal agents of this disease (Cacciola et al., 2007; Talhinhas et al., 2009; Sergeeva et al., 2010). Anthracnose is the most important disease of olive fruit worldwide, causing signifi cant yield losses and poor olive oil quality (Bompeix et al., 1988; Graniti et al., 1993; Moral et al., 2008). In previous studies it has been supposed that this variant (group B or genetic group A4), like other groups within C. acutatum s. l., may represent a distinct Colletrotrichum species, because it can be separated on the basis of morphological traits and multiple genetic markers. These include isozymes, random amplifi ed polymorphic DNA (RAPD)-polymerase chain reaction (PCR), restriction fragment length polymorphisms (RFLPs) of repetitive elements of nuclear DNA or A+T rich mitochondrial DNA (mtDNA), RFLPs of 1-kb intron of the glutamine synthetase (GS) gene and sequences of ITS-rDNA, ß-tubulin (tub 2) gene and intron 2 of both glutaraldehyde-3-phosphate dehydrogenase (G3PD) and GS genes (Vinnere et al., 2002; Guerber et al., 2003; Sreenivasaprasad and Talhinhas, 2005; Peres et al., 2008; MacKenzie et al., 2009; Shivas and Tan, 2009; Sergeeva et al., 2010). However, the hypothesis that group A4 can be regarded as a well-defi ned taxon has not been defi nitely demonstrated.

In the present study, a large population of isolates of C. acutatum A4, mostly obtained from olive in Italian regions where olive anthracnose is endemic, have been collected since 1992 and analyzed with a polyphasic approach to discern both the taxonomic status and the phylogenetic position of this genetic group within C. acutatum s. l. ITS and ß-tubulin 2 sequences of this collection of isolates were compared with GenBank deposited sequences of isolates of Colletotrichum, including C. acutatum A4 and C. acutatum sensu stricto (s. s.) of worldwide origin and from various hosts.

Materials and methods

Fungal isolates

Colletotrichum isolates examined in this study are listed in Table 1. The majority were obtained from drupes and leaves of olive with symptoms of anthracnose, collected between 1992 and 2011 in various regions of southern and central Italy, including Apulia, Calabria, Sardinia and Umbria. The bulk of isolates came from the Calabria and Apulia regions (southern Italy). All isolates were obtained from monoconidial cultures and stock cultures were maintained on potato dextrose agar (PDA, Oxoid Ltd, Basingstoke, UK) slants under mineral oil at 10-12°C in the collections of the Dipartimento di Gestione dei Sistemi Agroalimentari e Ambientali, University of Catania (Italy) and the Dipartimento di Gestione dei Sistemi Agrari e Forestali, Mediterranean University of Reggio Calabria (Italy). Isolates of C. gloeosporioides, C. musae, and C. circinans were used as outgroups for comparisons (Table 1).

Morphology of conidia and appressoria

Isolates of Colletotrichum were inoculated in the center of 9-cm-diameter Petri dishes containing PDA with a 5-mm-diameter plug, each taken from the margin of a 5-day-old actively growing colony kept at 24(±1)°C, and incubated under fl uorescent light for at least 7 days at 24(±1)°C to stimulate the conidiogenesis. Conidia were mounted in water and observed microscopically at ×1000 magnifi cation. For each isolate 100 conidia were randomly selected, and length, width and shape were recorded.

The observation of the morphology of conidia by scanning electron microscopy (SEM) was carried out by growing isolates on PDA at 24(±1)°C under continuous fl uorescent light as described above. After 7-10 days of incubation, plugs of 4×4 mm were fi xed in 2% glutaraldehyde in 0.1 M sodium- cacodylate buffer (EMS), pH 7.2, for 1 h at 4°C and then post-fi xed in 1% osmium tetroxide (EMS) for 1 h at 4°C. After dehydration in graded ethanol and critical point drying using CO2 (Emscope-CPD 750), the samples were attached by CCC carbon adhesive directly on the microscope stubs, coated with vacuum evaporated gold (Emscope-SM 300) and observed using a Field Emission Scanning Electron Microscope (FESEM).

Appressoria were produced in slide cultures on potato-carrot-agar (PCA, Smith and Onions, 1984) grown at 25°C for 7 days alternating natural light and darkness (Sutton, 1968) and on PDA in the dark (Cai et al., 2009).

Cultural characterization of isolates

To determine cardinal growth temperatures for isolates, 5-mm-diameter mycelium plugs taken from the margins of 5-day-old actively growing colonies at 24(±1)°C were transferred onto PDA and incubated at 5, 10, 15, 20, 24, 27, 30 or 35°C both in the dark and under continuous fl uorescent light. Colony diameters were measured daily for 7 days. Three replicates of each isolate were evaluated, and the experiments were repeated twice. Growth rate was calculated after a 7-day-incubation period as mean daily growth rate (mm day-1) for each temperature.

Molecular characterization

RAPD-PCR analyses

PCR reactions were performed using 16 decamer oligonucleotides (OPB-01, OPB-03, OPB-07, OPB-09, OPB-14, OPB-19, OPF-01, OPF-03, OPF- 04, OPF-06, OPF-09, OPF-10, OPF-11, OPF-13, OPF-15 and OPF-20) selected during preliminary investigations with a restricted number of isolates (data not shown). All primers were purchased from Operon Technologies Inc. (Alameda, CA, USA). Total DNA was extracted from fresh mycelium scraped from cultures grown on PDA for 7 days at 24°C using DNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). RAPD-PCR was carried out in 25 µl of reaction mixture containing 20 mM Tris HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2, 100 µM each dNTP, 0.2 µM primer, 5 ng genomic DNA and 1 U Taq DNA Polymerase (Invitrogen, Life Technologies, Carlsbad, CA, USA). Amplifi cation was performed in a Perkin-Elmer Cetus (Norwalk, CT, USA) GeneAmp PCR System 9600, starting with 2.5 min at 94°C, followed by 45 cycles consisting of 30 s at 94°C, 1 min at 36°C, 2 min at 72°C, and a fi nal step of 5 min at 72°C. Amplicons were analyzed by electrophoresis in 1.5% agarose gels containing SYBR Safe DNA gel stain (Invitrogen, Life Technology Corporation, Carlsbad, CA, USA) or ethidium bromide (0.5 µg ml-1) in Tris-acetate-EDTA (TAE) buffer. After separation, the bands were visualized on a UV transilluminator and the gels photographed using a digital camera. RAPD analyses were repeated at least twice per isolate and primer. A negative control using water instead of template DNA was included in all amplifi cations.

ITS-rDNA and ß-tubulin analyses

Genomic DNA was extracted from Colletotrichum isolates following the procedure described by Schena and Cooke (2006). The ITS1-5.8S-ITS2 region and a fragment of the ß-tubulin 2 gene comprised between exons 2 and 6 (Glass and Donaldson, 1995) were amplifi ed with primers ITS5 and ITS4 (White et al., 1990), and primers T1 (O'Donnell and Cigelink, 1997) and ßt2b (Glass and Donaldson, 1995), respectively. Amplifi cations were performed in a 25-µl reaction volume containing 1× PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1.5 mM MgCl2, 0.2 mM of each deoxyribonucleotide triphosphate, 0.5 µM of each primer, 10 ng template DNA, and one unit of Taq DNA Polymerase (Invitrogen). A negative control using water instead of template DNA was included in all PCR reactions. PCR reactions were performed in an automated thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer Cetus) programmed to perform 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 50 s at 58°C (ITS) or 60°C (ß-tubulin) and 1 min at 72°C. All reactions ended with 10 min at 72°C. Amplifi ed products were analyzed by electrophoresis as described above, and single bands of the expected size were purifi ed with the QIAquick PCR purifi cation kit (Qiagen) and sequenced with both forward and reverse primers by Macrogen Europe (Amsterdam, the Netherlands). The 'ChromasPro version 1.5' software (http://www.technelysium. com.au/) was utilized to evaluate reliability of sequences and to create consensus sequences. Non-reliable sequences in which either forward or reverse sequences contained doubtful bases were sequenced a second time.

Phylogenetic analyses

Cluster analysis to compare associations between bands of RAPD patterns obtained from Colletotrichum isolates was performed by using the PAST software ver. 2.09 (Hammer et al., 2001). The genetic relatedness between all isolates was represented as a dendrogram generated using the Dice similarity index (Dice, 1945) and the unweighted pair-group method with arithmetical averages (UPGMA) algorithm (Sneath and Sokal, 1973). Nodal support was assessed using bootstrap analysis from 1,000 replicates (Felsenstein, 1985).

ITS and ß-tubulin 2 sequences obtained in the present study (Table 1) and GenBank deposited sequences, selected as representative taxa of Colletotrichum (Table 2), were utilized to determine phylogenetic relationships between C. acutatum genetic group A4 and other species and genetic groups of C. acutatum s. l.

Multiple alignment of both ITS and ß-tubulin 2 sequences was carried out by CLUSTALW (Thompson et al., 1994) with a total of 109 and 101 nucleotide sequences, respectively. Molecular phylogeny estimation was performed by UPGMA distance-based, Maximum Likelihood (ML) and Bayesian character-based methods. Pair-wise genetic distance was estimated using the Kimura two-parameter (K2P) model (Kimura, 1980) with complete deletion option to treat gaps. The degree of statistical support for the nodes of the phylograms generated by UPGMA clustering was evaluated in 1,000 resample trees by the bootstrap interior-branch test (Sitnikova et al., 1995). ML analysis was conducted using K2P substitution model and Nearest-Neighbor-Interchange method with 1,000 bootstrap replicates. Analyses were performed using MEGA software ver. 5 (Tamura et al., 2011). Bayesian analysis was performed using MrBayes ver. 3.1.1 as implemented in TOPALi v2.5 (Milne et al., 2009). Four runs were conducted simultaneously for 200,000 generations with 10% sampling frequency and burn-in of 25%. Multi-alignment of datasets are accessible through TreeBase under the reference number S11685 (http://purl.org/phylo/treebase/phylows/ study/TB2:S11685).

Results

RAPD-PCR analyses

Reliable amplifi cation products were obtained from all isolates. A total of 376 different RAPD loci were detected consistently with 16 random primers in all isolates of Colletotrichum. The size of fragments ranged from 100 and 3,000 bp and each primer generated between two and 12 bands. Only major bands ranging from 200 to 2,000 bp amplifi able with high reproducibility in different PCR reactions were scored for consistency. All selected decamer primers generated polymorphic bands among the different species or molecular groups of Colletotrichum isolates (overall polymorphism of 98.72%) and monomorphic banding patterns within each of these groups (average monomorphism per group of 85.63%). The genetic distance based on the similarity index of Dice (1945) demonstrated suffi cient genetic divergence to discriminate the isolates analyzed in this study into different Colletotrichum species (Figure 1). RAPD patterns of Italian isolates from olive (including IMI 398854 and IMI 398855), rhododendron (AZJ), apple (MELA), olive isolates from Greece (CBS 193.32) and Montenegro (CGMUL), and the sweet cherry isolate from Norway (8689), which can be referred to the genetic group A4 of C. acutatum, were different from all other Colletotrichum isolates examined. They clustered together into a distinct group that was very well supported by bootstrap analysis (100% of generated trees), and clearly differed from the reference isolates of C. acutatum, C. simmondsii and C. fi oriniae, as well as from the isolates of C. gloeosporioides and C. musae used as outgroups. In turn, this cluster exhibited a small genetic variation and encompassed two different subgroups of isolates (86 and 49% bootstrap support values, respectively). The fi rst subgroup included an isolate from rhododendron (AZJ) sourced in Piedmont (Italy), an isolate from sweet cherry (8689) sourced in Norway and all olive isolates from Apulia (Italy). The second subgroup comprised an Italian isolate from apple (MELA), an olive isolate from Greece (CBS 193.32) and Montenegro (CGMUL) as well as the olive isolates from Calabria, Sardinia and Umbria.

Phylogenetic analysis

The identity of all isolates investigated in this study was confi rmed using ITS and ß-tubulin sequence data. The ITS dataset used for phylogenetic analysis included 501 sites with 68 (13.57%) potentially informative sites and a fi nal expected transition/transversion ratio (TI/TV) of 1.81. As for the ß-tubulin 2 gene, 766 sites containing 308 (40.21%) variable sites with a TI/TV ratio of 4.62 were identifi ed.

The ITS-based UPGMA phylogenetic tree of Colletotrichum isolates investigated in the present study revealed eight phylogenetic groups within C. acutatum s. l. (Figure 2) which were congruent with those of previously reported studies by Sreenivasaprasad and Talhinhas (2005) and Shivas and Tan (2009). Italian isolates from olive (e.g. IMI 398854 and IMI 398855), rhododendron (AZJ) and apple (MELA), as well as isolates from sweet cherry sourced in Norway (8689) and olive sourced in Montenegro (CGMUL) had ITS sequences identical to those of reference isolates (CBS 193.32, AJ748612; JG05, AJ409302; PT169, AJ748609). These isolates, ascribable to C. acutatum group A4 sensu Sreenivasaprasad and Talhinhas, formed a well-defi ned clade distinctly separated from the clades encompassing isolates of C. acutatum s. s., C. simmondsii, C. fi oriniae and C. acutatum genetic groups A1, A6, A7 and A8, as well as from C. gloeosporioides and C. musae (Figure 2). The mean genetic distance of C. acutatum group A4 from C. acutatum s. s., C. simmondsii, C. fi oriniae and C. acutatum genetic groups A1, A6, A7 and A8 was 0.009. The most closely related clade was the genetic group A7 of C. acutatum (68% bootstrap support) with a genetic distance of 0.003. The genetic distance of C. acutatum group A4 from C. gloeosporioides and C. musae, used as outgroups, was 0.113 and 0.103, respectively (99% bootstrap support). In terms of sequence divergence, C. acutatum group A4 showed the least divergence (0.28%) as compared to C. acutatum A7 and a divergence of 1.12% with C. acutatum, C. fi oriniae and C. acutatum A6 (Table 3). Higher levels of divergence were observed between C. acutatum group A4 and C. simmondsii, C. acutatum group A1, and C. acutatum group A8. On the whole, these levels of divergence were generally greater if compared with those differentiating the newly described species C. fi oriniae and C. simmondsii from other C. acutatum s. l. (Table 3).

The topology of the ß-tubulin 2 tree generated with UPGMA was similar to that of ITS (Figure 3). However, this gene showed greater polymorphism with a mean genetic distance of 0.053, enabling a major resolution for isolates clustering in C. simmondsii and C. fi oriniae clades. The genetic distance of C. acutatum group A4 from both C. gloeosporioides and C. musae was 0.29. All isolates of the genetic group A4 clustered together (100% bootstrap support) in a monophyletic clade clearly separate from other species of Colletotrichum, including C. acutatum s. s., C. simmondsii, C. fi oriniae and C. acutatum groups A1 and A6 (Figure 3). Mean pair-wise percent divergence values for ß-tubulin 2 gene were greater compared to those of the ITS regions, whereas patterns were very similar to those of the above described ITS sequences (Table 4).

Phylogenetic groups largely congruent with those of the above described UPGMA analysis were also obtained with the Maximum Likelihood and Bayesian analyses (data not shown).

Taxonomy

Analysis of ITS and ß-tubulin sequences supports the group A4 of C. acutatum as a separate species within the C. acutatum complex. This species corresponds also to group B of Lardner et al. (1999). The allocation and the formal description of this genetic group to the species rank besides the taxonomic signifi cance could also have epidemiological implications as well as quarantine relevance.

Colletotrichum clavatum G.E. Agosteo, R. Faedda & S.O. Cacciola, sp. nov. (Figure 4)

Etymology: clavatum refers to club-shaped conidia that are the dominant form for this species.

MycoBank number: MB561749

Coloniae in PDA cinereae et lanuginosae cum margine regulari albicante, mycelio aerio denso, sparsis fl occis albicantibus mycelii et luteis massis conidiorum sparsis sed saepius in media colonia. Aversum coloniae pallide cinereum ad pallide roseum. In agaro PDA post septem dies ad 24°C colonia est fere 40 mm diametro. Sclerotia et setae absunt. Conidia unicellularia, subcylindrata vel saepius subclavata, 10-(14.9)-17 × 4.0-(4.6)-5.0 µm, saepe in medio constricta, hyalina, levia, apice obtuso, basi angusta, brevi projectione annelli forma. Appressoria fusca unicellularia, levia, plerumque clavata vel lobata, saepe complexa 8- (11.5)-19 × 4.5-(5.5)-7.5 µm. Haec species differt ab aliis taxis generics aspectu coloniae, conidiis saepe clavatis et ITS et ß-tubulina ordine.

Isolates of C. clavatum on PDA show uniform colony morphology white to greyish pale salmon or light brown, dense aerial mycelium. In reverse, dull beige to pale salmon. Mycelium may produce pink conidial masses mainly in the centre of the colony (Figure 4). The conidiogenesis is stimulated by light, and under natural light colonies develop greyish concentric rings. Conidiophores are septate and bear enteroblastic, phialidic, hyaline conidiogenous cells and, in culture, conidia are produced after 7 days of incubation (Figure 5 A, B, C). Conidia developing also in the aerial mycelium are hyaline and unicellular, subcylindrical or more frequently clavate, smooth and thinwalled, measuring 10-(15) -17×4.0-(4.6)-5.0 µm, with a mean length/breadth (l/b) ratio 3.3 (±0.9). They have a rounded distal apex and often a light median constriction, a funnel-shaped base ending with a short ring-like projection (Figure 5 D). In slide cultures on PCA, hyphal appressoria are melanized, mostly regular in shape, brown, ovate to long clavate, sometimes lobate, often complex, that is, producing columns of several closely connected appressoria (Sutton, 1992), measuring 8- (11.5)-19×4.5-(5.5)-7.5 µm (Figure 4 D). Sclerotia and setae obsente. On PDA, mycelium grew between 10 and 30°C with an optimum at 24°C, whereas at 5 and 35°C no growth was observed. Radial growth rate at 24°C was between 3.8 and 8.2 mm per day, with an average (± S.D) of 5.9±1.0 mm day-1.

Typus: Italy: Rizziconi, Reggio Calabria (southern Italy), isolated from rotten olive fruit (Olea europaea L.), collected in an olive orchard (O. europaea) in the Gioia Tauro plain, Rizziconi, Reggio Calabria, October 1992, G. E. Agosteo and G. Magnano di San Lio. Holotype: OLDC10 (dried culture on PDA, herbarium of the Dipartimento di Gestione dei Sistemi Agrari e Forestali, Mediterranean University of Reggio Calabria). Ex-type living culture OL10. This strain is available at IMI, CABI Bioscience, Egham, Surrey, UK (IMI 398854) and the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, (CBS 130251), ITS sequence GenBank JN121126, ß-tubulin 2 sequence GenBank JN121213, DIGESA Culture Collection, Catania (Italy) and in the Culture Collection of the Dipartimento di Gestione dei Sistemi Agrari e Forestali, Mediterranean University of Reggio Calabria, Italy (F49).

Additional specimens examined (see Table 1). The earliest collection of isolates of C. clavatum examined was obtained from Olea europaea of the Calabria and Apulia regions in southern Italy starting from 1992. Additional isolates of the same species were then collected from other Italian regions and European countries and different hosts. Paratypes of C. clavatum deposited in International Collections are OL20 (=IMI 398855, CBS 130252) and CBS 193.32 (Figure 6).

Habitat: the list of all known hosts of C. clavatum includes Olea europaea, Fragaria × ananassa, Lycopersicon esculentum, Malus domestica, Ficus carica, Eriobotrya japonica, Feijoa sellowiana, Hepatica acutiloba, Sambucus nigra, Prunus dulcis, Rhododendron spp., Rubus sp., Ceanothus sp., Vitis sp., Juglans sp., Primula sp., Camellia sp. and Bergenia sp. (Sreenivasaprasad and Talhinhas, 2005)

Teleomorph: Unknown

Notes: Colletotrichum clavatum is distinct from other species of Colletotrichum in its colony morphology, shape and l/b ratio of conidia, ITS and ß-tubulin 2 sequences, and RAPD patterns. This new species is characterized by its club-shaped conidia with rounded distal apices and median constriction, funnel shaped base with a short cylindrical projection. In other species of Colletotrichum, conidia are mostly cylindrical with both ends rounded. Colletotrichum clavatum has conidia with a greater l/b ratio with respect to other species of this genus. Moreover, growth of isolates of C. clavatum is slower in comparison with that of isolates of other species of Colletotrichum.

Discussion

In this study, a polyphasic approach was used to confi rm that Colletotricum clavatum, formerly identifi ed as C. acutatum group B or A4, can be considered a distinct species within C. acutatum s. l. as hypothesized in previous studies (Sreenivasaprasad and Talhinhas 2005; Cacciola et al., 2007; Shivas and Tan, 2009; Sergeeva et al., 2010). The characterization of the causal agent of olive fruit anthracnose in Italy prompted us to allocate a large population of isolates collected over almost 20 years to the appropriate taxonomic status.

RAPD genomic fi ngerprinting and phylogenetic analysis of both ITS and ß-tubulin DNA sequences showed that this species constitutes a strongly supported monophyletic lineage clearly distinct from C. acutatum s. s. and other species or genetic groups previously referred to as C. acutatum s. l. These analyses are directly comparable with those of Sreenivasaprasad and Talhinhas (2005), who identifi ed eight phylogenetic groupings of the C. acutatum species complex, as well as with those of Shivas and Tan (2009) who recently revised the taxonomy of C. acutatum introducing two new species, C. fi oriniae and C. simmondsii.

Even though a multi-gene phylogenetic analysis has been suggested by several authors to give a better understanding of the relationships within Colletotrichum (Cai et al., 2009), RAPD-PCR is a method that provides a better overview of the entire genome (Laroche et al., 1995) and, as also demonstrated by Yang and Sweetingham (1998), this allowed better resolution for Colletotrichum than ITS sequences. RAPD analysis enabled us to highlight greater genetic intraspecifi c variability and to give better interspecifi c resolving power with respect to ITS and ß-tubulin 2 sequences. In fact, RAPD patterns of Colletotrichum isolates examined in this study, besides showing a very high interspecifi c polymorphism, made it possible to differentiate C. clavatum isolates into two welldefi ned subgroups. Italian isolates of C. clavatum from olive correlated with their geographic origin as all isolates collected in the Apulia region clustered into the same subgroup, which was distinct from the subgroup comprising isolates from other regions, including Calabria, Sardinia and Umbria. Thus, it could be speculated that populations of C. clavatum from olive established in the Calabria and Apulia regions, respectively, are undergoing an allotropic speciation process, or otherwise they have originated from different introductions, very probably from Greece or Albania (Ciccarone, 1950; Agosteo, 2010). To the best of our knowledge, the oldest living culture of this new species, an isolate from olive indicated here as a paratype (CBS 193.32), dates back to 1930 and was deposited at CBS by Lionello Petri who had received it from Jean Serejanni of the Benaki Phytopathological Institute of Kiphissia, Athens, Greece, (Petri, 1930; Biraghi, 1934).

Biometric characteristics of C. clavatum overlap with those of other Colletotrichum species, such as C. acutatum, C. fi oriniae and C. simmondsii; however, even though this new taxon was identifi ed primarily on the basis of molecular analyses, it shows morphological and physiological traits, including the shape of conidia and the colony morphology, which discriminate it from all other Colletotrichum species, confi rming that in this genus conidial morphology and cultural characters refl ect phylogeny more than host association (Than et al., 2008). Although, according to Sreenivasaprasad and Talhinhas (2005), group A4 fi ts into group B of Lardner et al. (1999), iso- lates of C. clavatum examined in the present study did not produce perithecia in vitro and the perfect stage has never been found in plants with natural infections. In contracts, isolates of group B were described as differentiating perithecia in culture.

Colletotrichum clavatum is a polyphagous and widespread pathogen in Europe, and is common as causal agent of anthracnose of olive in the Mediterranean basin (Agosteo et al., 2002; Talhinhas et al., 2002, 2005; Cacciola et al., 2007; Talhinhas et al., 2009; Sergeeva et al., 2010; Talhinhas et al., 2011). Recently, this pathogen was reported as causal agent of strawberry anthracnose in northern Europe (Damm et al., 2010; Van Hemelrijck et al., 2010). Moreover, our results confi rmed the previous study of Sreenivasaprasad and Talhinhas (2005) inferring that C. clavatum is associated with azalea anthracnose, an emerging disease of this ornamental fl ower plant in Europe (Vinnere et al., 2002; Bertetti et al., 2008), as well as cherry anthracnose in Norway (Børve and Stensvand, 2006; Cacciola et al., 2007). Colletotrichum clavatum has also been reported to occur in New Zealand and the USA (Johnston and Jones, 1997; Lardner et al., 1999; Sreenivasaprasad and Talhinhas, 2005). Furthermore, other ITS sequences accessioned by GenBank matching those of C. clavatum demonstrate that this species is present in other countries and in different hosts; however, these records are not associated to any publication and should be carefully verifi ed.

The re-assessment of the systematics of C. acutatum, which has led to the introduction of three novel species as well as to a better defi nition of C. acutatum s. s., has practical implications for biosecurity, quarantine, plant breeding and disease control (Shivas and Tan, 2009; Hyde et al., 2010). C. acutatum (teleomorph G. acutata) has, for example, been classifed as an organism of quarantine signifi cance in the European Community since 1993 and was included in the list A2 of the European and Mediterranean Plant Protection Organization (EPPO) as causal agent of anthracnose of strawberry. The causal agent of olive anthracnose has never been on the EPPO lists, as according to von Arx (1957) it has been referred to as G. cingulata (Bompeix et al., 1988; Graniti et al., 1993) or its anamorph C. gloeosporioides which is improperly considered a ubiquitous species (Phoulivong et al., 2010). By contrast, results of the present study show that in Italy the prevailing causal agents of strawberry and olive anthracnoses are C. simmondsii and C. clavatum, respectively, while C. acutatum s. s., which is widespread and causes anthracnose diseases of important crops in the Austral hemisphere, has been found only occasionally on potted oleander plants grown under greenhouse conditions in a nursery of ornamentals in Sicily (southern Italy). A precise and stable defi nition of species previously referred to collectively as C. acutatum s. l. will ultimately contribute to improve diagnosis and control of these important plant pathogens.

Acknowledgements

This research was funded by the University of Catania (PRA), the Mediterranean University of Reggio Calabria (PRIT) and MIUR (PRIN 2008). The authors would like to thank Prof. M. L. Gullino and Prof. A Garibaldi from the University of Torino (Italy) for providing the isolate AZJ from rhododendron; Dr. A Stensvand and Dr. V. Talgø from Bioforsk Norwegian Institute for Agricultural and Environmental Research (Norway) for providing the isolate 8689 from cherry; Dr. V. Sergeeva from the University of Western Sydney (NSW, Australia) for providing the isolates UWS 14, UWS 68, UWS 103, UWS 137, UWS 147, UWS 149 and UWS 166 from olive; Dr. B. Hall from South Australia Research and Development Institute, Plant Research Centre, Adelaide (SA, Australia) for providing the isolate 67 from almond, and Dr. H. Förster from the University of California, Davis (CA, USA) for providing the isolates 8 and 1765 from citrus, 1409 from papaya and 725 from strawberry.

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Accepted for publication: May 1, 2011