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Section of Feed and Food Microbiology, National Veterinary Institute, Ullevaalsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Trude Vrålstad
Trond Schumacher
Ascomycete Research group Oslo (ARON), Division of Botany and Plant Physiology, Department of Biology, University of Oslo, P.O. Box 1045 Blindern, 0316 Oslo, Norway
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ABSTRACT |
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A new genus and species is described to accommodate a newly discovered fungus pathogenic to Linnaea borealis. The fungus forms true sclerotia on stems and leaves of its host and apothecia arise singly or gregariously on the surface of ripe sclerotia. The new fungus was collected together with a stromatic conidiomal fungus that occurred on the same host. A putative teleomorph-anamorph connection of the observed taxa was ruled out by sequence comparison of the nuclear ribosomal internal transcribed spacer DNA sequences (ITS rDNA). Based on morphology and pathogenicity, the new fungus belongs in the family Sclerotiniaceae, Helotiales, Ascomycota. A phylogenetic analysis of ITS rDNA sequences from 26 taxa of the family Sclerotiniaceae was performed to conclude on the systematic position of the new fungus. The small tuberoid sclerotia, brownish subsessile to substipitate apothecia, four-spored asci, ellipsoid to isthmoid ascospores, inability to grow on PDA culture media and a number of ITS rDNA sequence autapomorphies characterize and distinguish the fungus from other taxa of the Sclerotiniaceae.
Key words: Botrytis, Caprifoliaceae, Ciboria, Ciborinia, internal transcribed spacer, ITS rDNA, phylogeny, ribosomal DNA, Sclerotinia, taxonomy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Holst-Jensen et al 1997b). The characters used to distinguish genera of the Sclerotiniaceae include the type and morphology of the sclerotial stroma, presence or absence of a mitosporic (macroconidial) anamorph, microanatomy of ascomata and the host and tissue specificity of infections (Whetzel 1945, Buchwald 1949, Kohn 1979, Schumacher and Kohn 1985, Batra 1991, Holst-Jensen et al 1997b, 1998, Schumacher 2000).
Several new species belonging in the Sclerotiniaceae are yet to be described, but the uncertain delimitation of core genera is a problem. The genera Ciboria Fuckel and Ciborinia Whetzel in particular are polyphyletic (Holst-Jensen et al 1997b, 2002), as probably is the type genus Sclerotinia Fuckel (Holst-Jensen et al 1998). While several new species could be described and accommodated within these genera as currently delimited (Schumacher 2000), only few species would be included if the genera were restricted to monophyletic entities. Because we believe that classification should reflect evolutionary relationships, we now restrict ourselves from describing new taxa with affinity to Ciboria and Ciborinia sensu lato. However, new taxa of the Sclerotiniaceae can and should be described if they are characterized by distinctive features, are clearly distinguishable from other taxa of the family and in particular if phylogenetic evidence support a specific classification. This could be either by linking them to type species of genera or to well-characterized genera, e.g., Myriosclerotinia N.F. Buchw. (Holst-Jensen et al 1998). If new taxa are sufficiently distinctive, another possibility is to erect new genera to host the new taxa, but this should be supported by phylogenetic evidence to avoid creation of paraphyletic taxa.
In Norway in late May 2000 we collected a number of specimens of a small apothecial fungus arising from true tuberoid sclerotia on leaves of Linnaea borealis (Table I; Figs. 1–4). Judged from gross morphology and host specificity, the fungus immediately was suspected to represent an undescribed taxon of the family Sclerotiniaceae sensu stricto (Holst-Jensen et al 1997b). The finding of a mitosporic anamorph of the Botrytis-type on L. borealis the same day at a separate locality led us to suspect a possible teleomorph-anamorph connection between the apothecial (teleomorph) and the conidiomal (Botrytis) fungus. We have not found previous records of any sclerotial fungus growing on Linnaea or other caprifoliaceous hosts. Although host specificity is typical for many sclerotiniaceous fungi, a number of oligo- or polytrophic species are known, e.g., in the genera Botryotinia Whetzel with Botrytis anamorphs, Monilinia Honey with Monilia anamorphs, and Sclerotinia (Kohn 1979, Batra 1991, Holst-Jensen et al 1997a, 1998, Carbone and Kohn 2001).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. In our attempts to obtain axenic culture isolates, ascospores were shot onto water agar (WA; 15 g agar, 1 L distilled H2O, autoclave 20 min) from fresh ascocarps, whereafter single spores and spore clusters were isolated under a stereo magnifier and transferred to potato-dextrose agar (PDA; Difco) and incubated at room temperature. This procedure is used successfully at ARON for all but a very few taxa of the Sclerotiniaceae (obligate biotrophic Ciborinia spp.). Axenic cultures also were prepared from mitospores of the Botrytis-anamorph, by direct transfer to PDA.
DNA sequences –
DNA was extracted from three sources: (i) fresh and dried apothecia; (ii) a dried sclerotium from which the apothecia had been detached; (iii) fresh axenic culture mycelium obtained from the Botrytis anamorph. Extraction was done with a CTAB procedure (Murray and Thompson 1980) with these modifications: (i) scaling down to eppendorf tubes (starting volume 300 µL extraction buffer); (ii) crushing the fungal tissues in the extraction buffer followed by freezing and heating once (10 min at -80 C followed by 10 min at +65 C); (iii) a single chlorophorm:isoamylalcohol extraction followed by precipitation with isopropanol and ethanol rinse. The nuclear ribosomal internal transcribed spacer region (ITS rDNA, including the ITS1, the 5.8S rRNA gene and the ITS2) was PCR amplified using the primers ITS5 and ITS4 (White et al 1990). PCR-amplifications were conducted in 40 µL volumes containing 19.5 µL 100 x diluted template DNA and 20.5 µL reaction mix (final concentrations: 4 x 250 µM dNTPs, 0.625 µM of each primer, 2 mM MgCl2 and 1 unit DyNAzymeTM II DNA polymerase [Finnzymes Oy, Espoo, Finland]) on a Genius Operator (Techne) thermal cycler. Ice-cold reaction tubes were preheated for initial denaturation of the template (4 min at 95 C), followed by 35 cycles (15 s denaturation at 94 C, 15 s annealing at 55 C and 40 s synthesis at 72 C) and a final elongation step (7 min at 72 C) before storage (4 C). The complete ITS rDNA-region was sequenced manually using the ThermoSequenase radio labelled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Ohio) with 
-33P-ddNTPs. Both strands were sequenced to confirm the sequence. The new sequence accessions are listed in Table I. For comparison of sequence similarity and to retrieve related sequences from the EMBL/GenBank/DDBJ sequence database, we performed a similarity search using the FastA program on the EMBL node (http://www.ebi.ac.uk/), probing with the full-length ITS rDNA sequence of the putative new taxon. The ITS rDNA sequences were visually aligned with available sequences of other Sclerotiniaceae retrieved from the EMBL/GenBank/DDBJ sequence database (Table I), using the freeware BioEdit version 5.0.9 (Hall 1999).
Phylogenetic analyses –
To determine whether the fungus could be linked with an existing genus of the Sclerotiniaceae, we performed a maximum parsimony analysis in PAUP* version 4.0b10 (Swofford 2001) and included sequences of all sequenced type species of genera of the Sclerotiniaceae: Botryotinia, Ciboria, Ciborinia, Coprotinia Whetzel, Dumontinia L.M. Kohn, Grovesinia M.N. Cline, J.L.Crane & S.D. Cline, Monilinia, Myriosclerotinia, Ovulinia F.A. Weiss, Pycnopeziza W.L. White & Whetzel, Sclerotinia, Stromatinia Boud., Valdensinia Peyronel and additional representatives of the genera, Ciboria, Encoelia P. Karst., Monilinia and Sclerotinia as well as of the form genera Botrytis and Sclerotium Tode : Fr. (Table I). A short region of the alignment (characters 95–123) was excluded because this region could not be unambiguously aligned. The aligned sequence matrix is available from TreeBase (Accession SN 1348). Parsimony analysis was carried out with these parameter settings: heuristic search with maxtrees = unlimited, random addition of sequences, 500 replicates to increase the chance of finding the most optimal tree island(s), swapping with the tree-bisection-reconnection (TBR) algorithm, save all minimal trees (MULPARS), collapse branches if maximum length is zero and accelerated transformation (ACCTRAN). To examine the support for inferred relationships, bootstrap analyses were performed (Felsenstein 1985). The robustness of inferred clades (Bremer 1988) was examined using the inverse constraints option. To reduce the number of most-parsimonious trees (MPTs) to be presented, we performed successive weighting (Farris 1969) with the maximum rescaled consistency index for each character over all the MPTs as the new weight (default in PAUP*).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b), a result corroborated by the present study. Even though these basally branching lineages of the MPTs suggest that long branch attraction (Felsenstein 1978) may affect the tree topology, the distinct and isolated status of the new taxon based on unique ITS rDNA sequence characters is evident. The sequence of the Botrytis anamorph grouped within the same clade as the other Botrytis/Botryotinia spp. (87% bootstrap support, Bremer support = 2). The rest of the tree is not very well resolved, as demonstrated by the low bootstrap and Bremer support values.
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Apothecia e sclerotiis orienta, subsessilia vel stipitata; discus cupulatus vel discoideus, colore pallide brunnea vel cinnamomea carnosus, receptaculo glabro vel minute pubescentia. Asci inoperculati, cylindraceo-clavati, apice incrassati, jodo caerulescentes, ad basim attenuati. Ascosporae uniseriatae, ellipsoideae, hyalinae, unicellulares; paraphyses tenues; conidia et spermatia non visa.
Apothecia arising from a sclerotial stroma, subsessile to stipitate, disks pale brown to cinnamon, fleshy, cupulate to discoid, receptacle glabrous to pruinous. Asci inoperculate, cylindric-clavate, with a thickened apex, bluing in iodine. Ascospores uniseriate, ellipsoid, hyaline, unicellular. Paraphyses slender. Conidia and microconidia wanting.
Apothecia solitaria vel plura in sclerotio, subsessilia vel brevistipitata, profunde cupulata, demum plus expansa, colore pallide brunnea vel cinnamomea, 0.6–3 mm diametro, stipes 0–1.5 mm longus. Asci cylindraceo-clavati, tetraspori, 60–75 x 4–7 µm. Ascosporae uniserialate ellipsoideae et isthmoideae, tetranucleatae, biguttulatae. Paraphyses tenues, simplices, basi ramosae, 70–85 x 1.5–2 µm, ad apicem paullum incrassatae ad 3.0–4.0 µm, ascos per 10 µm excedentes. Subhymenium et excipulum medullare e textura porrecta vel intricata, excipulum exterius 40–60 µm crassum, e cellulis angularibus vel subglobosis (textura globulosa).
Sclerotia nigra, ovata vel cylindracea, in laminis, petiolis et caulis hospitis formata.
Type species. Kohninia linnaeicola Holst-Jensen, Vrålstad & T. Schumach.
Etymology. To honor the significant contribution of Dr. Linda Kohn to the study of the Sclerotiniaceae and to reflect the host preference of the type species.
Apothecia arising one to several from the surface of the sclerotium, disk cupulate to applanate, pale brownish to cinnamon, 0.6–3 mm in diam, stipe lacking or very short to 1.5 mm long. Subhymenium brownish, of densely packed isodiametric to prismatic cells, 3–3.5 µm broad. Medullary excipulum about 40 µm thick at the base of the receptacle, gradually tapering toward the receptacular margin, composed of densely packed hyaline to pale brownish, thin-walled hyphae, 2–4 µm broad. Ectal excipulum 40–60 µm thick, hyaline, composed of thin-walled, isodiametric subangular to globose cells, commonly 8–15 µm diam, cells smaller toward the surface, there giving rise to brownish elongate-clavate hair-like rows of cells. Asci cylindric-clavate, four-spored, 60–75 x 4–7 µm, tapered below to a short stipe. Ascospores ellipsoid, inequipolar and constricted at the middle (isthmoid), tetranucleate, biguttulate, 7.0–9.5 x 2.5–4.5 µm. Paraphyses slender, branched from below, 70–85 x 1.5–2 µm, at the tip clavately widened to 3.0–4.0 µm, with brownish excudate on tips, projecting 5–10 µm above asci, forming an epithecium. Sclerotia tuberoid, blackish, with a white medulla, 1–5 x 0.5–3.5 mm, on leaf laminas, petioles and stems of the host.
Colonies not obtained from discharged ascospores on WA and PDA medium at room temperature within 3 wk.
Specimens examined. On last-year stems, leaf laminas and petioles of Linnaea borealis: NORWAY. Oppland. Ringebu. Geiteryggen south of the river Tromsa, UTM: NP 684 141, 26 May 2000, leg. A. Holst-Jensen, T. Vrålstad & T. Schumacher; teleomorph and sclerotial anamorph (ARON 2000/058-O HOLOTYPE); Oppland. Øyer. Rugsveen. UTM: NP 687945, 27 May 2000 leg. A. Holst-Jensen, T. Vrålstad & T. Schumacher teleomorph and sclerotial anamorph (ARON 2000/454-O); Oppland. Øyer. Bårdsengbekken. UTM: NP 702939, 27 May 2000 leg. A. Holst-Jensen, T. Vrålstad & T. Schumacher teleomorph and sclerotial anamorph (ARON 2000/455-O); NORWAY. Akershus. Feiring. 27 May 2000 leg. A. Holst Jensen & T. Vrålstad; culms and leaves of L. borealis infected by a Botrytis anamorph (ARON 2000/073-O).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Our observations clearly demonstrate the importance of ascertaining morphotype connections within this group of fungi based on evidence other than circumstantial field evidence, e.g., DNA sequences, as earlier pointed out (Holst-Jensen and Schumacher 1994).
Following the terminology of Whetzel (1945), the new species of Kohninia produces true sclerotial stromata, incorporating host tissues from leaves and culms of the host plant within sclerotial medulla, the sclerotia giving rise to short-stipitate apothecia, 4-spored asci, and irregularly, ellipsoid to isthmoid ascospores. The apparent lack of a conidiomal anamorph, inability to grow on PDA culture media, and a specific ITS rDNA sequence highly distinctive from other genera of the Sclerotiniaceae, further characterize the genus Kohninia.
Similar to our unsuccessful attempts to obtain axenic culture isolates of Kohninia, we and others have been unable to obtain cultures of Ciborinia spp. and have had little success with some species currently ascribed to Ciboria (Nawaschin 1893, Whetzel 1945, Buchwald 1947, Groves and Bowerman 1955, Holst-Jensen 1995). These problems might be associated with a putative obligate parasitic life history (Schumacher and Holst-Jensen 1997).
In a previous paper (Holst-Jensen et al 1998) we discussed the delimitation of Sclerotinia versus the closely related genera Botryotinia, Dumontinia, Grovesinia and Myriosclerotinia. We concluded, based on ITS nrDNA phylogeny, ecology and morphology, that to delimit truly monophyletic taxa Sclerotinia either might have to be very narrowly delimited (Sclerotinia sensu stricto) as proposed by Kohn (1979) or relaxed to include Botryotinia, Dumontinia and Grovesinia (Sclerotinia sensu lato). The first alternative implies exclusion of several taxa presently assigned to the genus Sclerotinia and would imply further that a number of small related genera need to be erected.
Our study concludes that the new fungus shares the highest ITS sequence similarity to Sclerotinia tetraspora (94.5% similarity) among all hitherto characterized taxa (data retrieved from EMBL/GenBank and unpublished data from ARON). Morphologically, S. tetraspora and the new fungus also share the feature of 4-spored asci, which is uncommon among the Sclerotiniaceae. The inclusion of S. tetraspora in Sclerotinia may be questioned based on axenic culture characteristics (sclerotia not fully detached from surface mycelium) and its consistent position as a basal branch to the clade referable to Sclerotinia sensu lato (Holst-Jensen et al 1998). In Fig. 5 of the present study, S. tetraspora is separated from Sclerotinia sensu stricto, although this placement is not well supported. The new fungus reported here is morphologically and ecologically distinct from S. tetraspora and Sclerotinia sensu stricto, and in the ITS rDNA phylogeny it occupies a much more distant position to Sclerotinia sensu stricto than S. tetraspora. The new taxon clearly is distinct from all other taxa of the Sclerotiniaceae, and it is of particular importance that it lacks affinity to the type species of the polyphyletic genera Ciboria and Ciborinia. If the new taxon is to be classified in Sclerotinia, the ITS rDNA phylogeny suggests that the genera Botryotinia, Ciboria sensu stricto, Coprotinia, Dumontinia, Grovesinia, Monilinia sensu stricto, Myriosclerotinia, Ovulinia, Pycnopeziza and Stromatinia also would have to be included in Sclerotinia sensu lato.
Although the recognition of separate genera for Botryotinia, Monilinia and Sclerotinia may be justified by their importance as crop pathogens, we would argue that classification also should reflect evolutionary patterns. Phylogenetic inference based on multiple genes and coalescent evolutionary history recently has resolved populations and species complexes in several economically important ascomyceteous taxa (cf. Taylor et al 2000, O'Donnell et al 2000, Carbone and Kohn 2001, Koufopanou et al 2001). Similar studies are needed to clarify whether the patterns observed with ITS and other rDNA sequence data reflect the underlying evolutionary history of the Sclerotiniaceae. In the meantime, we will continue to explore nature in search of new and interesting taxa and to describe new sclerotiniaceous taxa whenever we think that the conclusive evidence is sufficient, in the spirit of predecessors and colleagues such as M. Woronin, H.H. Whetzel, E.E. Honey, N.F. Buchwald, R.P. Korf, L.R. Batra and L.M. Kohn. The new genus described here is unlikely to be of any economical importance, but it serves to demonstrate that the diversity of the Sclerotiniaceae is yet to be fully appreciated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 29, 2003.
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LITERATURE CITED |
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