Ophiostoma kryptum sp. nov. from Larix decidua and Picea abies in Europe, similar to O. minus

Ophiostoma kryptum sp. nov. from Larix decidua and

Picea abies in Europe, similar to O. minus

Karin JACOBS1* and Thomas KIRISITS2

1Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada.2 Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF ), Universitat fur Bodenkultur Wien (BOKU ),Hasenauerstrasse 38, A-1190 Vienna, Austria.

E-mail : [email protected]

Received 27 January 2003; accepted 2 July 2003.

An unknown species of Ophiostoma was isolated from European larch (Larix decidua) infested by Tetropium gabrieli(Coleoptera : Cerambycidae) and Norway spruce (Picea abies) infested by Tetropium sp. in Austria. The fungus is similarto O. minus, but distinguished from it by the ecology, colony morphologies on OA and MEA, and phylogenetic analysis

of aligned DNA sequences of the ITS region of the rDNA operon and the partial b-tubulin gene. It is described hereas O. kryptum sp. nov. The new species readily produces perithecia with short necks and reniform ascospores, and hasHyalorhinocladiella and Leptographium-like anamorphs. Circumstantial evidence suggests that Tetropium spp. act as

vectors of O. kryptum. O. minus and O. kryptum represent additional examples of morphologically similar, yet geneticallyand ecologically distinct species in the genus Ophiostoma. The new combination, O. crenulatum comb. nov.(syn. Ceratocystiopsis crenulata), is also made.

INTRODUCTION

Species of Ophiostoma and related anamorph generaare economically important causal agents of blue stainin living conifers and lumber and also include speciescausing root, vascular wilt and vascular stain diseases(Brasier 1991,Wingfield, Seifert &Webber 1993, Jacobs& Wingfield 2001). Ophiostoma spp. are well-adaptedfor dispersal by insects, in particular bark beetles(Coleoptera : Scolytidae) and other phloem feeding andwood boring beetles (Nelson 1934, Bramble & Holst1940,Mathiesen-Kaarik 1953,Upadhyay 1981, Solheim1986, Wingfield et al. 1993, Jacobs et al. 2003). Severalmorphologically similar or indistinguishable fungalspecies, cryptic or sibling species in the broad definitionof the term (Kirk et al. 2001), have been recognized asdistinct taxa (Brasier & Kirk 1993, Brasier & Stephens1993, Brasier & Mehrotra 1995). The best-known ex-amples of sibling species in Ophiostoma are O. piceaeand O. quercus as well as O. ulmi, O. novo-ulmi andO. himal-ulmi, which are morphologically similar butreproductively isolated (Brasier 1993, Brasier & Kirk

1993, Halmschlager et al. 1994, Brasier & Mehrotra1995, Harrington et al. 2001).

Several strains of an Ophiostoma species resemblingO. minus were isolated from European larch (Larixdecidua) infested by Tetropium gabrieli and Norwayspruce (Picea abies), attacked by Tetropium sp. inAustria. Although this fungus is almost identical toO. minus based on teleomorph and anamorph charac-ters, its colony morphology on artificial media is dis-tinctively different. The habitat and insect associates ofthese strains are also unusual for O. minus, which hasthus far only rarely been reported from Norway spruce(Mathiesen 1950, Mathiesen-Kaarik 1953, Kotynkova-Sychrova 1966) and not from European larch. Thisstudy compares the strains from larch and spruce withO. minus and other species with similar morphologiesand assesses their phylogenetic relationships based onrDNA operon and partial b-tubulin gene sequences.

MATERIALS AND METHODS

Isolation of fungi

Strains were isolated from Larix decidua trees infestedby Tetropium gabrieli, alone or intermingled with thebark beetle Ips cembrae. Ascospores from perithecia

* Present address: Forestry and Agricultural Biotechnology Insti-tute (FABI), Department of Genetics, University of Pretoria,Pretoria, 0002, South Africa.

Mycol. Res. 107 (10): 1231–1242 (October 2003). f The British Mycological Society 1231

DOI: 10.1017/S0953756203008402 Printed in the United Kingdom.

or small pieces of bark were transferred to plates withmalt agar (MA, 2% malt, 1.6% agar, 100 mg lx1

streptomycin sulphate). Strains were also isolatedfrom the sapwood of a Norway spruce tree that hadbeen mass inoculated with the pathogenic blue stainfungus Ceratocystis polonica in May 1999 (Kirisits &Offenthaler 2002). The tree died in spring 2000 and wassubsequently infested by Tetropium sp. in the lowerpart of the bole and various bark beetle species else-where on the stem (Kirisits & Offenthaler 2002). Thetree was felled in Sept. 2000 and small pieces of bluestained sapwood were removed from the inoculatedstem section on the lower bole and placed on MAplates. Petri dishes were incubated at room temperatureand diffuse daylight. Pure cultures of the Ophiostomasp. were obtained by transferring mycelium or asco-spores from primary isolation plates to fresh MA.Seven isolates from larch, collected at four localitiesin Lower Austria (Rax, Hasendorf, Asperhofen, andKirchschlag) and six strains obtained from spruce(Kreisbach, Lower Austria), were available for detailedmorphological studies and molecular comparisons. Allisolates (Table 1) have been preserved in the culturecollection of the Institute of Forest Entomology, ForestPathology and Forest Protection (IFFF), Universitatfur Bodenkultur Wien (BOKU), and in the CanadianCollection of Fungal Cultures and herbarium(DAOM). Ten additional strains of the Ophiostoma sp.were isolated from L. decidua infested by T. gabrieliat Wartberg i. Murztal (Styria, Austria) in June 2002.

Here, T. gabrieli attacked living mature larch trees andcaused losses of 500 m3 wood between 2000 and 2001.These isolates are also maintained in the culture col-lection of IFFF-BOKU.

Morphological studies

All measurements and microscopic observations weremade from fungal structures grown on 2% MEA andOatmeal agar (OA) (Gams et al. 1998) and incubated inthe dark or in incident light at 25 xC. Fungal structureswere mounted on slides in 85% lactic acid and exam-ined using phase or differential interference contrastmicroscopy. Measurements are reported as the maxi-mum and minimum values of 50 measurements as wellas the mean ¡standard deviation. Colour codes followKornerup & Wanscher (1978).

Phylogenetic analysis

Strains (Table 1) were grown on commercial PotatoDextrose agar (PDA) for 10 d in the dark or in incidentlight at 25 x. DNA extractions from pure cultureswere prepared with the UltraCleanTM Microbial DNAkit (MoBio Laboratories, Carlsbad, CA). Successfulisolation of DNA was confirmed on 1% agarose gels.Amplification of the ITS1, ITS2 (internal transcribedspacer regions) and 5.8S region of the rDNA operonand partial b-tubulin genes was done using standardprotocols for PCR reactions. PCR reactions were

Table 1. Strains of Ophiostoma kryptum and O. minus used in the study.

Species

Collection

accession nos.aGenBank

accession nos.

Date of

isolation Origin Host Associated insect Collector(s)

O. minus DAOM 212686 AY305690

AY212686

1989 Canada None B. Grylls &

K. A. Seifert

O. kryptum DAOM 229701

(IFFF K6/3/6)

AY305685

AY304436

6 Oct. 2000 Austria Picea abies Tetropium sp. T. Kirisits

DAOM 229704

(IFFF K6/3/5)

AY304425 6 Oct. 2000 Austria Picea abies Tetropium sp. T. Kirisits

IFFF K6/3/2 AY304428

AY305687


DAOM 229703

(IFFF K6/3/3)

AY304433 6 Oct. 2000 Austria Picea abies Tetropium sp. T. Kirisits

IFFF K6/3/1 AY305688

AY304430


IFFF K6/3/4 AY304432 6 Oct. 2000 Austria Picea abies Tetropium sp. T. Kirisits

DAOM 229702

(IFFF BW/1)

AY305686

AY304434

22 July 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits &

M. J. Wingfield

DAOM 229705

(IFFF Hasd/3)

AY304426 22 July 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits &

M. J. Wingfield

IFFF Rax/2 AY304427 27 May 995 Austria Larix decidua Tetropium gabrieli T. Kirisits

IFFF Hasd/1 AY304437 22 July 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits &

M. J. Wingfield

IFFF Hasd/2 AY304429 22 July 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits &

M. J. Wingfield

IFFF Rax/3 AY302231

AY305689

27 May 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits

IFFF Asper/1 AY304435 22 July 1995 Austria Larix decidua Tetropium gabrieli T. Kirisits &

M. J. Wingfield

a DAOM, Canadian Collection National Fungus Herbarium and culture collection, Ottawa; IFFF, culture collection of the Institute of

Forest Entomology, Forest Pathology and Forest Protection, Universitat fur Bodenkultur Wien (BOKU).

Ophiostoma kryptum sp. nov. 1232

performed using Ready-To-Go (RTG) PCR beads(Amersham Pharmacia Biotech, Piscataway, NJ).Primers used in the amplification reactions were NS7and ITS4 (White et al. 1990) for the ITS region andT10 and T222, that amplifies two short introns, ashort exon at the 5k-end of the main coding exon ofthe BenA gene (O’Donnell et al. 2000, http://www.crl.umn.edu/scab/primers.html). PCR products werepurified using the UltraCleanTM PCR cleanup kit(BIO/CAN Scientific, Mississauga, ON) and sequencedusing the Big dye terminator cycle sequencing premixkit (Applied Biosystems, Foster City, CA) on an ABIPRISM310 automatic sequencer (PerkinElmerAppliedBiosystems, Wellesley, MA). Primers NS7, ITS2, ITS3and ITS4 were used to sequence both strands of the ITSregion (White et al. 1990). Primers T10, T222, T12and T2 (O’Donnell et al. 2000) were used to sequenceboth strands of the beta-tubulin genes. Sequencecontigs were assembled using Sequencher v. 4.0.5.(Gene Codes Corporation, Ann Arbor, MI) and acomputer generated alignment (Wisconsin PackageVersion 10.1, Genetics Computer Group (GCG),Madison, WI; Canadian Bioinformatics Resource,http://www.cbr.nrc.ca/) of the resulting sequences andreference sequences from GenBank was importedand manually adjusted in PAUP* v.4.0b8 (Swofford1999). Bases from the 3k end of the small ribosomalsubunit and the 5k end of the large ribosomal subunitwere excluded in the analysis of the ITS data in orderto align with sequences of other Ophiostoma speciesfrom GenBank. Phylogenetic relationships were in-ferred using a heuristic search in PAUP* v.4.0b8.Characters were treated as unweighted in the analysisand gaps were treated as a 5th base. Heuristic searcheswere performedwith tree-bisection-reconnection (TBR)branch swapping. Starting trees were obtained throughstepwise addition. The resulting trees were used to ob-tain a consensus tree. Confidence levels were estimatedusing a Bootstrap analysis (1000 replicates) with the‘fast ’-stepwise addition option.

RESULTS

Phylogenetic analysis

Amplification of the ITS region of a strain of Ophio-stoma minus (DAOM 212686) and the strains fromspruce and larch resulted in single fragments of ap-proximately 900 base pairs in length. Amplificationof the partial b-tubulin gene resulted in fragments ofapproximately 1000 base pairs in the case of O. minus,while the strains from larch and spruce had singlefragments of approximately 950 base pairs. The alignedITS dataset consisted of 615 characters. 441 characterswere constant, 73 parsimony uninformative, and 101parsimony informative. The aligned partial b-tubulindataset consisted of 750 characters. 426 characterswere constant, 90 parsimony uninformative, and 234parsimony informative. Heuristic analysis of the ITS

dataset, resulted in eight most parsimonious trees(length=293) with similar topologies. Ophiostoma ipswas used as outgroup (Fig. 1). Heuristic analysis of thepartial b-tubulin dataset resulted in three most parsi-monious trees with similar topologies (length=752).O. triangulosporum was used as outgroup (Fig. 2).

In the tree derived from analysis of the ITS dataset,the strains from larch and spruce in Europe clustertogether, but separately from O. minus (Fig. 1). Thestrains from spruce and larch also formed a groupoutside species considered to be part of the O. piceaecomplex (Harrington et al. 2001). Analysis of the par-tial b-tubulin data set confirms the distinctivenessof the strains from larch and spruce from O. minus(Fig. 2). This indicates that, although they have similarmorphologies, the strains from spruce and larch andO. minus represent distinct taxa. In both cases, thebranches are supported by high bootstrap values(Figs 1–2).

DAOM 229705 (IFFF Hasd/3)

AF198243 O. catonianum

AF198244 O. ips

DAOM 229704 (IFFF K6/3/5)

100

100

100

100

100

96

O.kryptum

IFFF Rax/2

IFFF Rax/3

IFFF K6/3/2IFFF Hasd/2IFFF K6/3/1

IFFF K6/3/4DAOM 229703 (IFFF K6/3/3)IFFF Asper1

DAOM 229702 (IFFF BW/1)DAOM 229703 (IFFF K6/3/6)IFFF Hasd/1

AF198231 O. floccosumAY194504 O. floccosum

AF198237 O. querciAF198239 O. querciAF198238 O. querci

AF198233 O. himal-ulmiAF198234 O. himal-ulmi

AF198235 O. novo-ulmiAF198236 O. novo-ulmiAF198232 O. ulmi

AY194482 O. tetropiiAY194485 O. tetropii

AY194507 O. tetropiiAF198226 O. piceae

AF198227 O. piceaeAF198228 O. canum

AF198229 O. canum160 53 O. setosumAF198230 Pesotum cupulatum

AY304438 O. minusAF234834 O. minus

5 changes

Fig. 1. One of the eight most parsimonious trees generated

through heuristic analysis of the ITS dataset (293 steps,RI=0.902, CI=0.0737, HI=0.263). Branches with 100%support in a consensus tree are indicated in bold. Bootstrap

values are indicated above the branches.

K. Jacobs and T. Kirisits 1233

O. minus and the strains from spruce and larch alsoshow differences in their colony morphologies onartificial media. While O. minus has darkly pigmentedcolonies (Kornerup & Wanscher 1978: pl. 5F2)(Figs 21–22), the strains from spruce and larch inEurope have colonies that are almost colorless,with dark patches where perithecia develop on MEA(Fig. 19) and OA (Fig. 20). Morphological and mol-ecular comparison with other species of Ophiostomaled us to conclude that the strains from spruce andlarch represent a previously undescribed species ofOphiostoma. These strains were distinguished from O.minus based on the differences in sequence data, colonymorphologies on agar media as well as ecologicaldifferences.

TAXONOMY

Ophiostoma kryptum Jacobs & Kirisits, sp. nov.(Figs 3–22)

Etym. : From the Greek adjective ‘kryptos ’, hidden.

Bases peritheciales atrae, globosae et glabro-tunicatae,inornatae vel sparsissima ornatione, (40–)87¡14(–110) mm

diametro, collum fuscum vel atrum, leve, (46–)71¡11(–91) mm longum, (24–)30¡4(–39) mm super basim globo-

sam, (4–)6¡1(–8) mm latum ad apicem, hyphae ostiolaresadestis. Asci prototunicati, hyalini, evanescentes. Ascosporaereniformes, aseptatae, hyalinae, curvatae et non in vaginainvestitae, 3–5r1.0 mm. Anamorpham Leptographium:

Conidiophora evenientia singulatim vel catervatim, erecta,macronematosa, mononematosa, levia, subbrunnea velhyalina, (36–)63¡17(–93) mm longitudine, massa conidica

exclusa, rhizoidea absentia. Stipes subbrunneus vel hyalina,cylindricus, simplex, 0–1 septatus, (7–)10¡3(–17) mm longus.Secundus vel tribus seriebus ramorum cylindricorum; 2–4

metulae primariae. Cellulae conidiogenae discretae, 2–3 perramum, exigue attenuatae a basi ad apicem, cylindricae, rec-tae, (8–)20¡4(–28) longae et 1–2 mm latae. Synanamorpham

Hyalorhinocladiella : Conidiophora hyalina, simplicia, even-ientia singulatim ariis myceliis, conidiophora (7–)18¡11(–65) mm longa et 1–3 mm lata, conidia formantus per-currentur in cellis conidiogenis. Conidia hyalina, aseptata,

obovoidea vel allantoidea, apice rotundato et basibus sub-truncatis, 2–6r1–3 mm.Typus : Austria : Lower Austria : Kreisbach, sapwood of

dead Picea abies infested by Tetropium sp. after mass inocu-lation with Ceratocystis polonica, 6 Oct. 2000, T. Kirisits(DAOM 229701 – holotypus).

Perithecial bases black, globose and smooth, (40–)87¡14(–110) mm diam, with sparse basal hyphalornamentation. Perithecial necks dark brown to black,cylindrical with a slight apical taper, smooth, (46–)71¡11(–91) mm long, (24–)30¡4(–39) mm at the base,(4–)6¡1(–8) mm wide at the apex, ostiolar hyphaeoccasionally present, slightly pigmented at the base,hyaline towards the apex, (9–)18¡5(–24) mm long.Asci prototunicate, hyaline, evanescent. Ascosporesreniform in side view, aseptate, hyaline, not investedin a sheath, 3–5r1.0 mm.

Anamorphs : (1) Leptographium-like : Conidiophoresoccurring singly or in groups, mostly on aerial mycelia,macronematous, mononematous, (36–)63¡17(–93) mmin length excluding the conidial mass, rhizoid likestructures absent. Stipes olivaceous, simple, 0–1 septate,(7–)10¡3(–17) mm long, 2–4 mm wide below primarybranches. Conidiogenous apparatus with 2 to 5 seriesof cylindrical branches. Primary branches, 2–4, olivace-ous, smooth, cylindrical aseptate, (6–)9¡1(–13) mmlong and 1–3 mm wide, arrangement of the primarybranches on the stipe-type B (more than two branches;Jacobs & Wingfield 2001). Conidium developmentannellidic and delayed secession of conidia gives a falseimpression of sympodial proliferation. Conidiogenouscells discrete, 2–3 per branch, cylindrical, taperingslightly at the apex, (8–)20¡4 (–28) mm long and1 mm wide. (2) Hyalorhinocladiella : conidiophoresoccurring singly on aerial mycelia, conidiophore (7–)18¡11(–65) mm and 1–2 mm wide. Conidia of bothanamorphs hyaline, aseptate, obovoid with truncatebases and rounded apices, 2–6r1–3 mm. Conidialdroplet hyaline at first, becoming white with age.

Colonies 26 mm diam in 6 d at 25 x on 2% MEA.Colonies on 2% MEA white, floccose, perithecia

100

DAOM 229702 (IFFF BW/1)

DAOM 229701 (IFFF K6/3/6)

100

100

100

100

90

O.kryptum

IFFF Rax/3

IFFF K6/3/2

IFFF K6/3/1

AY305691 O. floccosum

AY305692 O. floccosum

AY305693 O. querci

AY305711 O. novo-ulmi

AY305712 O. novo-ulmi

AY305701 O. tetropii

AY305702 O. tetropii

AY305694 O. piceae

AY305695 O. piceae

AY305699 O. canum

AY305700 O. canum

AY305703 O. setosum

AY305690 O. minus

10 changes

100

99 AY305696 O. piceae

AY305697 O. piceae

AY305698 O. piceae

100

AY305713 O. triangulosporum

AY305704 O. piliferum







Fig. 2. One of the three most parsimonious trees generatedthrough heuristic analysis of the b-tubulin dataset (752 steps,

RI=0.884, CI=0.747, HI=0.253). Branches with 100%support in a consensus tree are indicated in bold. Bootstrapvalues are indicated above the branches.


Figs 3–13. Light micrographs of Ophiostoma kryptum (DAOM 229701, IFFF K6/3/6). Figs 3–5. Perithecia with short necks.Ostiolar hyphae occasionally present. Bar=50 mm. Fig. 6. Reniform ascospores without sheaths. Bar=10 mm. Figs 7–11.Hyalorhinocladiella anamorph with annellidic conidium development. Bar=10 mm. Fig. 12. Leptographium-like anamorph,

showing an almost synnematous appearance in some cases. Bar=10 mm. Fig. 13. Obovoid to almost clavate conidia.Bar=10 mm.


abundant. Colony becoming darker (Kornerup &Wanscher 1978: pl. 5F6) with age in patches whereperithecia form. Colony margin laciniate. Colonycolour on OA, colorless when young, becoming ta-bacco brown (Kornerup & Wanscher 1978: pl. 5F6)where perithecia are formed with abundant white aerialmycelia. Colony margin laciniate.

Habitat and vectors : Ophiostoma kryptum occurs inthe bark and the wood of Norway spruce (Picea abies)infested by Tetropium sp. and European larch (Larixdecidua) infested by Tetropium gabrieli, where it likelythrives as a weak parasite or saprophyte. Circumstan-tial evidence suggests that Tetropium species are specificvectors of O. kryptum, since perithecia and conidio-phores were only observed in and around galleriesand pupal chambers of the insects in the bark and woodof infested trees (Figs 23–28).

Distribution : Ophiostoma kryptum is at present onlyknown from a few localities in Lower Austria andaround Wartberg i. Murztal (Styria, Austria), but it issuspected to follow the distribution range of its insectvectors in Europe and Asia. Together with Tetropium

fuscum, O. kryptum might have been introduced intoAtlantic Canada, although it has so far not beenrecorded there (Jacobs et al. 2003).

Additional specimens examined : Austria : Lower Austria :Kreisbach, sapwood of dead Picea abies infested by Tetro-

pium sp. after mass inoculation with Ceratocystis polonica,6 Oct. 2000, T. Kirisits (DAOM 229704, IFFF K6/3/5) ;Kirchschlag, Bucklige Welt ; outer bark of Larix decidua

infested by Tetropium gabrieli, 22 July 1995, T. Kirisits &M. J. Wingfield (DAOM 229702, IFFF BW/1); Kreisbach,sapwood of dead Picea abies infested by Tetropium sp. aftermass inoculation with Ceratocystis polonica, 6 Oct. 2000,

T. Kirisits (DAOM 229703, IFFF K6/3/3) ; Hasendorf ; outerbark of Larix decidua infested by Tetropium gabrieli, 22 July1995, T. Kirisits & M. J. Wingfield (DAOM 229705, IFFF

Hasd/3).

DISCUSSION

The ophiostomatoid fungi associated with economi-cally important and potentially devastating insects arewell-known and have been documented in numerous

Figs 14–18. Line drawings of Ophiostoma kryptum (DAOM 229701, IFFF K6/3/6). Fig. 14. Perithecium with ostiolar hyphae.

Bar=50 mm. Fig. 15. Reniform ascospores. Bar=10 mm. Fig. 16. Leptographium-like anamorph. Bar=10 mm.Fig. 17. Hyalorhinocladiella anamorph. Bar=10 mm. Fig. 18. Obovoid conidia. Bar=10 mm.


studies. However, in the case of secondary pests, suchas Tetropium spp., very little is known regarding theirfungal associates (Jacobs et al. 2003). Although thesesecondary insects usually do not cause highly signifi-cant problems in their native habitat, they can be aserious threat when introduced into new areas, as in thecase of T. fuscum in Canada (Smith & Hurley 2000).

In the present study, phylogenetic analysis of theDNA sequences of the ITS region of the rDNA operonand the partial b-tubulin gene made it possible to rec-ognize Ophiostoma kryptum from spruce and larchin Europe as an undescribed species, distinct fromO. minus. That these fungi are different was suspectedbecause of differences in habitat and insect associates,as well as their distinct colony morphologies. O. minusis predominantly found on pine species attacked byTomicus piniperda in Europe (Mathiesen 1950,Mathiesen-Kaarik 1953, Lieutier et al. 1989, Solheim &Langstrom 1991) as well as Dendroctonus frontalis and

Dendroctonus brevicomis in North America (Hedgcock1906, Bramble & Holst 1940, Mathre 1964, Moser1985, Perry 1991) where it is considered to be a vascularstain pathogen and an economically important stain-ing organism of timber (Munch 1907, Nelson 1934,Bramble & Holst 1940, Solheim & Langstrom 1991,Uzunovic et al. 1999, Gorton & Webber 2000). Incontrast, O. kryptum is found in Europe on dying anddead spruce and larch trees infested by Tetropiumspecies. O. kryptum is morphologically most similar,to the well-characterized O. minus (Hedgcock 1906,Hunt 1956, Upadhyay 1981, Gorton & Webber 2000),and the two taxa are essentially indistinguishablemicromorphologically. In common with O. minus,O. kryptum is characterized by a Hyalorhinocladiellaanamorph, perithecia with short necks and reniformascospores without sheaths. Some researchers havesuggested the presence of a Leptographium-like ana-morph in O. minus (Hunt 1956, Upadhyay 1981). We

Figs 19–22. Colony morphology of Ophiostoma kryptum (DAOM 229701, IFFF K6/3/6) and O. minus on MEA and OA.Fig. 19. Light coloured colonies of O. kryptum on MEA. Fig. 20. Greyish colony of O. kryptum on OA. Fig. 21. Dark colonieswith patches of white aerial mycelia of O. minus on MEA. Fig. 22. Dark colonies with patches of white aerial mycelia ofO. minus on OA.


observed a Leptographium-like anamorph in O. kryp-tum, although the conidiophores are more complexand synnemata-like than those described for O. minus.The conidiophores of the Hyalorhinocladiella ana-morphs of O. kryptum and O. minus are similar inlength and they also have similar conidial shapes andsizes (Table 2). The perithecia of O. kryptum haveshorter necks than those of O. minus, but the rangesof dimensions of neck lengths of the two speciesoverlap considerably. Ostiolar hyphae are present inO. minus, while perithecia of O. kryptum only rarelyhave these structures. The ascospore shapes and sizesof O. kryptum correspond to those reported for

O. minus. Mathiesen (1950), Mathiesen-Kaarik (1953)and Kotynkova-Sychrova (1966) reported O. minusfrom P. abies, infested by Tetropium sp. However,because this is an unusual habitat for O. minus, it ispossible that the fungus was actually O. kryptum.

In a recent re-evaluation of the taxonomic status ofO. minus, Gorton & Webber (2000) recognized threetaxa in this group. O. minus and O. pseudotsugae (syn.Ceratocystis pseudotsugae), previously considered asynonym ofO. minus, are suggested to be separate taxa,based on differences in RAPD banding patterns andminor morphological differences. Also, O. minus ap-pears to be a homothallic species, while,O. pseudotsugae

Figs 23–28. Ophiostoma kryptum on European larch infested by Tetropium gabrieli (Wartberg i. Murztal, Styria, Austria).Fig. 23. Perithecia in a pupal chamber in the wood. Fig. 24. Abundant perithecia and ‘sclerotium-like ’ structures in the outerbark. Fig. 25. Perithecia in an insect gallery around the entrance hole of a larva into the wood. Fig. 26. Perithecia around

a live pupa of T. gabrieli in a pupal chamber in the bark. Fig. 27. Part of a pupal chamber in the wood with numerousperithecia. Fig. 28. Perithecia and ‘sclerotia-like ’ structures in the outer bark.


Table 2. Comparison of Ophiostoma kryptum with similar species of Ophiostoma based on morphological and ecological characters.

Characters O. kryptum O. minus O. crenulatum O. minutum O. ranaculosum O. neglectum

Anamorph Hyalorhinocladiella Hyalorhinocladiella Hyalorhinocladiella Hyalorhinocladiella Hyalorhinocladiella Hyalorhinocladiella

Synanamorph Leptographium-like Leptographium None reported Synnematous

(Leptographium?)

None reported Synnematous

Conidiophore length 7–65 mm 55(–70) mm 5–35(–55) mm 6–22(–55)r1–3.5 mm Not reported Not reported

Conidium shape Ellipsoid to obovoid Clavate, ellipsoid to

obovoid

Ellipsoid, ovate

to clavate

Ellipsoid or cylindrical Clavate Ellipsoidal, obovoid

or clavate

Conidium size 2–6r1–3 mm 2.5–6r1.5–3.5 mm 2–5r1–2.5 mm 2–5.5r1–2 mm 3–8r1–1.5 mm 3–6r1–3 mm

Perithecial base diam 40–100 mm 50–120 mm 50–100 mm 50–87.5 mm 40–150 mm 55–100 mmNeck length 46–91 mm 55–160 mm 100–200 mm 45–150 mm 12–60 mm 65–130 mm

Neck base 24–39 mm 15–25(–33) mm 18–30 mm 18–40 mm 15–25 mm 21–35 mm

Neck apex 4–8 mm 5–12(–16) mm 6–11 mm 6–21 mm Not reported 12–18 mm

Ostiolar hyphae Occasionally present Sometimes lacking Present Mostly present Absent Present

Ascospore shape Crescent to reniform Crescent to sickle Lunate (falcate

with sheath)

Falcate Falcate, tadpole shaped Cucullate

Ascospore size 3–5r1 mm 2.5–5.5r1–1.5 mm 2.5–4r1 mm 6.5–13r1–2 mm 9–15r0.5–3 mm 3.5–4r1.5–2 mm

Sheath Absent Absent Present Present Present Present

Host Larix decidua

Picea abies

Pinus

Pseudotsuga

Pinus Pinus

Picea

Larix

Pinus Picea abies

Insect vectors Tetropium spp. Dendroctonus spp.

Tomicus spp.

Ips spp.

Orthotomicus proximus

Hylastes ater

Pissodes pini

Tetropium sp.

Not known Dendroctonus spp.

Ips spp.

Crypturgus cinereus

Crypturgus pusillus

Dryocoetes autographus

Gnathotrichus materiarius

Hylurgops palliatus

Ips acuminatus

Ips typographus

Ips sexdentatus

Orthotomicus laricis

Pissodes pini

Pityogenes chalcographus

Polygraphus poligraphus

Tomicus minor

Tomicus piniperda

Trypodendron lineatum

Dendroctonus spp. Hylurgops palliatus

Crypturgus cinereus

Crypturgus pusillus

Dryocoetes autographus

Ips typographus

Pityogenes chalcographus

Trypodendron lineatum

Known geographic

distribution

Europe (Austria) Europe, North America North America Europe, USA, South

Africa, Asia

USA Europe (Germany)

References Hedgcock (1906),

Mathiesen-Kaarik (1953),

Hunt (1956)

Olchowecki & Reid

(1974)

Hunt (1956), Kirschner

(1998), Kirisits et al.

(2000), Mathiesen (1950,

1951), Mathiesen-Kaarik,

1953, Yamaoka et al.

(1997)

Bridges & Perry

(1987)

Kirschner &

Oberwinkler (1999)

K.JacobsandT.Kirisits

1239

is heterothallic. Furthermore, Gorton & Webber(2000) recognized two separate taxa, currentlyrecognized as O. minus, based on differences in RAPDbanding patterns as well as small morphologicaldifferences. The first taxon represents strains that arehomothallic, while the second one consists of isolatesthat are heterothallic. It is unlikely that O. kryptumis the same species as O. pseudotsugae, as the latter isconsidered heterothallic (Gorton & Webber 2000),while the former is homothallic. The ITS sequencesof strains of O. kryptum neither resemble those ofheterothallic strains of O. minus that Gorton &Webber (2000) recognised as distinct from the homo-thallic strains, nor those of homothallic strains ofO. minus (unpubl.).

In common with O. kryptum and O. minus, severalother Ophiostoma spp. have perithecia with shortnecks and Hyalorhinocladiella anamorphs (Table 2).However, morphological characters are sufficient todistinguish O. kryptum from the Ophiostoma spp. withsuperficially similar morphologies (Table 2). These taxainclude O. crenulatum1, O. minutum, O. neglectum andO. ranaculosum (Mathiesen 1951, Olchowecki & Reid1974, Upadhyay 1981, Bridges & Perry 1987, Benade,Wingfield & Van Wyk 1996, Kirschner & Oberwinkler1999). O. crenulatum, O. minutum, and O. ranaculosumhave, until recently been accommodated in Cerato-cystiopsis and have ascospores with falcate sheaths(Mathiesen 1951, Upadhyay 1981, Bridges & Perry1987) (Table 2). O. kryptum is easily distinguished fromthese species based on its reniform ascospores lackingsheaths. Besides other characters, O. kryptum is distin-guished from O. neglectum because of the cucullatesheaths around the ascospores in the latter species(Kirschner & Oberwinkler 1999), in contrast to theascospores without sheaths in the former species(Table 2).

O. kryptum shares some characters with O. tetropii,the most common species of Ophiostoma associatedwith Tetropium sp. on spruce in Europe (Mathiesen1951, Solheim 1986) and T. fuscum in Canada (Jacobset al. 2003). O. kryptum and O. tetropii have similaranamorphs (Hyalorhinocladiella and Leptographium-like) and both are homothallic with abundant peri-thecial development on oatmeal agar. O. tetropii canbe distinguished from O. kryptum by the larger peri-thecia with long necks, ornamented with abundantostiolar hyphae. Thus far, O. tetropii has not beenreported from European larch infested by T. gabrieli inEurope.

Our observations and isolation results provide cir-cumstantial evidence that Tetropium spp. act as vectorsof O. kryptum, since this fungus was only isolated from

bolts infested by these cerambycid insects. In addition,structures of O. kryptum are very common in breedinggalleries of T. gabrieli on European larch, and thisfungus is probably the most abundant fungal associateof this cerambycid. The biology of Tetropium spp.makes it difficult to understand how these insectsmight transmit O. kryptum and other ophiostomatoidfungi to their host trees and maintain a permanentsymbiosis with them. These cerambycid species hidetheir eggs in bark cracks and underneath bark scaleson the surface of the bark (Schimitschek 1929, Hellrigl1974). During oviposition by Tetropium spp., the barkis not wounded, which would be essential for success-ful inoculation of associated blue-stain fungi. Phoreticmites or nematodes might act as secondary vectors ofOphiostoma spp. to trees, as it has been shown in otherinsect-fungal systems (Wingfield & Blanchette 1983,Moser 1985, Moser, Perry & Solheim 1989). The modeof transmission of O. kryptum and other ophio-stomatoid fungi by Tetropium spp. remains poorlyunderstood and deserves further study.

Besides occurring in insect galleries and pupalchambers (Figs 23–27), perithecia and conidiophoresof O. kryptum were common in the outer, dry part ofthe bark of larch infested by T. gabrieli (Figs 24, 28).The outer bark is an unusual habitat for a speciesof Ophiostoma, although Munch (1907: 541–543) alsoreported and illustrated perithecia of O. minus in theouter bark of infected pine trees, as in O. kryptumon larch. As described for O. minus, O. kryptum pro-duces, according to the terminology of Munch (1907),‘sclerotia-like pillars ’ that lift off the inner bark fromthe sapwood surface and the outer from the inner bark(Fig. 28). Thereby hollows are created, in which peri-thecia and conidiophores of O. kryptum are numer-ously formed (Figs 24, 28). Nothing is known regardingthe pathogenicity of O. kryptum on larch and spruceor its abilty to cause stain in lumber. No signs of blue-stain were seen on larch trees infested by T. gabrieli,and this leads us to assume that O. kryptum is a sapro-phyte or very weak pathogen.

ACKNOWLEDGMENTS

This study was motivated by cultures obtained in 1995 by T.K. and

Mike J. Wingfield in the course of collecting ophiostomatoid fungi

from European larch in Austria. We thank Mike for his support

and interest during all phases of the work that formed part of this

study. We are also grateful to him and Keith A. Seifert for critical

reviews of earlier versions of this paper. We thank Keith and Gerry

Louise-Seize for making their unpublished b-tubulin sequences

available to us, and James Reid for the loan of the type ofCeratocystis

crenulata. We acknowledge the financial assistance to K.J. from

the National Research Foundation (South Africa) and a Strategic

Grant from the Natural Sciences and Engineering Research Council

of Canada (NSERC) to Colette Breuil (University of British

Columbia, Vancouver). T.K. received financial support from the

Special Research Program Forest Ecosystem Restoration (SF008),

funded by the Austrian Science Foundation and the Austrian Minis-

try of Agriculture and Forestry. This paper is part of a broader study

of ophiostomatoid fungi on insect-infested larch in Austria and

Scotland.

1 Ophiostoma crenulatum (Olchowecki & Reid) Jacobs, comb. nov.

Basionym: Ceratocystis crenulata Olchowecki & Reid, Can. J. Bot.52 : 1681 (1974). Syn.: Ceratocystiopsis crenulata (Olchowecki &Reid) Upadhyay, Monogr. Ceratocystis and Ceratocystiopis : 124(1981).


REFERENCES

Benade, E., Wingfield, M. J. & Van Wyk, P. S. (1996) Conidium

development in the Hyalorhinocladiella anamorphs of Cerato-

cystiopsis minuta-bicolor and Ophiostoma minus. Canadian Journal

of Botany 74 : 891–897.

Bramble, W. C. & Holst, E. C. (1940) Fungi associated with

Dendroctonus frontalis in killing shortleaf pines and their effect

on conduction. Phytopathology 30 : 881–899.

Brasier, C. M. (1991) Ophiostoma novo-ulmi sp. nov., causative agent

of the current Dutch elm disease pandemics. Mycopathologia

115 : 151–161.

Brasier, C. M. (1993) The genetic system as a fungal taxonomic tool:

gene flow, molecular variation and sibling species in the ‘Ophio-

stoma piceae–Ophiostoma ulmi ’ complex and its taxonomic and

ecological significance. In Ceratocystis and Ophiostoma : taxo-

nomy, ecology and pathogenicity (M. J. Wingfield, K. A. Seifert &

J. F. Webber, eds): 77–92. American Phytopathological Society

Press, St Paul, MN.

Brasier, C. M. & Kirk, S. A. (1993) Sibling species withinOphiostoma

piceae. Mycological Research 97 : 811–816.

Brasier, C. M. & Stephens, T. M. (1993) Temperature-growth re-

sponses distinguish the OPC and OPH sibling species within

‘Ophiostoma piceae ’. Mycological Research 97 : 1416–1418.

Brasier, C. M. & Mehrotra, M. D. (1995) Ophiostoma himal-ulmi

sp. nov., a species of Dutch elm disease fungus endemic to the

Himalayas. Mycological Research 99 : 205–215.

Bridges, J. R. & Perry, T. J. (1987) Ceratocystiopsis ranaculosus

sp. nov. associated with the southern pine beetle. Mycologia 79 :

630–633.

Gams, W., Hoekstra, E. S. & Aproot, A. (1998) CBS Course

of Mycology. 4th edn. Centraalbureau voor Schimmelcultures,

Baarn.

Gorton, C. & Webber, J. F. (2000) Reevaluation of the status of

the bluestain fungus and bark beetle associate Ophiostoma minus.

Mycologia 92 : 1071–1079.

Halmschlager, E., Messner, R., Kowalski, T. & Prillinger, H. (1994)

Differentiation of Ophiostoma piceae and Ophiostoma quercus

by morphology and RAPD analysis. Systematic and Applied

Microbiology 17 : 554–562.

Harrington, T. C., McNew, D., Steimel, J., Hofstra, D. & Farrell, R.

(2001) Phylogeny and taxonomy of the Ophiostoma piceae complex

and the Dutch elm disease fungi. Mycologia 93 : 111–136.

Hedgcock, G. G. (1906) Studies upon some chromogenic fungi

which discolor wood.Missouri Botanical Garden Annual Report 17 :

59–111.

Hellrigl, K. (1974) Cerambycidae, Bockkafer. In Die Forstschadlinge

Europas. Vol. 2. Kafer. (W. Schwenke, ed.) : 130–202. Paul Parey

Verlag, Hamburg.

Hunt, J. (1956) Taxonomy of the genus Ceratocystis. Lloydia 19 :

1–58.

Jacobs, K. & Wingfield, M. J. (2001) Leptographium Species: tree

pathogens, insect associates and agents of blue-stain. American

Phytopathological Society Press, St Paul, MN.

Jacobs, K., Seifert, K. A., Harrison, K. J. & Kirisits, T. (2003)

Identity and phylogenetic relationships of ophiostomatoid fungi

associated with invasive and native Tetropium spp. (Coleoptera :

Cerambycidae) in Atlantic Canada.Canadian Journal of Botany 81 :

316–329.

Kirisits, T., Grubelnik, R. & Fuhrer, E. (2000) Die okologische

Bedeutung von Blauepilzen fur rindenbrutige Borkenkafer. In

Mariabrunner Waldbautage 1999 – Umbau sekundarer Nadelwalder

(F. Muller, ed.) : 117–137. FBVA-Berichte III, Schriftenreihe der

Forstlichen Bundesversuchsanstalt, Wien.

Kirisits, T. & Offenthaler, I. (2002) Xylem sap flow of Norway spruce

after inoculation with the blue-stain fungus Ceratocystis polonica.

Plant Pathology 51 : 359–364.

Kirk, P. M., Cannon, P. F., David, J. C. & Stalpers, J. A. (2001)

Ainsworth & Bisby’s Dictionary of Fungi. 9th edn. CABI Publishing,

Wellington.

Kirschner, R. (1998) Diversitat mit Borkenkafern assoziierter

filamentoser Mikropilze. PhD dissertation, Germany, Eberhard-

Karls-Universitat, Tubingen.

Kirschner, R. & Oberwinkler, F. (1999) A new Ophiostoma species

associated with bark beetles infesting Norway spruce. Canadian

Journal of Botany 77 : 247–252.

Kornerup, A. & Wanscher, J. H. (1978) Methuen Handbook of

Colour. Eyre Methuen, London.

Kotynkova-Sychrova, E. (1966) Mykoflora chodeb kurovcu v

Ceskoslovensku. Ceska Mykologie 20 : 45–53.

Lieutier, F., Yart, A., Garcia, J., Ham, M. C., Morelet, M. &

Levieux, J. (1989) Champignons phytopathogenes associes a deux

coleopteres scolytidae du pin sylvestre (Pinus sylvestris L.) et etude

preliminaire de leur agressivite envers lkhote. Annales des SciencesForestieres 46 : 201–216.

Mathiesen, A. (1950) Uber einige mit Borkenkafern assoziierte

Blauepilze in Schweden. Oikos 2 : 275–308.

Mathiesen, A. (1951) Einige neue Ophiostoma-Arten in Schweden.

Svensk Botanisk Tidskrift 45 : 203–232.

Mathiesen-Kaarik, A. (1953) Eine Ubersicht uber die gewohnlichsten

mit Borkenkafern assoziierten Blauepilze in Schweden und einige

fur Schweden neue Blauepilze. Meddelanden fran Statens Skogs-

forskningsinstitut 43 : 1–74.

Mathre, D. E. (1964) Survey ofCeratocystis spp. associated with bark

beetles in California. Contributions from the Boyce Thompson

Institute 22 : 353–362.

Moser, J. C. (1985) Use of sporothecae by phoretic Tarsonemusmites

to transport ascospores of coniferous bluestain fungi. Transactions

of the British Mycological Society 84 : 750–753.

Moser, J. C., Perry, T. J. & Solheim, H. (1989) Ascospores hyper-

phoretic on mites associated with Ips typographus. Mycological

Research 93 : 513–517.

Munch, E. (1907) Die Blaufaule des Nadelholzels. Naturwissens-

chaftliche Zeitschrift fur Land- und Forstwirtschaft 5 : 531–573.

Nelson, R. M. (1934) Effect of bluestain fungi on southern pines

attacked by bark beetles. Phytopathologische Zeitschrift 7 :

326–353.

O’Donnell, K., Kistler, H. C., Tacke, B. K. & Casper, H. H. (2000)

Gene genealogies reveal global phylogeographic structure and

reproductive isolation among lineages of Fusarium graminearum,

the fungus causing wheat scab. Proceedings of the National Acad-

emy of Sciences, USA 97 : 905–910.

Olchowecki, A. & Reid, J. (1974) Taxonomy of the genus Cerato-

cystis in Manitoba. Canadian Journal of Botany 52 : 1675–1711.

Perry, T. J. (1991) A Synopsis of the Taxonomic Revisions in the genus

Ceratocystis including a review of Blue-staining species associated

with Dendroctonus bark beetles. [General Technical Report SO-86.]

USDA, Forest Service, Southern Forest Experiment Station,

New Orleans.

Schimitschek, E. (1929) Tetropium gabrieli Weise und Tetropium

fuscum F. – Ein Beitrag zu ihrer Lebensgeschichte und Lebens-

gemeinschaft. Zeitschrift fur Angewandte Entomologie 15 : 229–334.

Smith, G. & Hurley, J. E. (2000) First North American record of

the Palearctic species Tetropium fuscum (Fabr.) (Coleoptera : Cer-

ambycidae). The Coleopterists Bulletin 54 : 540.

Solheim, H. (1986) Species of Ophiostomataceae isolated from Picea

abies infested by the bark beetle Ips typographus. Nordic Journal of

Botany 6 : 199–207.

Solheim, H. & Langstrom, B. (1991) Blue-stain fungi associated with

Tomicus piniperda in Sweden and preliminary observations on

their pathogenicity. Annales des Sciences Forestieres 48 : 149–156.

Swofford, D. L. (1999) PAUP*: phylogenetic analysis using parsi-

mony (*and other methods). Version 4.0. Sinauer Associates,

Sunderland, MA.

Upadhyay, H. P. (1981) A monograph of Ceratocystis and Cerato-

cystiopsis. University of Georgia Press, Athens, GA.

Uzunovic, A., Yang, D. Q., Gagne, P., Breuil, C., Bernier, L., Byrne,

A., Gignac, M. & Kim, S. H. (1999) Fungi that cause sapstain

in Canadian softwoods. Canadian Journal of Microbiology 45 :

914–922.


Wingfield, M. J. & Blanchette, R. A. (1983) The pine-wood nema-

tode, Bursaphelenchus xylophilus, in Minnesota and Wisconsin:

insect associates and transmission studies. Canadian Journal of

Forest Research 13 : 1068–1076.

Wingfield, M. J., Seifert, K. A. & Webber, J. F. (1993) Ceratocystis

and Ophiostoma: taxonomy, ecology and pathogenicity. American

Phytopathological Society Press, St Paul, MN.

White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and

direct sequencing of fungal ribosomal RNA genes for phylo-

genetics. In PCR Protocols: A Guide to Methods and Applications

(A. M. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds) :

315–322. Academic Press, San Diego.

Yamaoka, Y., Wingfield, M. J., Takahashi, I. & Solheim, H. (1997)

Ophiostomatoid fungi associated with the spruce bark beetle

Ips typographus f. japonicus in Japan. Mycological Research 101 :

1215–1227.

Corresponding Editor: D. L. Hawksworth


Documents

Ophiostoma kryptum sp. nov. from Larix decidua and Picea abies in Europe, similar to O. minus