247 A new Ophiostorna associated with bark infesting Norway spruce

R. Kircchner and F. Oberwinkler

Abstract: During a survey of fungi associated with bark beetles in Germany, an undescribed specles of Ophiostoma was isolated that differs from the other species of the genus by having pigmented, aseptate, convergent ostiolar hyphae, cucullate, sheathed ascospores, al],d a Hyalorhinocladiella ar,amorph, The species is described as Ophiostoma neglectum Kirschner & Oberwinkler. It is rarely associated with primary bark beetles but often associated with secondary bark beetles mainly infesting Norway spruce.

I Key wordsi Ophiostoma neglectum, Hyalorhinocladiella, secondary bark beetles, Picea abies, Pinus sylveslris, conidial t develoPment

R6sum6 : Au cours d'un survol des champignons associ6s aux insectes des €corces en Allemagne, les auteurs ont isol6 une espöce d'Ophiostoma qui difföre des autres espöces du genre par la prdsence de pigments, d'hyphes ostiolaires aseptes et convergents, de spores cucull6es et enveloppees et d'un anamorphe de type Hyalorhinocladiella. Les a.ute!trs d6crivent cette espöce comme I'Ophiostoma negleclum Kfuschner and Oberwinkler Elle est rarement associde avec les insectes primaires de 1'6corce, mais souvent associde avec les insectes secondaires de 1'6corce, surtout ceux qui infestent 1'6pinette de Norvdge.

Mots clis I Ophiostoma neglectum, Hyalorhinocladiella, insectes secondaires de l'6corce, Picea abies, Pinus sylvestris, d6veloppement conidial.

[Traduir par Ia R6daction]

lntroduction tles' galleries in the phloem and sapwood of the infested tlees. Bark beetles are known to be associated with specific The diversity of ophiostomatoid fungi associated with fungi. Many of these are ophiostomatoid fungi, mainly spe- some bark species in Europe was investigated by cies of Ophiostoma H. & P Sydow, Cerqtocystis Ellis & Grosmann (1931) in Germany, Siemaszko (1939) in Poland, Halst., and Cefttocystiopsis Upadhyay & Kendrick, which Mathiesen-Käärik (1953) in Sweden, Kotynkovä-Sychrovä are vectored by bark beetles to new host trees (Mathiesen- (1966) in the former Czechoslovakia Republic, Solheim iäärik t953: Solheim 1986). Ceratocystis spp. differ mor- I (1986) in Norway, Harding (1989) in Denmark, L6vieux et ' phologically from the two other mentioned genera by their al. (1989) in France, and Pashenova et al. (1995) in Russia. Chalara (Cotda) Rabenh. anamorphs (de Hoog and Scheffer Nevertheless, galleries are still a poorly explored 1984). Ceratocystiopsrs spp. differ from species of the genus habitat of the fungi. Ophiostoma by having falcate ascospores (Upadhyay and During a recent survey of fungi associated with bark bee- Kendrick 1975). Species of Ophiostoma have ascospores of tles in Germany, several new species were isolated different shapes and often have anamorphs that can be ac- (Kirschner 1998). One was described as Phialocephala commodated in the following genera: Hyalorhinocladiella trigonospora by Kirschner and Oberwinkler (1998). Another Upadhyay & Kendrick, Sporothrix Hektoen & Perkins, new species is presented here as a member of Ophiostoma, Leptographium Lagerberg & Melin, alrfi. Graphiunt Corda (Seifert et al. 1993). Some ophiostomatoid fungi are virulent plant pathogens Materials and methods and assist aggressive bark beetles in overwhelming host tlee Fiom autumn 1994 to autumn 1996, bark samples of Norway resistance (Chdstiansen and Solheim 1990), whereas others spruce (Picea abies (L.) Karst.) and Scots pine (Pi,us are weak plant pathogens or saprophytes. They all depend rylreslris L.) containing adult bark beetles were collected near Bad on dispersal by bark beetles and sporulate in the bark bee- Waldsee, Langenau, Schluchsee, and Tübingen in Baden- Württemberg, and near Riedlhütte (Bayrischer Wald) and Oberjoch in Bavaria, all in southem Gemany. The beetles werc identified as cinereus (Hrbst.), C. pusillus (Gyll.), Dryocoetes auto- Received June 3, 1998. graphus (Ratz.), Hylurgops palliatus (Gyll.), Ips tlpographus (L,), R. Kirschnerr and F, Oberwinkler. Lehrstuhl Spezielle Pityogenes chalcographus (L.), ar,d Trypodendron lineatum (Oli- BotanikMykologie, Universität Tübingen, Auf der vier). Living beetles were individually placed in Petri dishes con- Morgenstelle l, D-'120'76 Tübingen, Germany. taining autoclaved pieces of spruce phloem embedded in water agar. Purc cultures of the new fungal species were obtained by lAuthor to whom all correspondence should be addressed. tansfering ascospores extruded at the tip of the ftuit bodies with a e-mail: [email protected] sterile needle to Petri dishes with autoclaved pieces of spruce twigs

Can. J. Bot. 71: 247-252 11999) O 1999 NRC Canada 248 Can. J. Bot. Vol. 77, 1999

Table 1. Infestation by Ophiostoma neglectum ol batk beetles collected frorn Picea abies and Pinus sylvestris in southem Gemany from 1994 to 1996.

Species No- infested 7. infested Total no- examined

Crypturgus cituereus 1 0.8 127 Crypturgus pusillus 49 15 Dryocoetes autographus 130 39 334 Hllurgops palliatus 130 30 431 Ips typographus 6 0.6 1071

P itlo B e ne s chal c o graphus 8 0.8 9'16 Trypodendron lineatum 7 5 137

embedded in l.5Ea watet agar and to Petri dishes containing hyalina cucullata, 3.54 >< 1.5-2 [rn a latere visae, cir- 2Eo malt extact agat (20 g Difco malt extmct per 1 L water). culares, l-1.5 Um diametro, cum vagina hyalina triangulari Growth diameters were measured from thrce cultures growing aspectu terminali, in filo gelatinoso exkusae. Coloniae in on malt extract agar in the dark at 23"C. For light microscopy, agaro malto l0 mm diamero post 12 dies äd 23"C, albae, fresh material was mounted in water. To compare the new fungus sine mycelium aedum. Conidiophorae in substrato naturali with a similar species, a culture of Ophiostoma brevicolla (David- saepe aggregatae. hyalinae. ereclae. mononematosae. stipes son) de Hoog & Scheffer (CBS 795.73) was examined by light mi- brevissimus, apparatus conidiogenus ex usque tribus ser- croscopy. For scanning electon microscopy (SEM), matedal was rcmoved from a pure culture of CBS 100596 (from ex-type stain iebus metularum et cellularum conidiogenarum compositus. of the new Ophiostoma species) on autoclaved spruce twigs em- Metulae 5-6 x 2 Ftm, cellulae conidiogenae annellidicae, bedded in water agar, frxed rn 2Vo glutaraldehyde in 0.1 M cylindricae, 2V27 x l-2 Um. Conidia hyalina, aseptata, cla- cacodylate buffer for several days, postfixed in l7o osmium yata,345 x 1-1.5 pm, in massa hyalina aggregata. Con- tetoxide in 0.1 M cacodylate buffer for I h, washed with distilled idiophorae in agaro malto simplices vel ex stipe brevissimo water, and dehydrated in a graded ethanol sedes (Rieder and et cellulis conidiogenis compositae, conidia hyalina, asep- Schmidt 1987). The material was subsequently critical point dried, tata, ellipsoidea, obovoidea vel clavata, 3-6 x 1-3 pm. coated with gold palladium, and examined using a Cambddge Slereoscan 250 MK 2 scanning electron micro\cope. EryMo]-ocy: 'fhe epithet neglect m refers to the inconspic- uous perithecia growing in a cryptic habitat. Hor-orypus: CBS 100596 (cultura sicca), de Hylurgope Results palliato ex trur,co Piceae abietis lecto isolata, Germania, Baden-Württemberg, Tübingen, 10.09.1995, R. Kirschner. Originating from individual bark beetles that were placed in Petri dishes containing autoclaved spruce phloem pieces Ophiostoma neglectum CBS 100597, de Pityogene chal- embedded in water agar, different fungi developed and spor- cographo ex tunco Piceae abietis lecto isolata, Germania, ulated after about I month. Among these, a previously un- Baden-Württemberg, Bad Waldsee, 12.09.1994, R. Kir described Ophiostoma species was found. The new species schner. has a Hyalorhinocladiella anamorph and cucullate asco- spores and is therefore included in the genus Ophiostoma. cuLruRAE vrvAE: Ophiostoma neglectum CBS 100596. The species was obtained from bark beetles mainly infesting globose, Picea abies. At leasl sEo of the examined individual beetles Perithecia superficial on the substratum, bases ir- (1-1.5 pm pale of the four bark beetle spectes Crypturgus pusillus, Dry- regularly covered with thin hyphae thick), ocoetes autogrqphus, Hylurgops palliatus, and Trypoden- brown, 55-100 pm in diameter, necks dark brown, 65 pm dron lineatunt were associated with the new Ophiostoma sp, 130 !m long, 2l-35 [m wide at the base, 12-18 wide at (Table 1). This Ophiostoma sp. was detected from less than the apex (Fig. l), ostiolar hyphae convergent, stiff, brown, lqo of the individuals from the other three species of bark aseptate, thick-walled, 20-59 En long, 2-3 Um wide at the gradually to the acute apex (Fig. cla- beetles, Crypturgus cinereus, Ips typographus, al],d Pityo- base, tapering 2). Asci (Fig. genes chalcographus. A description of the fungus as a new vate, up to 16 pm long and 5 Um wide, evanescent 3). sheath, Ophio stoma species follows. Ascospores cu ed, cucullate, with hyaline, cucullate 1.5-2 x 354 pm in side view (including sheath), circular, Ophiostoma neglectum Kirschner & Oberwinller sp.nov. 1-1.5 pm in diametet with hyaline, triangular sheath in end (Figs. 1-10) view (Fig. 4), exüuded as a gelatinous filament (Fig. l). Colonies on malt extract agar 10 mm in diameter in Perithecia superficialia, bases globosae, irregulariter or- 12 days at23"C, white, without aerial mycelium and without näta angustatis (l-1.5 Um diametro) hyphis, pallide brun- perithecia. Conidiophores on natural media often aggre- neae, 55-100 [m diametro, colla atrobrunnea,65-130 Um gated, hyaline, erect, mononematous, with a very short stipe longa, ad basim 2l-35 Urn, ad apicem 12-18 Um lata, hy- and up to thrce series of metulae and conidiogenous cells phae ostiolares convergentes, rigidae, brunneae, aseptatae, (Fig. 5b). Conidiogenous cells annellidic, cylindrical, 1-2 x parietibus crassis, 20-59 Um longae, ad basim 2-3 pm latae, 2V27 W. Conidia (Fig. 5a) hyaline, one-celled, clavate, 1- sensim acuminatae. Asci clavati, ad 16 Um longi, 5 pm lati, 1.5 x 3-4.5 pm, aggregated in a slimy head. Conidiophores evanescentes. Ascosporae curvatae, cucullatae, cum vagina in malt extract agar simple or composed, with a very short

O 1999 NRC Canada Krrschner and oberwinkler 249

Figs. 1-4. Teleomorph of Ophiostoma neglectum. Fig. l. Perithecium with ascospores extruded in a filament. Scale bar = 20 pm. Fig. 2. Convergent, pigmented, aseptate ostiolar hyphae. Fig. 3. Clavate, young asci. Fig. 4. Ascospores with cucullate sheaths in side view and triangular sheaths in end view. Figs. 2-4. Scale bar = l0 pm. qffiw

E^ Da P 1s- C §q

1 2 stipe and conidiogenous cells (Fig. 6ä), conidia hyaline, nellations are irregularly swollen, causing distortions of the one-celled, ellipsoidal, obovoid or clavate, 1-3 x 3-6 !m conidiogenous cell. (Fig. 6a). The mode of conidiogenesis was not clearly visible using Discussion light microscopy. Some very inconspicuous scars seemed to be present at the tips of the conidiogenous cells (Figs. 5ä, 6ä). Denticles indicating a sympodial conidiogenesis were The anamorph of the new species has hyaline, mono- not found by either light or electron microscopy. By SEM, nematous conidiophores. The annellated conidiogenous cells annellations at the distal parts of the conidiogenous cells of this anamorph, seen by SEM, differ from annellated were visible (Figs. 7-10). The segmerts between the an- conidiogenous cells without distortions known from other

@ 1999 NRC CaMda Can. J. Bot. Vol. 77, 1999

Figs, 5 and 6. Anamorph of Ophiostoma neglectutu. Scale bar = 10 |rm. Fig. 5. Hyalorhinocladiella anamorph on spruce twig piece§ embedded in water agar. Conidia (.r) and penicillately branched conidiophore (ä). Fig. 6. Hyalorhinocladiella anamorph on malt extract agar. (a) Conidia. (ä) Reduced conidiophores.

k,, O 5

")o %go

hyphomycetes with annellidic conidiogenesis, e.8., Lepto- conidiogenous cells with denticles were predominant graphium spp. (Wingfield 1985) and lhe Hyalorhinoclad- (Benade et al. 199'7). The deviating type of annellidic iella atamorph of Ophiostoma ips (Rumb.) Nannf. (Benade conidiogenous cells was regarded as an intermediate stage et al. 1995). A very similar type of annellidic conidiogenous between exclusively sympodial and exclusively annellidic in cells was found in the Hyalorhinocladiella anamorphs of which most conidia are produced by annellidic conidio- Ceratocystiopsis minuta-bicolor (Davidson) Upadhyay & genesis and few conidia by sympodial conidiogenesis Kendrick and Ophiostoma mizus (Hedgcock) H. & P. (Benade et al. 1996, 1,997). Although intermediates exist be- Sydow (Benade et al. 1996). Similar annellidic conidio- tween annellidic and sympodial conidiogenesis in both the genous cells were also detected in the Sporothrix anamorph anamorph gerrcta Hyalorhinocladiella ar.d Sporothrlx, the of Ophiostoma nigrocarpum (Davidson) de Hoog, but two genera can be separated by predominantly annellidic

O 1999 NRC Canada K rschner and Oberwinkler 251

Fig§. 7-10. Hyalorhinoclatlielh anamorph of Ophiostoma neglectum (frorn ex-type culture) seen by SEM. Fig. ?. Distal parts of several conidiogenous cells producing teminal conidia. Scale bar = 2 pm. Fig. 8. Conidiogenous cells with the sites of last conidium secession visible (arrowheads). pm. Scale bar = 4 Fig. 9. Distal part of a conidiogenous cell with distorted annellations. Scale bar = 2 pm. Fig. 10. Apex of young conidiogenous cell terminally producing a conidium. Scale bar = I p6.

conidiogenesis in H,-aktrltinockrclie1Lr and predominantly Ophiostoma neglectLLm differs from other species of the sympodial conidiogenesis in Sporotlu.tr. Because no conidio- genus by the combination of the following three characteris genous cells with denticles werc detected in the anamorph of tics: pigmented, aseptate, convergent ostiolar hyphaei the new Ophiosto,r?r7 species, annellidic conidiogenesis is cucullate, sheathed ascospores; and Hyalorhinocladiella arra- apparently predominant. Therefore, this anamorph is accom- morph. The species is similar to Cerqtocystiopsis minuta modated in Hl,alorltinocLadiellct. (Siemaszko) Upadhyay & Kendrick in having aseptate, con- The conidiophores of this anamorph genus were charac- vergent ostiolar hyphae and a Hyctlorhinocladielkt ara- tedzed as "simple, reduced or hardly distinguishable from morph, but the ascospores of the former are cucullate and the vegetative hyphae" (Upadhyay 1981). This is the case in those of the latter are falcate (Upadhyay 1981). Ophiostoma cultures growing on rich media like malt extract agar neglectum is similar to O. brevicolla because of the conver- (Fig.6ä). On natural media, however, the conidiophores of gent ostiolar hyphae and curyed, sheathed ascospores, but the HyalorlinocladieLla anamorph are more complex, i.e., O. brevicollq differs by its septate ostiolar hyphae (R. the conidiophores are composed of a shoft, hyaline stipe and Kirschner and F. Oberwinkler, personal obseryations on a penicillately branched conidiogenous head (Fig. 5D). Thus strain CBS 795.73) and its pigmented Leptographium ari,a- apart from the lack of pigmentation and the short stipe in morph (Upadhyay 1981). The ascospores of O. brevicolla comparison with the large conidiogenous head, these co- are 6-'1.5 trrm long including sheath (Upadhyay 1981) and nidiophores exhibit a similar structure as those of Lepto- are therefore longer than those of O. neglectum. graphium. The conidial development in both genera is identical (Benade et al. 1996). Similar, complex conidio- Ecology phores were also described for the Hyalorhinocladiella arla' Ophiostoma neglectum was found to be associated with morph of O. minus (Hedgcock) H. & P Sydow developing bark beetles (Table l) that preferably äest Picea abies on the natural substratum (Münch 1907). (Postner 1974). The fungus was not detected from bark bee-

O I ggq NRC Cänaila 252 Oan. J. Bot. Vol.77, 1999 tles predominantly infesting other trees , e.9., Ips sexdentatus Christiansen, E., and Solheim, H. 1990. The bark beetle-associated (Boem.) and Orthotomicus laricis (F.) infesting Plnrs syl- blue-stain fungus Ophiostoma polonicum can kill various 436-446. vesrris (Kirschner 1998). Therefore, O. neglectum seems lo spruces and Douglas-fir Eur J. For. P^thol. 2t vetsts have a preference for Picea abies phloem and sapwood. de Hoog, G.S., and Scheffeq R.J. 1984. Ceratocystis Among the spruce-infesting bark beetles associated Ophiostoma'. a reappraisal. Mycologia, 76: 292-299. 1931. Beiträge zur Kenntnis der Lebensgemein- with O- neglectum, Ips typograPhus and Pityogenes chalco- Grcsmann, H. Borkenkäfem und Pilzen. Z. Pamsitenkd. 3: 56- graphus arc primary, i.e., aggressive bark beetles that can in- schaft zwischen to2. fest healthy living trees (Rudinsky 1962). T\e other beetle Grünwald, M. 1986. Ecological segregation of bark beetles species in the list are secondary bark beetles that rarely in- (Coleoptera, Scolytidae) of spluce. J. Appl. Entomol. 101: 176- fest living trees (Postner 197 4). Table 1 shows that the fun- 187. gus was rarely associated with the mary bark beetles lps P Harding, S. 1989. The influence of mutualistic blue §tain fungi on and Pityogenes chalcographus because less typographus bark beetle dynamics. Ph.D. thesis, Department of Zoology, carried O. neglectum. In contast, tham lVo of these beetles Royat Veterinary and Agricultural Univer§ity, Copenhagen, the secondary the fungus was very often associated with Denmark. pall- bark beetles Dryocoetes autographus arÄ Hylurgops Kirsch[er. R. 1998. Dive$ität mit Borkenkäfem assoziierter beetles. idrrr, being carried by at least 307o of the individu4l filamentöser Mikropilze. Ph.D. thesis, Fakultät für Biologie These beetles preferably colonize dead tre€s in a shady and Universität Tübingen, Tübingen, Gemany. moist environment (Grünwald 1986). Because of the com- Kirschner, R., and Oberwinkler, F. 1998. Phialocephala trigono' quto' paratively close association of O. neglectum with D, spora, a new hyphomycete §pecies associated with conifer- graphus atd, H. palliatus, the fungus seems to have icolous bark beetles. Sydowia, 50: 205-212. substralum requirements like those of these two bark beetle Kotynkovä-sychrovä, E. 1966. Mykoflora chodeb kurovcu v species. To a lesser extent, the fungus was also associated Ceskoslovensku. Ceska Mykol. 20t 45-53. with the other secondary bark beetles CryPturgus spp. arld L6vieux, J., Lieutie( J., Moser, J.C., and Perry, T.J. 1989. Trans- Trypotlendron lineatum. h most cases, Crypturgus spp. es- portation of phytopathogenic fungi by the bark beetle lPr tablish their galleries using galleries of other bark beetles sexdentatus Boerner and associated mites J. Appl. Entomol. (Postner 1974). In our collections, Crypturgus spp. were 108: 1-11. very often associated with primary bark beetles. Crypturgas Mathiesen-Kä?idk, A. 1953. Eine Übeßicht über die gewöhntichen spp, are assumed to carry a fungus flora similar to that of mit Borkenkäfem assoziierten Bläuepilze in Schweden Medd. their host beetles and therefore are not regularly associated Statens Skogsforskninginst. 43: l-74. with O. neglectum. Trypodendron lineaturu is an ambrosia Münch, E. 1907. Die Btauftiule des Nadelholzes. Naturwiss. Land- beetle preferably associated with an ambrosia fungus (Post- Forstwin.5:531-573. rrcr 197 4). Pashenova, N.V, Vetrova, VP., Maffenina, R.M., and Sorokina, Because of the close associatiot of O. neglectum wilh E.N. 1995. Ophiostomataceae fungi in larch bark beetle galler- secondary bark beetles infesting spruce trees, this fungus aP- ies. Un Russian.l Lesovedenie, 6: 62-68. Die pears to be a pure saprophyte predominantly colonizing dead Postner, M. 1974. Scolytidae (= Ipidae), Borkenkäfer. In Edited W. trunks of Norway spruce, probably lacking plant pathogenic Forstschädlinge Europas. Band 2: Käfer. by pp. 334482. capabilities. Schwenke. Paul Parey, Hamburg, Germany. Rieder, N., and Schmidt, K. 1987. Morphologische Arbeits- methoden in der Biologie. VCH Publisheß, Weinheim, Ger- Acknowledgements many. We thank H. Schoppmann for the work with SEM, F. Rudinsky, J.A. 1962. Ecology of Scolytidae. Annu. Rev. Entomol. Albrecht for helping with the photographic work, M. Pie- 7:327 348. A penbring for criticalty reading the manuscript, and S. Wood Seifert, K.A., Wingfield, M.J., and Kendrick, W.B. 1993. species ol Cerutocystis, Ophiostoma, and I. Reinhold for conecting the English text. C. Turnau nomenclator for described atrd Sphaeronaemella. In and S. Harding kindly provided literature not available in Cerotocystiopsis, Ceratostomella Ceratocystis al,d Ophiostoma'. taxonomy, ecology, and pathoge- Germany. The study was supported by the DFG-project Or- nictty. Edited by M.J. Wingfield, K.A. Seifert, and J.F Webber. ganismische Interaktionen in Waldökosystemen. APS Press, St. Paul, Minn. pp. 269-288. Siemaszko, W. 1939. Zespoty grzyb6w towarzyszacych komikom References polskim. Plarta Pol. 7: l-54. isolated from Benade, E., Wingfield, M.J., and van Wyk, P.S 1995. Conidium Solheim, H. 1986. Species of Ophiostomataceae development ln the Hyalothinocladiella anamorph of Picea abies infestedby the bark beetle lpr ,rpographus. Nord. l. Ophiostoma !pr. Mycologia, 87l. 298-303. Bot. 6t 199-207 . Benade, E., Wingfield, M.J., and van Wyk, PS. 1996. Conidium Upadhyay, H.P. 1981. Ceratocystis and Ceratocystiopsis. Univer- development in the Hralorhinocladiella anamotphs of sity of Georgia Press, Athens, Ga. Ceratocystiopsis tuinuta-bicolor artd Ophiostotha minus. Can. l. Upadhyay, H.P, and Kendrick, W.B. 1975. Prodromu§ for a rcvi- (Micrcascales, its Bot. 74: 891-89'l . sior. of Ceratocystis Ascomycetes) and Benade, E., Wingfield, M.J., and van Wyk, PS. 1997. Conidium conidial states. Mycologia, 67: 798-805. development it Sporothtitc anamolphs of Ophiostoma. Mycol. Wingfield, M.J. 1985. Reclassification of Vefiicicladiella based on Res- 101: 1108-1112. conidiat development. Trans. B. Mycol. Soc.85: 81-93.

@ 1999 NRC Canada