New Leptographium Species from Indonesia and Eastern North America

595

Mycoscience 41: 595--606, 2000

New Leptographium species from Indonesia and Eastern North America

Karin Jacobs", Michael J. Wingfield" and Dale R. Bergdahl21 1)Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, Republic of South Africa 2)Department of Forestry, School of Natural Resources, University of Vermont, Burlington, Vermont, 05405, U.S.A.

Accepted for publication 21 September 2000

Leptographium species have predominantly been described from North America, Canada and Europe. These fungi generally occur on conifers and many cause blue-stain of lumber. Most Leptographium species are also associated with insects and in particular, bark beetles (Coleoptera: ScolytidaeJ. Recently, an unknown species of Leptographium was isolated from pine infested with an Ips sp. in Indonesia. In addition, two unknown species have been collected from red spruce (Picea rubra) and balsam fir (Abies balsamea) roots from high elevation sites in Eastern North America. The latter isolates are unusual in that they are associated with the feeding wounds made by the conifer swift moth Korsche/fellus graci/us (Lepidoptera: Hepialidae), which is a habitat unique for species of Leptographium. Comparison with known Leptographium species has revealed that the isolates from Indonesia and those from Eastern North America represent three previously un described taxa. They are, therefore, described in this study as L. pineti sp. nav, L. abietico/ens sp. nov. and L. peucophi/um sp. nav.

Key Words-conifer swift moth; conifers; Ips; Leptographium.

Species of Leptographium Lagerb. & Melin are generally known due to their role in causing black-stain root dis- characterised by dark mononematous conidiophores with ease on pine (Pinus spp.) and Douglas-fir (Pseudotsuga complex conidiogenous apparatuses (Kendrick, 1962; menziesii (Mirb.) Franco) (Kendrick, 1962; Harrington 1964; Wingfield, 1993). Numerous conidia are and Cobb, 1987; Harrin9ton, 19881. produced from conidiogenous cells through percurrent Most of the Leptographium species described from proliferation (Kendrick, 1962). Delayed secession of the North America have been associated with insects. Ex- conidia can lead to the false appearance of sympodial de- ceptions include L. antibioticum (W. B. Kendrick) M. J. velopment (Van Wyk et aI., 1988). Leptographium spe- Wing!., L. brachiatum (W. 8. Kendrick) M. J. Wing!. and cies are generally associated with conifers (Lager berg et O. trinacriforme (Leptographium anamorph) (A. K. Par- aI., 1927; Kendrick, 1962; Harrington, 1988), with only ker) T. C. Harr. {Parker, 1957; Kendrick, 19621. The as- a few exceptions described (Davidson, 1942; 1958; sociation of Leptographium species with bark beetles is 1971; 1976; Jooste, 1978; Weber et al.. 1996), Most well recognised, and various hypotheses exist regarding species of Leptographium are carried by insects, especial- the relationships between the fungi and these insects ly, bark beetles (Coleoptera: ScolytidaeJ and they sporu- (Craighead, 1928; Harrington, 1988, Six and Paine, late profusely in galleries of these insects (Lagerberg et 1996). A common view is that most species are ac- aI., 1927; Leach et al.. 1934; Harrington, 1988). cidental contaminants of bark beetles and that they are Species of Leptograph;um have been recorded from generally saprophytic (Harrington, 1988). In some many parts of the world and many species have been ac- cases, they might serve as a source of nutrition for the in- cidentally introduced into new areas along with bark bee- sect larvae (Six and Paine, 1996) and their role as patho- tles (Wingfield and Marasas, 1980). However, the gens has been extensively recorded, although in some majority of species are native to the Northern hemisphere cases this is also disputed {Wingfield et aI., 1995; and especially North America and Europe, where most Krokene and Solheim, 1996, 19981. conifers and their bark beetle pests originate (Lagerberg In Europe and Asia, many Leptographium species et al.. 1927; Rumbold, 1936; Goidanich, 1936; Parker, have been associated with bark beetles and particularly 1957; Kendrick, 1962; Robinson-Jeffrey and Grin- species of Ips (Solheim, 1986; Van der Westhuizen et aI., chenko, 1964; Kendrick and Molnar, 1965; Robinson- 1995; Yamaoka et aI., 1997; Jacobs et ai, 19981. From Jeffrey and Davidson, 1968; Griffin, 1968; Davidson, studies conducted on conifers infested with Ips species 1971; Morelet, 1988; Jacobs et aI., 1997). Amon9 the in Japan, two new Leptographium species were recently North American species, the three varieties of L. described. However, these species were not found to wagener; (W. B. Kendrick) M. J. Wingf. are probably best be associated with similar insects in Europe (Wingfield et 596 K. Jacobs et at. aI., 1994; Van der Westhuizen et aI., 1995; Jacobs at aI., ameters of colonies were computed as an average of 1998). Apart from O. penieil/atum (Grosmannl Sie- eight readings. maszko and O. piceaperdum (Rumbold) Arx. that are as- Cycloheximide tolerance of the same isolates sociated with I. typographus in Europe ISolheim. 19861, representing the three species that were used in the tem- various Leptographium species from east Asia have not perature studies, was determined by growing them on been recorded elsewhere in the world (Yamaoka et aI., 2% MEA amended with different concentrations of cyclo- 1997). Two interesting examples include. L. {arids K. heximide (0, 0.05, 0.1,0.5, 1,2,5 and 5 g/l) in Petri dish- van der Westh., M. J. Wingf. & Yamaoka and L. aenig- es. Dishes were incubated in the dark at 25°C for 8 d maticum K. Jacobs. M. J. Wingf. & Yamaoka, from Larch and the colony diameters determined from two measure- (Larix spp.), associated with I. cembrae and I. ments taken at 90° to each other. Five replicates for typographus, respectively (Van der Westhuizen et ai, each cycloheximide concentration were included, and 1995; Jacobs et aI., 1998). growth was determined based on averages of ten di- In recent years, a collection of isolates of Lep- ameter measurements. tographium species has emerged from Pinus merkusii infested with Ips sp. in Indonesia as well as balsam fir Results (Abies balsamea (L.) Mill.) and red spruce (Picea rubens Sarg.) in North America associated with damage to roots The Leptographium species from P. merkusii infested by the conifer swift moth, Korscheltel/us gracil/us with Ips sp. in Sumatra is characterised by short robust (Lepidoptera: Hepialidae). The main objective of this conidiophores with dark stipes and short conidiogenous study was to examine these isolates and to provide ap- apparatus made up of two to three series of branches. propriate names for them. These isolates are, furthermore, characterised by their short conidiophores that produce small, obovoid conidia. Materials and Methods Comparison with all other Leptographium species revealed that these isolates do not resemble any known tax- Galleries of an Ips sp., commonly infesting P. merkusii on, and we conclude that they represent a previously trees in Northern Sumatra, Indonesia, were examined un described species, which is described as follows: and the dominant fungus in these galleries was a Lep- tographium species. Red spruce and balsam fir roots Leptographium pineti K. Jacobs & M. J. Wingf., sp. wounded by the conifer swift moth, Korcheltel/us graci/- novo Figs. 1-7 Ius, collected on White Face Mountain, New York. USA Coloniae in 2% MEA ad 25°C optime crescents, et were also found to be infested with Leptographium spe- post 6 dies 15 mm diam. attingentes atro-olivaceae, mar- cies. Conidial masses from these fungi were transferred gine integra, sub 5°C vel supra 30°C non crescents, ex from the apices of conidiophores to 2% malt extract agar mycelia vegetativo immerso vel emerso pallide oliva ceo (MEA) (20 9 8iolab malt extract, 20 9 8iolab agar and vei hyalino laevi non constricto 12.0-12.5-4.01-6.01 pm 1000 ml distilled waterl plates amended with 0.05 g/l cy- diam. constantes, sine hyphis aeriis. Conidiophora sin- cloheximide. Resulting colonies were transferred to guta, e mycelio rectangulatim exorientia, erecta, macro- clean 2% MEA plates and incubated at 25°C until the on- nematosa, mononematosa, 100-202(-210) pm longa; set of sporulation., Fungal structures for microscopic ex- stipites cylindracei, simplices, olivacei, leaves, 2-4-septa- amination were mounted on glass slides in lactophenol. ti, 150-)70-1251-150) pm longi, infra ramos primaries Fifty measurements of each relevant morphological struc- 5.0-7,5 pm lati, ad baslm (5.0-16.0-10 pm lati et haud ture were made and ranges and averages computed. tumidi. Apparatus conidiogeni 17-74 pm longi ex ramis Colours of the colonies and fungal structures were deter- cylindricis, 2-3 seriatis compositi; rami primarii 2-3, pal- mined using the colour charts of Rayner (1970). Cul- lide olivacel cel hyaline, aseptati, (10-)13-17(-20) x 3.0- tures have been stored in the collection of the Forestry 5.0(-6.0) pm; rami secundarii hyaline, aseptati, (7.0-) and Agricultural Biotechnology Institute (FABI), Universi- 9.0-121-15) x 2,0-4,0 pm; rami tertiatoo hyaline, asep- ty of Pretoria (CMWL as well as in the culture collection tat;, 15.0-17.0-9.01-15) x 2.0-3.0; structura rhizoidei- of the National Collection of Fungi, Pretoria, South Africa formi absens. Cellulae conidiogenae discretae, 2-3 per (PPRI). ranum, cylindricae, sursum leviter attenuatae, holoblasti- The optimal temperatures for growth of isolates cae, 6.0-16 x 2.0 pm. Conidia aseptata, oblonga vel representing three distinct Leptographium species (CMW obovoidea, ad apicem apparatus conidiogeni in massa 3831; PPRI 6911 and CMW 3832; PPRI 6910 from in- guttulata mucilagiosa accumulata, 2.0-3.0 x 1.0 pm. donesia, CMW 2865; PPRI 6917 and CMW 2866; PPRI Colonies with optimal growth at 25°C on 2% MEA, 6918 from balsam fir, CMW 2875; PPRI 6920 and CMW reaching 15 mm in diam in 6 d. No growth below 5°C or 2876; PPRI 6922 from red sprucel were determined by above 30°C. Able to withstand high concentrations of inoculating eight MEA plates for each temperature with a cycloheximide with a 12% reduction in growth on 0.1 gll 0.6 mm diameter agar disk taken from the actively grow- cycloheximide after 6 d at 25°C in the dark. Colony ing margins of a fresh isolate. Plates were incubated at dark olivaceous (19" f). Colony margin smooth. Hyphae submerged or on top of solid medium with no temperatures ranging from 5 to 35 ° C at 5°C intervals. Colony diameters were measured on the fourth and the aerial mycelium, light olivaceous to hyaline, smooth, not eighth day after commencement of the trail, and the di- constricted at the septa, (2.0-13.01-6.0) pm in diam. New Leptographium species 597

Figs. 1-6. Leptographium pineti fCMW 3831). 1. Conidiophore with a dark olivaceous stipe and complex conidiogenous apparatus (Bar= 50 pm). 2. Complex conidiogenous apparatus IBar= 10 pm). 3. Conidiogenous cells showing false sympodial conidiogenesis IBar= 10 pm). 4. Conidiogenous cells showing false sympodial conidiogenesis fBar= 5 pm). 5. Conidia IBar= 10 pm). 6. Conidia (Bar= 1 pm). 598 K. Jacobs et al.

A 7 B Fig. 7. Leptographium pinet; (CMW 3831). A. Habit sketch of the conidiophore (Bar= 50 pm). B. Conidiogenous apparatus (Bar=10pm). C. Conidia (Bar=10pml.

Conidiophores occurring singly, arising directly from (Figs. 2, 7b). Conidiogenous cells discrete, 2-3 per the mycelium, erect. macronematous, mononematous, branch, cylindrical, tapering slightly at the apex, (6.0-1 100-202{-21 01 I'm in length, rhizoid-like structures oc- 10(-161I'm long and 21'm wide. Conidium develop- casionally present. Stipe olivaceous~ smooth, cylindri.. ment occurring through replacement wall building with cal, simple, 2-4-septate, (50-199(-150) I'm long (from holoblastic ontogeny and percurrent proliferation and first basal septum to below primary branches), (4.0-) deJayed secession giving a false impression of sympodial 5.0(-7.51 pm wide below primary branches, apical cell proliferation (Minter, Kirk and Sutton, 1982; 1983; Van Wyk et al.. 19881 (Figs. 3, 4). Conidia, aseptate, obo- not swollen, (5.0-)7.5{-10) I'm wide at base, basal cell not swoJ1en (Figs. 1, 7a). Conidiogenous apparatus 30- void, 2.0-3.0 x 1.0 I'm (Figs. 5, 6, 7c). Conidia ac- 70 long, excluding the conidial mass, with 2 to 3 series of cumulating in slimy droplets at the apex of conidiogenous cylindrical branches, 2-3 primary branches, light apparatus. olivaceous to hyaline, smooth, cylindrical, aseptate, (1 0-) Teleomorph state: not known Holotype: PREM 56391 (dried specimen). from 9alle- 15(-201 I'm long and 3.0-5.0(-6.0) I'm wide, secondary branches hyaline, aseptate, (7 .0-)9.0-12(-15) I'm ries of Ips sp. under the bark of P. merkus;;, collected by long, 2.0-4.0 pm wide, tertiary branches hyaline, asep- M. J. Wingfield, Samosir Island, Sumatra, Indonesia, tate, (5.0-)7.0-9.0{-151I'm long, 2.0-3.0 I'm wide March 1996. New Leptographium species 599

Additional specimens: PREM 56351, PREM 56354, ameter. PREM 56392, PREM 56355, PREM 56353, PREM Conidiophores occurring singly or in groups of up to 56352, PREM 56351 (dried specimens), from 9alleries of six, arising directly from the mycelium, erect, macro- Ips sp. under the bark of P. merkusii, collected by M. J. nematous, mononematous, 1120-1160-195(-3601 I'm in Wingfield, Samosir Island, Sumatra, Indonesia, March length, rhizoid-like structures present. Stipe dark 1996, olivaceous. smooth, cylindrical, simple, 2-11-septate, Etymology: From the Latin noun pinetum: a pine 172-192-2391-264) I'm long Ifrom first basal septum to stand. This specific epithet refers to the habitat of the below primary branches), 3.0-6.0 pm wide below prima- fungus on Pinus spp. ry branches, apical cell not swollen, 4.0-7.0 pm wide at base, basal cell not swollen (Figs. 8, 14al. Conidio~ The isolates from both A. ba/samea and P. rubra, genous apparatus (32-155--681-104) long, excluding the were characterised by dark conidiophores and a high conidial mass, with 3 to 4 series of cylindrical branches, degree of tolerance to cycloheximide, which is similar to 2-3 primary branches, olivaceous to light olivaceous, other Leptographium species. The isolates from A. ba/- smooth, cylindrical, aseptate, 18.0-) 12-151-31) I'm lon9 samea, are characterised by optimal growth at low tem- and 13.0-)3.5(-5.01 I'm wide, secondary branches light peratures and slow growing colonies. These isolates are oliva ceo us to hyaline, aseptate, 7.0-12(-15) pm long, further characterised by dark, medium-length conidio- 2.0-4.0 pm wide, tertiary branches hyaline, aseptate, ph ores with rhizoids at their bases. The conidia of these (6.0-)7.0-1 01-121 I'm long, 2.0-4.0 I'm wide, quaterna- isolates are broadly ellipsoidal to obovoid. The Lep- ry branches aseptate, hyaiine, 15.0-)7.0-9.01-101 I'm tographium sp. from A. ba/samea, superficially resembles long, 2.0-4.0 I'm wide (Figs. 9, 14bl. Conidiogenous certain Leptographium species. It could, however, be cells discrete, 2-3 per branch, cylindrical, tapering slight- distinguished from these and is thus described as fol- ly at the apex, (8.0-111-15(-23) I'm long and 2.0- lows: 3.0 pm wide. Conidium development occurring through replacement wall building with holoblastic ontogeny and Leptographium abieticolens K. Jacobs & M.J. Wingf., percurrent proliferation and delayed secession giving a sp, nov, Figs, 8-14 false impression of sympodial proliferation (Minter et al., Coloniae in 2% MEA 15°C optime crescents et post 1982; 1983; Van Wyk et aI., 19881 (Figs. 10, 111. 14dies 18 mm diam. attingentes, atro-olivaceae (19"f); Conidia, aseptate, broadly ellipsoidal to obovoid, (4.0-) margine integra, sub 5°C vel supra, 25°C non crescents, 5.0-7.0x2.0-3.0I'm (Figs. 12, 13, 14cl. Conidia ac- ex mycelio vegetativo immerso vel emerso pallide cumulating in slimy droplets at the apex of conidiogenous olivaceo vel hyalino laevi non constricto, 1.0-6.0 pm apparatus. diam, constantes hyphis aeriis abundantibus formantes. Teleamorph state: not known Conidiophora singula vel usque ad sex gregaria, e mycelio Holotype: PREM. 56336 (dried specimen), from A. rectangulatim exorientia, erecta, macronematosa, ba/samea roots wounded by K. graci/us, collected D. R. mononematosa, (120-1160-195(-3601I'm longa; sti- Bergdahl, White Face Mountain, New York, USA, August pites cylindricei, ieaves, 2-11-septati, (72-)92-2391- 1990. 264) pm longi, infra ramas primaries 3.0-6.0 pm lati, ad Additional specimens: CMW 2875 (live isolatel. basim 4.5-7.5.um lati, non tumidi. Apparatus conidia- PREM56337 (dried specimenl, from A. balsamea roots geni (32-)55--68(-104) I'm longi, ex ramis cylindricis 3- wounded by K. graci/us, collected D. R. Bergdahl, White 4-seriatis compositi; rami primarii, 2-3, olivacei vel pal- Face Mountain, New York, USA, August 1990. lide olivacei, aseptato, 18.0-)12-14(-31) x 3,0-5.0 I'm; Etymology: From the Latin noun abies: fir and Latin rami secundarii pallide olivacei vel hyaline, aseptati, 6.0- verb inco/ere: to inhabit. This specific epithet refers to 12(-15) x 2.0-3.0 pm; rami tertiarii hyaline, 8septati, Abies which is the only known host of this species. 16.0-)7.5-10(-121 x 2.0-4.0 I'm; rami quartii hyaline, aseptati, (5.0-)7.0-9.01-101 x 2.0-4.0 I'm; structura The Leptographium species from red spruce has long rhizoideiformes adest. Cellulae conidiogenae discretae, conidiophores with 3 to 4 series of cylindrical branch- 2-3 per ranum, cylindricae, sursum, leviter, attenuatae, es. Isolates were also found to display slow growth in holobalsticae 18.0-)11-15(-23) x 2.0-3.0 I'm. Conidia culture and low optimal growth temperature similar to L. aseptata, late ellipsoidea vel obovoidea, ad apicem abietico/ens. Comparison with other Leptographium apparatus conidiogeni in massa guttulata mucilaginosa species has shown that this fungus resembles L. proce- accumulata 14.0-)5.0(-7.01 x 2.0-3.0 I'm. rum, but could be distinguished based on longer conidio- Colonies with optimal growth at 15°C on 2% MEA, phores, lower optimal growth temperature and differ. reaching 18 mm in diam in 14 d. No growth below 5 °C ences in colony morphology. It is thus described as fol. or above 25°C. Able to withstand high concentrations lows: of cycloheximide with a 17% reduction in growth on 0.1 gll cycloheximide after 6 d at 15°C in the dark. Leptographium peucophiJum K. Jacobs & M. J. Wingf., Colony dark olivaceous (19"fl. Colony margin smooth. sp. novo Figs. 15--21 Hyphae submerged or on top of solid medium with abun- Coloniae in 2% MEA 15°C optime crescents et post dant aerial mycelium, light olivaceous to hyaline, smooth, 10 dies 10 mm diam. attingentes, atro-olivaceae (19'"f); not constricted at the septa, (1.0-)3.01-6.0II'm di- margine laciniato, sub 100e vel supra, 300e non 600 K. Jacobs et af.

Figs. 8-13. Leptograph;um abieticolens (CMW 2865). 8. Conidiophore with a dark olivaceous stipe and complex conidiogenous apparatus (Bar= 50 pm). 9. Complex conidiogenous apparatus IBar= 10 pm!. 10. Conidiogenous cells showing false sympodial can idiogenesis (Bar= 10 pm). 11. Conidiogenous cells showing false sympodial conidia genesis (Bar=5 pm). 12 Conidia (Bar=10pm). 13. Conidia (Bar=1 pm), New Leptographium species 601

14 A

Fig. 14. Leptographium abietico/ens (CMW 2865). A. Habit sketch of the conidiophore (Bar=20pm). B. Conidiogenous apparatus (Bar= 10pm). C. Conidia fBar= 10 pm). 602 K. Jacobs at al.

Fig. 15-20. Leptographium peucophl1um (CMW 28761. 15. Conidiophore with a dark olivaceous stipe and complex conidiogenous apparatus (Bar=50 pm). 16. Complex conidiogenous apparatus (Bar= 10 pm). 17. Conidiogenous cells showing false sympodial conidiogenesis !Bar= 10 pm). 18. Conidiogenous cells showing false sympodial con idiogenesis (Bar= 1 pm), 19. Conidia (Bar= 10 pm). 20. Conidia (Bar= 1 ,urn). New Leptographium species 603

A 21 B Fig. 21. Leptographium peucophila (CMW 2876). A. Habit sketch of the conidiophore (Bar= 10 .urn). B. Conidiogenous apparatus (Bar= 10 pm). C. Conidia (Bar= 10 pm). crescents,ex mycelia vegetativo immerso vel emerso ti, (7.0-)8.0-10(-13) x 2.0-3.0 I'm; structura rhizoidei- olivaceo vel hyalino laevi non constricta, 2.0-3.0,um formes adest. Cellulae conidiogenae discretae, 2-3 per diam, sine hyphis aeriis. Conidiaphara singula vel bina, e ranum, cy1indricae, sursum, leviter, attenuatae, holobal- mycelio rectangulatim exorientia, erecta, macronemato- sticae (8.0-19.0-17(-201 x 2.0 I'm. Conidia aseptata, sa,mononematosa, (230-)31 0-352(-5201 I'm longa; sti- hyaline, obovoidea, ad apicem apparatus conidiogeni in pites cylindricei, simplices, atro-olivacei, leaves. 3-7-sep- massa guttulata mucilaginosa accumulata 3.0-6.0 x 2.0- tati. 170-255(-420),urn longi, infra ramos primaries 3.0- 3.0 I'm. 8.0 pm lati, ad basim 4.5-11.um lati, non tumidi. Colonies with optimal growth at 20°C on 2% MEA, Apparatus conidiogeni 40-96(-120) pm longi, ex ramis reaching 10 mm in diam in 10 d. No growth below 10°C cylindricis 3-4-seriatis compositi; rami primarii, 2-3, or above 30°C Able to withstand high concentrations of olivacei, cylindrici, aseptati, 10-25 x 3.0-8.0 pm; rami cycloheximide with no reduction in growth on 0.1 g/I cy- secundarii pall ide olivacei vel hyaline, aseptati, (7.0-)8.0- cloheximide after 6 days at 25°C in the dark. Colony 17 x 2.0-5.0 pm; rami tertiarii hyaline, aseptati, (7.0-) dark olivaceous (19" f). Colony margin laciniate. 8.0-13(-15) x 2.0-4.0 pm; rami quartii hyaline, asepta- Hyphae submerged or on top of solid medium with no 604 K. Jacobs et al.

aerial mycelia, olivaceous to hyaline. smooth, not con- with the relatively longer conidiophores of the latter spe- stricted at the septa, 2.0-3.0 pm in diam. cies (100-210 pm). Leptographium pineti is also charac- Conidiophores occurring singly or in pairs, arising terised by small obovoid conidia (2-3 pm long) compared directly from the mycelium. erect, macronematous, to the large (8-17 pm) oblong conidia of O. robustum mononematous, (230-)310-352(-5201 pm in length, (Robinson-Jeffrey and Davidson, 1968). rhizoid-like structures present. Stipe dark olivaceous, Leptographium ca/ophylli J. F. Webber, K. Jacobs & smooth, cylindricai,simple, 3-7-septate, (170-)255- M. J. Wingf. is another Leptographium species that is 270(-420) pm long (from first basal septum to below pri- morphologically similar to L. pineti (Webber et aI., 1999). mary branchesl. (3.0-)5.5(-8.0) pm wide below primary The most striking difference between these fungi lies in branches, apical cell not swollen; (4.5-17.0(-11.01 pm their very different hosts. Leptographium ca/ophylli is wide at base, basal cell not swollen (Figs. 15, 21 a). known only from the non-coniferous Ca/ophyllum in- Conidiogenous apparatus 40-96(-120110ng, excluding ophyllum L. (Takamaka), which is a native of various the conidial mass, with 3 to 4 series of cylindrical branch- tropical islands. Leptographium ca/ophylliis also charac- es; 2-3 primary branches, olivaceous, smooth, cylindri- terised by optimum growth temperature of 30°C, com- cal. aseptate, 9.0-25.0 pm long and (3.0-)4.0-6.0(- pared with the optimum of 25°C of L. pineti. Morpho- 8.0) pm wide, secondary branches light olivaceous to logically, these species can also be distinguished by the hyaline, aseptate, (7.0-)8.0-13(-17) pm long, 2.0- short (41-100pml and long (100-210pml conidio- 5.0 pm wide, tertiary branches hyaline, aseptate, (7.0-) phores of L. ca/ophylli and L. pineti, respectively. Fur- 8.0-11 (-15) pm long, 2.0-4.0 pm wide, quaternary thermore, L. ca/ophylli also has considerably larger branches aseptate, hyaline, (7.0-18.0-10(-131 pm long, conidia (3-7 pm) (Webber et aI., 19991 than those of L. 2.0-3.0pm wide (Figs. 16, 21bl. Conidiogenous cells pineti (2-3 pm). discrete, 2-3 per branch. cylindrical, tapering slightly at Several Leptographium species are found in associa- the apex, (8.0-113.0(-20.0) pm long and 2.0 pm wide. tion with Ips spp. on spruce, larch and pine in Europe and Conidium development occurring through replacement Japan. These are L. penicillatum Grosm., L. piceaper- wall building with holoblastic ontogeny and percurrent dum K. Jacobs, M. J. Wingf. & Crous, L. laricis and L. aew proliferation and delayed secession giving a false impres- nigmaticum, respectively (Solheim, 1986; Van der sion of sympodial proliferation (Minter et aI., 1982; Westhuizen et al" 1995; Jacobs et al" 1998). Lep- 1983; Van Wyk et aI., 19881 (Figs. 17, 18), Conidia, tographium pineti can easily be distinguished from these aseptate, obovoid, 3.0-6.0 x 2.0-3.0 pm (Figs. 1g, 20, species by its small obovoid conidia and short robust 21 c). Conidia accumulating in slimy droplets at the apex conidiophores. This is in contrast to the large allantoid of conidiogenous apparatus. conidia and long conidiophores associated with L. penicil- TeJeomorph state: not known. latum (Grosmann, 1931). This is also different from the Holotype: PREM 56591, from P. rubra roots wound- larger obovoid conidia and considerably longer conidio- ed by K. gracillus, collected: D. R. 8ergdahl, White Face phares associated with L. piceaperdum, L. laricisand L. Mountain, New York, USA, August 1g90. aenigmaticum (Rumbold, 1936; Van der Westhuizen et Additional specimens: PREM 56390 (herbarium al" 1995; Jacobs et aI., 19981. Of the three species, specimenl CMW 2875, CMW 2839 (live specimen!. from only L. piceaperdum has been associated with Pinus spp. P. rubra roots wounded by Korsche/tellus graci/us, col- (Griffin, 1968; Hutchison and Reid, 1988). lected: D.R. Bergdahl, White Face Mountain, New York, Leptographium abieticolens morphologically resem- USA, August 1990 bles L. antibioticum (W. B. Kendrick) M. J. Wingf. Lep- Etymology: From the Greek noun peuce: spruce and tographium antibioticum was described by Kendrick Greek adjective phi/os: loving. This species refers to (1962) and is characterised by its ability to produce anti- Picea, which is the host of this fungus. biotic substances in culture. Leptographium abieticolens can easily be distinguished from L. antibioticum Discussion based on its darker stipes and considerably more complex conidiogenous apparatus. These species can fur- Leptographium pineti most closely resembles the Lep- ther be distinguished based on their different optimal tographium anamorph of O. robustum (Rob.-Jeffr. & growth temperatures. Leptographium abietico/ens R. W. Davids.) T. C. Harr. Although O. robustum is simi- grows optimally at 15°C, in contrast to L. antibioticum, lar to L. pineti and also originated from Pinus spp., it has which grows optimally at 25-300C. Leptographium been described only from Canada and is associated with abieticolens is characterised by its 2 to 3 primary branch- the bark beetles in the genus Oendroctonus (Robinson- es, whereas up to 5 primary branches have been ob- Jeffrey and Davidson, 1968). This is in contrast to L. served in isolates of L. antibioticum. Leptographium pineti, which is associated with Ips sp. in a very distinct abieticolens also can be distinguished from L. anti- geographical area. The two species can also be distin- bioticum by its larger, broad ellipsoidal conidia (4-7 pm), guished by the presence of a teleomorph in O. robustum in contrast to the smaller obovoid to oblong conidia (2.5- and no evidence of perithecia associated with L. pineti. 5 pml in the latter species (Kendrick, 19621. The Leptographium anamorph of O. robustum can be dis- Leptographium peucophilum morphologically resemw tinguished from L. pineti by the considerably shorter (31- bles L. procerum (W.8. Kendrick) M. J. Wingf. These 116 pm) conidiophores in the former species, compared two species can, however, easily be distinguished by New Leptograph;um species 605 colony appearance. Isolates of L. procerum are charac- Davidson, R. W. 1971. New species of Ceratocystis. MycoJo- terised by concentric rings of conidiophores on 2% MEA gia 63: 5-15. in Petri dishes at 25°C. This character is not observed Davidson, R. W. 1976. Sapwood staining fungi from two tree in L. peucophilum which is also considerably slower species. Mem. N. Y. Bot. Gard. 28: 45-49. growing than L. procerum. Furthermore, the conidio- Goidanich, G. 1936. II genera di Ascomiceti "Grosmann~ G. phares of L. procerum are slightly longer (aver- Goid. Bollettino delia R. Statione di Patologia Vegetale- age=408,um) than those of L. peucophilum (aver- Roma N. S. 16: 26~60. Griffin, H. D. 1968. The genus Ceratocystis in Ontario. Can. age=330 ,urn). Both of these species are characterised by the presence of rhizoids and 2 to 3 primary branches J. Bot. 46: 689-71 B. Grosmann, H. 1931. Contributions to the knowledge concern- in the conidiogenous apparatus. Comparison of the ing the life partnership between bark beetles and fungi. Z. conidia revealed that both species have obovoid conidia Parasitenkd. 3: 56-102. that are between 3 and 6.um long. Harrington, T. C. 1988. Leptographium species, their distribu- The occurrence of L. abieticolens and L. peucophi- tions, hosts and insect vectors. In: Leptographium root dis- lum on conifers is not unusual although their association eases on conifers. (ed. by Harrington, T. C. and Cobb, witt, moth damage is unique for Leptographium. The lar. F. W.), pp. 1-39, APS Press, St. Paul, Minnesota. val stage of this moth feeds on the roots of many plant Harrington, T. C. and Cobb, F. W. (Jr.). 1987. Leptographium species, including spruce and fir. The fungi appear to wageneri var. pseudotsugae, var. nov., cause of black stain enter through the wounds caused by the feeding habits root disease on Douglas-fir. Mycotaxon 30: 501-507. of the swift moth. It is not known whether L. abietico- Hutchison, L. J. and Reid, J. 1988. Taxonomy _of some poten- tial wood-staining fungi from New Zealand 1. lens or L. peucophilum, are pathogenic, although large Ophiostomataceae. New Zealand J. Bot. 26: 63-81. areas of discoloration are usually associated with the Jacobs, K., Wingfield, M. J. and Bergdahl, D. R. 1997. A new feeding wounds caused by moth larvae. species of Ophiostoma from North America, similar to Almost nothing is known of the ecology of L. Ophiostoma penieil/atum. Can. J. Bot. 75: 1315-1322. abieticolens and L. peucophila. It seems unlikely that Jacobs, K., Wingfield, M. J, Wingfield, B. D. and Yamaoka, Y. the conifer swift moth would be able to carry these fungi 1998. Comparison of Ophiostoma huntii and O. eu- directly, as the adult insects never enter roots directly. raphioides and description of O. aenigmaticum sp. novo It is possible that these are soil inhabiting Leptographium Mycol. Res. 102: 289-294. species that are able to colonise wounds made by the Jooste, W. J. 1978. Leptographium reconditum sp. novo and moth larvae. They may also be endophytes of spruce observations on conidiogenesis in Verticicladiella. Trans. and fir, respectively, that are adapted to sporulate and Br. Mycol. Soc. 70: 152-155. grow in wounded tissue. Another hypothesis is that Kendrick, W. B. 1962. The Leptographium complex. Ver- they are carried by phoretic mites vectored by the conifer ticicladiella Hughes. Can. J. Bot. 40: 771-797. Kendrick, W. B. 1964. The Leptographium complex. swift moth. This hypothesis would be supported by the Scopularia Venusta Preuss. Can. J. Bot. 42: 1119-1122. fact that a close association is known to exist between Kendrick, W. B. and Molnar, A. C. 1965, A new Ceratocystis phoretic mites on bark beetles and a Pyxidiophora fungus and its Verticicladiella imperfect state associated with the (Blackwell et aI., 1986). Such secondary vectorship is bark beetle Dryocoetus confusus on Abies lasiocarpa. Can. thought to playa role in the association of Ophiostoma J. Bot. 43: 39-43. species and long horn beetles (Coleoptera: Cerambyci- Krokene, P. and Solheim, H. 1996. Fungal associates of five dae). The adult insects never enter wood, but Ophiosto- bark beetle species colonizing Norway spruce. Can. J. ma spp. are commonly found sporulating in the galleries Forest. Res. 26: 2115-2122, of their larvae (Wingfield, 19871. Krokene, P. and Solheim, H. 1998. Pathogenicity of four blue- stain fungi associated with aggressive and non aggressive Acknowledgements-We thank the National Research Foun~ bark beetles. Phytopathology 88: 39-44. dation (NRF), members of the Tree Pathology Co-operative Lagerberg, T., Lundberg, G.and Melin, E. 1927. Biological and Programme (TPCP) and the school of National Resources, practical researches into blueing in pine and spruce. Svens- University of Vermont for financial support. We also thank ka Skogsvardsf6reningens Tidskrift 25: 145-272, 561- Prof. J. P. van der Walt for suggesting the names of the new 691. species as well as Dr. Hugh Glen for providing the Latin diag- Leach, J. G., Orr, L. W. and Christensen, C. 1934. The inter- relationships of bark beetles and blue-staining fungi in felled noses. Norway pine timber. J. Agr.Res. 49: 315-341. Minter, D. W., Kirk, P. M. and Sutton, B, C. 1982. Holoblastic Literature cited phialides. Trans. Br. Mycol. Soc. 79: 75-93. Minter, D. W., Kirk, P. M. and Sutton, B. C. 1983. Thamc Blackwell, M., Bridges, J. R., Moser, J. C. and Perry, T. J. 1986. phialides. Trans. Br, Mycol. Soc. 80: 39-66. Hyperphoretic dispersal of a Pyxidiophora anamorph. Morelet, M. 1988. Observations sur trois Deuteromycetes Science 232: 993-995. infeodes aux pins. Annales de la Societe des Sciences Craighead, F. C. 1928. Interrelation of tree-killing bark beetles Naturelles et d' Archeologie de Toulon et du Var 40: 41-43. {Dendroctonus! and blue-stain. J. Forest. 26: 886-887. Parker, A. K. 1957a. Europhium, a new genus of the asco- Davidson, R. W. 1942. Some additional species of Cer- mycetes with a Leptographium imperfect state. Can. J. atostomel/a in the United States. Mycologia 34: 650-662. Bot. 35: 173-179. Davidson, R. W. 1958. Additional species of Ophiostoma- Rayner, R. W. 1970. A mycological colour chart. Common. taceae from Colorado. Mycologia 50: 661-670. wealth Mycological Institute and British Mycological Socie- 606 K. Jacobs et al.

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