Leptographium longiclavatum sp. nov., a new species associated

with the mountain pine beetle, Dendroctonus ponderosae

Sangwon LEE, Jae-Jin KIM and Colette BREUIL*

Department of Wood Science, University of British Columbia, Vancouver, B.C. V6T 1Z4, Canada.E-mail : breuil@interchg.ubc.ca

Received 15 January 2005; accepted 22 June 2005.

The mountain pine beetle, Dendroctonus ponderosae, and its fungal associates are devastating the lodgepole pine forestsin British Columbia, Canada. During our fungal survey, an unknown Leptographium species has been consistently

isolated from both D. ponderosae and infested lodgepole pine (Pinus contorta var. latifolia). This Leptographium specieshas similar morphology with the Leptographium anamorph of Ophiostoma clavigerum whose association with theD. ponderosae is well known. However, thorough morphological comparisons showed that this fungus is distinct from

all the other Leptographium species described in the literature, and especially from O. clavigerum. Comparison ofDNA sequences of multiple loci and the profiles by the PCR-RFLP marker also confirmed that this Leptographiumspecies represents an undescribed taxon. Based on its distinct morphological, physiological characteristics and

phylogenetic position, we describe it as L. longiclavatum sp. nov.

INTRODUCTION

For the last twelve years, an epidemic of the mountainpine beetle, Dendroctonus ponderosae has spread over10.1 M ha in British Columbia (Council of Forest In-dustries 2004). This bark beetle has infested 173.5 M m3

of lodgepole pine (Pinus contorta var. latifolia), one ofthe most prevalent and commercially valuable treespecies. The epidemic is driven by mutually beneficialrelationships between the beetle and its fungal associ-ates (Paine, Raffa & Harrington 1997). D. ponderosaemechanically damages trees by feeding on the phloem,while the fungi carried by the beetle discolor the sap-wood and disrupt the transportation of water to thetree crown (Reid 1961). The infested trees are killed bythe simultaneous actions of the beetle and fungi.

To better understand the effects of fungi related withD. ponderosae outbreaks, it is important to identifyexisting or new fungal associates. Many bark beetles(Coleoptera, Scolytidae) that infest coniferous treescarry ophiostomatoid fungi, especially species withLeptographium anamorphs (Harrington 1988, 1993Wingfield & Gibbs 1991). These fungi are best knownas agents that stain the sapwood of conifers (Wingfield,Seifert & Webber 1993, Jacobs & Wingfield 2001), andthey are casually or specifically associated with insect

vectors (Harrington 1988). Leptographium is one of themost common anamorphs of Ophiostoma species. It istypically characterized by mononematous conidio-phores with darkly coloured stipes and a series ofbranches at the apices. Many Leptographium speciesare weak pathogens; only a few are strong pathogenssuch as L. wageneri (Harrington & Cobb 1983,Wingfield, Capretti & Mackenzie 1988). Of thedescribed Leptographium species, only L. clavigerum,the asexual stage of O. clavigerum, has been known asan associate of D. ponderosae (Robison-Jeffrey &Davidson 1968, Whitney & Farris 1970). O. huntii, aspecies with a Leptographium anamorph, has also beenisolated from old galleries of the trees attacked by D.ponderosae (Solheim 1995, Whitney 1971). However,this fungal species has not been isolated from the D.ponderosae (Whitney & Farris 1970), and it was sug-gested that the fungus might have been introduced byother insects, such as secondary bark beetles or mites.

During our ecological survey in 2001–2004, anunidentified Leptographium species was isolated fromboth D. ponderosae and D. ponderosae-infested lodge-pole pines in British Columbia. The fungus showedmorphological similarity to O. clavigerum, especially inthe shape of the conidia.

This paper describes the unknown Leptographium sp.associated with D. ponderosae, using its distinct mor-phological and physiological characteristics, and its* Corresponding author.

Mycol. Res. 109 (10): 1162–1170 (October 2005). f The British Mycological Society 1162

doi:10.1017/S0953756205003588 Printed in the United Kingdom.

phylogenetic relationships to closely related species.The phylogenetic trees were constructed using the par-tial regions of rDNA and three protein-coding genes:b-tubulin, actin, and glyceraldehyde-3-phosphatedehydrogenase (GPD).

MATERIALS AND METHODS

Fungal isolation

The fungi were isolated from Dendroctonus ponderosae,stained sapwood, or bark of lodgepole pines, which hadbeen infested for less than one year. The sampling wasconducted at Kamloops, Princeton, and Williams Lakein British Columbia in 2001. To isolate the fungi fromD. ponderosae, the beetles were collected under the barkof infested trees and washed with 1 ml of 0.01% (v/v)Tween 20 solution (Sigma-Aldrich, Oakville, ON). Thediluted suspension was spread on 2% OMEA (33 gOxoid malt extract agar, 10 g Oxoid agar and distilledwater in total volume of 1000 ml) amended withampicillin at 50 mg mlx1 and incubated at room tem-perature for 2–3 d. The separated colonies on the plateswere sub-cultured on 2% OMEA individually and in-cubated at room temperature. The fragments of bark,and stained sapwood were also aseptically removed andplaced on 2% OMEA/amp. The hyphal tip was sub-cultured to 2% OMEA and incubated at room tem-perature. The purity of the cultures used in this studywas ensured through single spore isolation. All isolatesare maintained in the Department of Wood Science,University of British Columbia, and have also beendeposited at the Canadian Collection of Fungal Cul-tures (DAOM; Agriculture and Agri-Food Canada,Ottawa).

Cultural and microscopic characteristics

Cultural appearances were observed on both 2%OMEA and 2% DMEA (20 g Difco malt extract, 15 gDifco agar, and distilled water in total volume of1000 ml) plates. The colony colour was described fol-lowing Methuen handbook of colour (Kornerup &Wanscher 1961). The growth rates were examined attemperatures ranging from 5–35 xC, at five-degree in-tervals. The colony diameters of three replicates on 2%OMEA plates were measured along two perpendicularlines two and four days after inoculation. The radialgrowth rates were subsequently calculated. The toler-ance to cycloheximide was assessed by measuring fun-gal growth on 2% OMEA containing 0.05, 0.1 and0.5% of cycloheximide (Sigma-Aldrich) at 25 x. Formicroscopic characteristics, two to three-week oldfungal cultures, grown at room temperature on 2%DMEA, were observed with a Zeiss (Axioplan II) lightmicroscope. Each microscopic value was obtained fromthe average of at least 50 measurements. The con-idiophore was also observed using scanning electronmicroscopy (SEM) as described by Lee et al. (2003).

DNA extraction, PCR, sequencing,and PCR-RFLP marker

The genomic DNA was extracted from the mycelium offungi, which were grown for 3–4 d on 2% OMEAplates overlaid with cellophane sheets (Bio-Rad,Mississauga, ON). The DNA extraction and PCRamplifications of rDNA (ITS2 and partial 28S) and thepartial regions of three protein-coding genes (actin, b-tubulin, and GPD) were performed as described Limet al. (2004). The primer sets used in this study wereas follows: ITS3/LR3 for amplifying rDNA (Vilgalys& Hester 1990, White et al. 1990), Lepact F/Lepact Rfor actin gene (Lim et al. 2004), T10 (O’Donnell et al.2000)/BT12 (Kim et al. 2003) for b-tubulin gene,and GPD5-ex2 (5k-ATTGGCCGYATCGTCTTCCG)/GPD-int (5k-TTGCCGTTAAGCTCTGGAAT) forGPD gene. PCR amplicons were purified usingQiaquick Gel Extraction Kit (Qiagen) and sequencedwith an ABI 3700 automated sequencer (Perkin-Elmer, Foster City, CA) at Macrogen (Seoul). The O.clavigerum specific PCR-RFLP marker was tested oneleven isolates of this new Leptographium species listedin Table 1, following the method described by Lee et al.(2003).

Phylogenetic analysis

22 strains representing eight named species were ana-lyzed phylogenetically (Table 1). The sequences werealigned using the ClustalW algorithm (Higgins, Bleasby& Fuchs 1991) and optimized using the PHYDITprogram version 3.2 (http://plasza.snu.ac.kr/yjchun/phydit/). Multiple alignment parameters were ‘gapopen penalty of ten’ and ‘gap extension penalty ofone’. The phylogenetic analyses of individual loci andcombined sequence of four loci were performed withPAUP*4.0b10 (Swofford 2002). A partition homogen-eity test was carried out with PAUP*4.0b10 to evaluatewhether rDNA and three protein-coding genes couldbe combined. The most parsimonious tree was searchedwith heuristic search option in which all characterswere of type ‘uncord’, all characters had equal weightand gaps were treated as missing. The tree-bisection-reconnecting (TBR) was applied as branch swappingalgorithm and ‘MulTrees ’ option was in effect. Thestability of branches was evaluated by bootstrapanalysis with 1000 replications (Felsenstein 1985). Fullheuristic search option was chosen for bootsrapping.Based on the previous studies (Lim et al. 2004),O. huntiiand L. lundbergii were assigned as outgroups.

RESULTS

Taxonomy

The Leptographium species isolated from Dendroctonusponderosae exoskeletons and D. ponderosae-infestedlodgepole pines is typical of the genus, having well-developed long conidiophores and masses of conidia

S. Lee, J.-J. Kim and C. Breuil 1163

Table 1. Cultures used in this work and GenBank accession numbers for sequences.

Species Isolatea Hostb Origin Isolation source Collector/Year

GenBank accession no.c

Actin ITS2 & LSU b-tubulin GPD

L. longiclavatum SL-Kw1436 PC Kamloops, B.C., Canada Sapwood infested by D. ponderosae S. Lee, 2001 AY816679 AY816686 AY288934 AY816693

SL-Kp11 PC Kamloops, B.C., Canada Bark infested by D. ponderosae S. Lee, 2001 AY816680 AY816687 AY816712 AY816694

SL-W001 PC Williams Lake, B.C., Canada D. ponderosae S. Lee, 2001 AY816681 AY816688 AY288936 AY816695

SL-Pw5 PC Princeton, B.C., Canada Sapwood infested by D. ponderosae S. Lee, 2001 AY816682 AY816689 AY288935 AY816696

C187 PP Yosemite Valley, CA, U.S.A. Phloem infested by D. ponderosae D. Owen, 1984 AY816683 AY816690 AY816713 AY816697

SL-K215 PC Kamloops D. ponderosae S. Lee, 2001 – – AY288931 –

SL-Kw439 PC Kamloops Sapwood infested by D. ponderosae S. Lee, 2001 – – AY288932 –

SL-Kw442 PC Kamloops Sapwood infested by D. ponderosae S. Lee, 2001 – – AY288933 –

SL-Wg403 PC Williams Lake Gallery of D. ponderosae S. Lee, 2001 – – AY288937 –

SL-Ww405 PC Williams Lake Sapwood infested by D. ponderosae S. Lee, 2001 – – AY288938 –

SL-Ww407 PC Williams Lake Sapwood infested by D. ponderosae S. Lee, 2001 – – AY288939 –

L. lundbergii UAMH9584 PS Uppland, Sweden Board A. Mathiesen-Kaarik AY544585 AY544603 AY263184 AY816698

L. pyrinum DLS879 PA Pinaleno Mtns., AZ, U.S.A. Dendroctonus adjunctus D. L. Six AY544586 AY544604 AY263185 AY816699

CMW3889 PJ CA, U.S.A. Unknown D. L. Six AY544587 AY544605 AY544621 AY816700

L. terebrantis UAMH9722 PC Sooke, B.C., Canada Unknown J. Reid AY544588 AY544606 AY263192 AY816701

C418 PP Blodgett, CA, U.S.A. Associated with D. brevicomis T. C. Harrington AY544589 AY544607 AY263191 DQ082862

AU98Pr2-155 PC Princeton, B.C., Canada Sapwood A. Uzunovic AY544590 AY544608 AY544622 –

O. aureum ATCC16936 PC Invermere, B.C., Canada Ascocarps in bark beetle-infested tree R. C. R.-Jeffrey/

R. W. Davidson

AY544592 AY544610 AY263187 AY816702

AU98Pr2-169 PC Princeton, B.C., Canada Sapwood A. Uzunovic AY544594 AY544612 AY263188 AY816703

O. clavigerum ATCC18086 PP Cache Creek, B.C., Canada Tree attacked by Dendroctonus sp. R. C. R.-Jeffrey/

R. W. Davidson

AY544595 AY544613 AY263194 AY816704

C843 UN Nevada Mtns., CA, U.S.A. D. jeffreyi D. L. Six AY544596 AY544614 AY263196 AY816705

SL-Kw1407 PC Kamloops, B.C., Canada Sapwood infested by D. ponderosae S. Lee, 2001 AY544597 AY544615 AY263195 AY816706

SL-St.J11 PC Fort St. James, B.C., Canada D. ponderosae S. Lee, 2002 AY816684 AY816691 AY263201 AY816707

SL-Wg602 PC Williams Lake, B.C., Canada Gallery of D. ponderosae S. Lee, 2001 AY816685 AY816692 AY263205 AY816708

O. huntii UAMH4997 PC Invermere, B.C., Canada Bark beetle galleries R. C. R.-Jeffrey AY544599 AY544617 AY349023 DQ082861

UAMH4825 PC Westcastle, Alta., Canada Sapwood between beetle galleries A. Tsuneda AY544600 AY544618 AY544625 AY816709

O. robustum CMW668 PP McCall, ID, U.S.A. Ambrosia and Dendroctonus spp. R. C. R.-Jeffrey/

R. W. Davidson

AY544601 AY544619 AY263190 AY816710

CMW2805 PP ID, U.S.A. Unknown T. Hinds AY544602 AY544620 AY263189 AY816711

a UAMH, the University of Alberta Microfungus Collection and Herbarium, Devonian Botanic Garden, Canada; DLS, the culture collection of D. L. Six, University of Montana, U. S. A.; CMW, Culture

Collection Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; C, Culture Collection of T. C. Harrington, Iowa State University, U. S. A.; ATCC, American Type

Culture Collection, Manassas, VA, U. S. A.; AU- and SL-isolates, Breuil’s Culture Collection, University of British Columbia, Canada.b PC, Pinus contorta ; PP, Pinus ponderosa ; PS, Pinus sylvestris ; PA, Pinus arizonica ; PJ, Pinus jeffreyi ; UN, Unknown.c Accession numbers of sequences obtained in this study presented in bold.

Leptographium

longicla

vatum

sp.nov.,anew

sapsta

iningfungus

1164

carried in moist drops at their apices. The fungus is ableto tolerate high concentrations of cycloheximide, atypical feature of the Ophiostoma species and theiranamorphs. The most distinct characteristics of thisfungus are its clavate conidia and long conidiophores.The shapes of conidia are much more homogenousthan in O. clavigerum, which also produces clavate andother shapes of conidia. The Leptographium species(Figs 1–9) can be clearly distinguished from theLeptographium anamorph of O. clavigerum by thelength of the conidiophores (Figs 10–11), colony colourand margin, optimal growth rate and the stability ofanamorph shape as well as conidia shapes (Figs 12–13).Therefore, we consider that this fungus is a new speciesand provide the following description.

Leptographium longiclavatum S. Lee, J.-J. Kim & C.Breuil, sp. nov. (Figs 1–9)

Etym. : longiclavatum refers to the long clavateconidia.

Crescit optime ad 25 x, tum 7.3 mm per diem in 2% ‘OMEA’.Non crescit infra 5 x vel supra 35 x. In ‘OMEA’ cum alio 0.05,0.1 et 0.5% ‘cycloheximide’, crescit ad 25 x alium 7.3, 7.7 et

5.8 mm per diem. Coloniae in ‘DMEA’ effusae, extendentes,olivaces.Hypharum parietes laeves, hyphae 2–5 mm diametro.Conidiophorae singulae vel aggregatae, mononematae, mac-

ronematae, in basibus cum rhizoideis, interdum in hyphis

aeriis factae. Stipites erecti, brunnei, simplices, 3–20 septati,75–1202 (medius=752¡329) mm longi, basi 8–11 (medius=10¡1.5) mm lati. Apparatus conidiogenus 40–451 (medius=280¡137) mm longus, massa conidiali exclusa; ramis pri-mariis bisulcus vel ternis, medio-brunneis, ramis centralibusaegre quam aliis maioribus, 25–45 (medius=34¡4)r2.5–3 mm; ramis secondariis laete brunneis, 25–35 (27¡2)r2.5–3 mm; ramis tertiariis hyalinis, 25–30 (26¡2)r2.5–3 mm. Cellulae conidiogenae discretae, apicem versus

angustatae, 35–50 (medius=39¡8) mm longae. Evolutio

112 3

9

4

5 6 7 8

Figs 1–9. Leptographium longiclavatum (SL-Kw1436) Fig. 1. Colony grown on 2% DMEA for 5 d at 20 xC. Figs 2–3.Light micrographs of long conidiophores with clavate conidia. Figs 4–5. Scanning electron micrographs of conidiogenous

apparatus with clavate conidia. Fig. 6. Scanning electron micrograph of conidiogenous cells showing annellations(arrows). Fig. 7. Scanning electron micrographs of clavate conidia. Figs 8–9. Light micrographs of conidia.Bars: Fig. 2=100 mm; Fig. 3=50 mm; Figs 4–5=20 mm. Fig. 6=5 mm. Figs 7–8=10 mm; and Fig. 9=20 mm.

10 11 13

12

Figs 10–13. Ophiostoma clavigerum (ex-holotype, ATCC-18086). Figs 10–11. Light micrographs of conidiophores with

different shape of conidia: clavate (Fig. 10); oblong (Fig. 11).Figs 12–13. Light micrographs of various conidia: ovoid tosubglobose (Fig. 12) and oblong, ovoid, or peanut-shaped

with a truncated end (Fig. 13). Bars: Figs 10–11=50 mm;Fig. 12=10 mm; and Fig. 13=20 mm.

S. Lee, J.-J. Kim and C. Breuil 1165

conidii per aedificationem parietis supplementariae ontogeniaholoblastica et proliferatione percurrenti. Conidia hyalina,

0–4 septata, clavata vel obclavata, 10–85 (medius=28¡18)r3–6 (medius=4¡1.5). Conidia alia, parva: hyalina, ovoi-dea vel clavata 2.5–7 (medius=5¡2.5) r2–4 (med-

ius=3¡1.5) mm.

Typus : Canada : British Columbia : Kamloops: Opax Mt.,on Pinus contorta var. latifolia, Nov. 2001, S. Lee SL-Kw1436

(DAOM 234192 – holotypus).

Growth. Optimal growth temperature 25 x with agrowth rate of 7.3 mm dx1 on 2% OMEA. No growth

(A) rDNA

O. huntii

SL-Pw5

SL-Kp11

SL-W001

SL-Kw1436

C187

CMW3889DLS879

UAMH4997UAMH4825

UAMH9584 L. lundbergii 0.5 changes

75

64

C843

SL-Kw1407

SL-Wg602

SL-St.J11

ATCC16936AU98Pr2-169

65

62

UAMH9722C418

AU98Pr2-155

CMW668CMW2805

ATCC18086

L. longiclavatum

L. pyrinum

O. clavigerum

O. aureum

L. terebrantis

O. robustum 59

(C) Actin

UAMH9584 L. lundbergii1 change

100

100 UAMH4997

UAMH4825 O. huntii

SL-Kw1407

C418

UAMH9722

AU98Pr2-155

CMW668

CMW2805

DLS879

CMW3889

ATCC16936

AU98Pr2-169

92

76

69

75

85

SL-Kw1436

SL-Kp11SL-Pw5SL-W001

C187

64

98

97

SL-St.J11SL-Wg602

ATCC18086C843

85

87

O. robustum

O. aureum

O. clavigerum

L. longiclavatum

L. pyrinum

L. terebrantis

(D) GPD

UAMH9584 L. lundbergii 5 changes

100

DLS879

AU98Pr2-16995

CMW668

CMW280591 O. robustum

ATCC16936

C843ATCC18086

SL-Kw1407SL-St.J11SL-Wg602

O. clavigerum

CMW388986 L. pyrinum

SL-Kw1436

SL-Kp11SL-Pw5

SL-W001

C187

95L. longiclavatum

O. aureum

UAMH9722

C418 L. terebrantis

UAMH4825

UAMH4997 O. huntii

(B) β-tubulin

L. longiclavatum

O. huntii

UAMH9584 L. lundbergii

100

UAMH4997

UAMH482599

CMW2805

CMW668

SL-Pw5

DLS879

CMW3889

ATCC16936AU98Pr2-169

ATCC18086SL-Kw1407

C843SL-St.J11

SL-Wg602

AU98Pr2-155

C418UAMH9722

O. clavigerum

L. terebrantis

O. robustum

SL-W001

SL-Kp11

SL-Kw1436

C187

L. pyrinum

O. aureum

99

99

61

74

62

82

5 changes

Fig. 14. One of the most parsimonious trees for each of the four gene datasets using Leptographium lundbergii and Ophiostomahuntii as outgroups. Numbers above branches are bootstrap values with 1000 replications and shown when greater than 50%.(A) ITS2 and partial 28S region of rDNA; among total 714 aligned characters, 699 characters were constant, 10 were variable

and 5 were parsimony-informative characters. Three most parsimonious trees were generated with tree length 15, consistencyindex (CI) 1.0, Homoplasy index (HI) 1.0, retention index (RI) 1.0. (B) b-tubulin; 679 out of 794 aligned characters wereconstant and 64 were parsimony-informative characters. One most parsimonious tree with a tree length 139, CI 0.9281, HI

0.0719 and RI 0.9206 was generated. (C) Actin; 758 out of a total 841 characters were constant and 56 parsimony-informative.The number of most parsimonious trees was four and they had tree length 97, CI 0.9072, HI 0.0928 and RI 0.9167. (D) GPD;543 total characters with 461 constant characters and 47 parsimony-informative characters produced five most parsimonioustrees, which had tree length 99, CI 0.9293, HI 0.0707 and RI 0.9205.

Leptographium longiclavatum sp. nov., a new sapstaining fungus 1166

was found below 5 x or above 35 x. On 2% OMEAamended with 0.05, 0.1, and 0.5% cycloheximide, thegrowth rates at 25 x were 7.3, 7.7 and 5.8 mm dx1, re-spectively. Cultures on DMEA, colonies with sinuateedges, effuse, spreading, olive (1E4; Kornerup &Wanscher 1961).Hyphae, submerged in the medium, oraerial, smooth-walled, 2–5 mm diam. Conidiophoressingle or in groups up to seven, mononematous, mac-ronematous without rhizoids at their bases, sometimesproduced on aerial hyphae. Stipes erect, brown, simple,3–20 septate, 75–1202 (mean=752¡329) mm long, and8–11 (mean=10¡1.5) mm wide at the base. Conidio-genous apparatus 40–451 (mean=280¡137) mm longexcluding conidial mass ; primary branch mediumbrown, two to three (mostly two), central branchesslightly larger than the others, 25–45 (mean 34¡4)r2.5–3 mm; secondary branches pale brown, 25–35(27¡2)r2.5–3 mm; tertiary branches hyaline, 25–30(26¡2)r2.5–3 mm; branches in 3–10 series, the ulti-mate branches being conidiogenous cells. Con-idiogenous cells discrete, tapering distally, 35–50(mean=39¡8) mm long. Conidium development in themanner of holoblastic ontogeny and percurrent pro-liferation with delayed secession giving a falseimpression of sympodial proliferation as described byWingfield (1993). Conidia accumulating in light cream-coloured mucilaginous masses at the apices of theconidiophores, two types : large conidia, hyaline, 0–4septate, clavate to obclavate, 10–85 (mean=28¡18)r3–6 (mean=4¡1.5) mm; smaller conidia, hyaline,

aseptate, ovoid to clavate, 2.5–7 (mean=5¡2.5)r2–4(mean=3¡1.5) mm, produced on smaller con-idiophores on aerial mycelia, also occasionally pro-duced on the large clavate conidia.

Additional specimens examined : Canada : British Columbia:Williams Lake : D. ponderosae, August 2001, S. Lee,

SL–W001 (DAOM 234191); Kamloops : Pinus contorta,October 2001, S. Lee, SLxKp11 (DAOM 234190);Princeton : P. contorta, July 2001, S. Lee, SL–Pw5 (DAOM

234189).

L. longiclavatum

O. huntii

UAMH9584 L. lundbergii

5 changes

SL-St.J11SL-Wg602

SL-Kw1407

ATCC18086

C843

L. terebrantis

CMW668

CMW2805

SL-Kw1436SL-Kp11SL-Pw5SL-W001

C187

DLS879CMW3889

ATCC16936AU98Pr2-169

100

100

96100

100

58

65

100

59

99

59

67

O. robustum

O. clavigerum

L. pyrinum

O. aureum

UAMH4997

UAMH9722

C418

UAMH4825

52

92

Fig. 15. One of the most parsimonious tree based on the combined datasets of four loci : rDNA (ITS2 and 28S), b-tubulin,actin, and GPD. The tree was rooted with Leptographium lundbergii and Ophiostoma huntii as outgroups. Bootstrap valuesfrom 1000 replication were indicated above the branches when greater than 50%. Among total 2892 aligned characters,

2597 characters were constant and 172 were parsimony-informative characters. Two most parsimonious trees (tree length 352,CI 0.9205, HI 0.0795 and RI 0.9157) were generated.

M1 M2 1 2 3 4 5 6 7 8

490 bp

730 bp

238 bp

750 bp750 bp

500 bp500 bp

250 bp250 bp

Fig. 16. Agarose gel PCR-RFLP of the partial b-tubulin genedigested with HinfI. Lanes: M1, 1 kb DNA marker; M2,100 bp DNA marker: 1, Leptographium longiclavatum (SL-Kw1436) ; 2, L. longiclavatum (SL-W001); 3, L. longicla-

vatum (SL-Pw5); 4, L. longiclavatum (C187); 5, Ophiostomaclavigerum (ATCC18086); 6, O. clavigerum (C843); 7, O.clavigerum (SL-Wg602) ; and 8, O. clavigerum (SL-Kw1407).

S. Lee, J.-J. Kim and C. Breuil 1167

Phylogenetic analysis and PCR-RFLP marker

Leptographium longiclavatum was also recognized as aphylogenetic species (Taylor et al. 2000) based on aconcordance of multiple gene genealogies. RibosomalDNA (ITS2 and 28S) and partial regions of three pro-tein-coding genes (b-tubulin, actin and GPD) wereanalyzed. The sequences generated in this work and thereference sequences are listed in Table 1.

In all parsimonious trees generated with the rDNAand protein-coding genes, L. longiclavatum isolateswere consistently grouped in a single clade, which wasclearly separated from the other species. The ITS2 andpartial 28S regions of rDNA generated trees, in whichthe in-group taxa were separated into four clades :L. longiclavatum,O. clavigerum,L. terebrantis/L. pyrinum,and O. robustum/O. aureum (Fig. 14A). Clade resol-utions were better in the protein-coding gene trees thanin the rDNA tree. The aligned partial b-tubulin genesequences contained four exons and three introns, whilepartial actin gene region included two exons and oneintron. Although a strain (C187) of L. longiclavatumshowed sequence variations in these two genes,all isolates of L. longiclavatum formed a single clade,which was resolved to the base of O. clavigerum,L. terebrantis and O. robustum clades (Figs 14B–C).The tree of the partial GPD gene which contains oneintron and one exon, showed that the five speciesemerging from a polytomy included O. clavigerum,L. terebrantis, O. robustum, L. pyrinum, andL. longiclavatum (Fig. 14D). Regardless of the differ-ences in the branch resolutions between gene trees, theseparation of L. longiclavatum was consistent.

To obtain more robust phylogenetic information, thecombined sequences of rDNA, b-tubulin, actin, andGPD genes were analyzed. We aligned 2892 characters,of which 2597 were constant and 172 were parsimony-informative, and two most parsimonious trees weregenerated. Both trees showed basically the same top-ology, in which a distinct clade of L. longiclavatumwas basal to of O. clavigerum, L. terebrantis andO. robustum clades. The clades of L. longiclavatum andO. clavigerum were strongly supported with high boot-strap values, 100% and 99% respectively (Fig. 15). Inprevious work, we reported a PCR-RFLP markerbased on the b-tubulin sequence that can differentiateO. clavigerum from other morphologically similarspecies (Lee et al. 2003). In this work, the marker alsodistinguished L. longiclavatum from the most morpho-logically similar species, O. clavigerum (Fig. 16). Thedigested fragments of the b-tubulin gene amplicon(838 bp) of L. longiclavatum were 51, 57, and 730 bp.In contrast, the fragments from the 836 bp amplicon ofO. clavigerum were 51, 57, 238, and 490 bp.

DISCUSSION

The new species Leptographium longiclavatum was iso-lated from Dendroctonus ponderosae, bark, and stainedT

able2.MorphologicalcomparisonsofLeptographium

longiclavatum

andphylogeneticallyclosely

relatedspecies.

L.longiclavatum

L.pyrinum

aL.terebrantisb

O.aureum

cO.clavigerum

b,c

O.robustum

a,b,c

Region

NorthAmerica

USA

NorthAmerica

NorthAmerica

NorthAmerica

NorthAmerica

Host

Pinuscontorta,

P.ponderosa

P.ponderosa,

P.jeffreyi

P.ponderosa,P.contorta,

P.sylvestris,P.taeda,

P.bank

siana,P.resinosa,P.edulis,

P.strobus,Pseudotsuga

menziesii

P.contorta,

P.ponderosa,

P.edulis

P.contorta,P.monticola,

P.ponderosa

P.ponderosa

Insect

association

Dendroctonus

ponderosae

D.adjunctus

D.frontalis,D.terebrans,

D.valens,Hylobiusradicis,

H.rhizophagus,Hylurgopsporosus

D.ponderosae,

Dendroctonussp.,

H.porosus

D.ponderosae

Dendroctonussp.

Conidiophore

length

110–1650mm

118–393mm

143–509mm

100–1350mm

mononem

atous:

100–300mm

synnem

atous:

500–1150mm

31–116mm

Conidium

shape(two

types:L,large;S,sm

all)

L:clavate

toobclavate

S:ovoid

toclavate

oblong

(pear-shaped)

obovoid

withtruncate

ends

androundapices

oblongwithtruncate

endsandroundapices

L:clavate

toobclavate

S:cylindricalto

clavate

orellipsoidal

oblongwithtruncate

endsandroundapices

Conidium

size

(lengthrbreadth)

L:10–85r3–6mm

S:2.5–7r2–4mm

5–12r4–6mm

4–10r2–3mm

5.5–12.5r2–4mm

L:12.5–85r2–6mm

S:2–4r1–2.5

mm

3–7r

2–6mm

Teleomorph

absent

absent

absent

Ophiostoma

Ophiostoma

Ophiostoma

Rhizoids

absent

present

absent

absent

present

absent

Optimum

temp.forgrowth

(xC)

25

25

25

20

25

25

aJacobsandWingfield(2001).

bRobinson-Jeff

rey&

Davidson(1968).

cUpdhyay(1981).

Leptographium longiclavatum sp. nov., a new sapstaining fungus 1168

sapwood of infested lodgepole pines at several sites inBritish Columbia. This fungus was more commonlyisolated when the trees were not too dry. Its frequencyon the beetle body surface was relatively low (data notshown). We also found this fungus in the mycangia ofD. ponderosae collected in the USA from isolates(TF20-2, HV7, HR1141) provided by Diana L. Six(University of Montana). It appears that L.longiclavatum is associated with D. ponderosae asspecifically as O. clavigerum and O. montium (Whitney& Farris 1970).

The most distinct morphological characteristic ofL. longiclavatum is its clavate conidia. Amongst specieswith a Leptographium anamorph, only O. clavigerumwas previously known to have septate clavate conidia,so this conidium shape has been used as a quickdiagnostic feature for O. clavigerum. Given this,L. longiclavatum could have been easily misidentifiedas O. clavigerum ; this was the case for C187 which wasoriginally reported as O. clavigerum. Although clavateconidia are common to L. longiclavatum and O. clavi-gerum, O. clavigerum also produces conidia of othershapes that vary from oblong to peanut-shaped witha truncated end, ovoid, and subglobose (Robinson-Jeffrey & Davidson 1968, Tsuneda & Hiratsuka 1984,Lee et al. 2003). Non-clavate conidia are moreabundant than clavate conidia in O. clavigerum (Leeet al. 2003). The conidiophores of L. longiclavatum areconsiderably longer than those of O. clavigerum andany other Leptographium spp. (Jacobs & Wingfield2001). Furthermore, the teleomorphs and synnematousanamorphs observed inO. clavigerum (Upadhyay 1981)were not found in L. longiclavatum. In addition to thesemicroscopic characteristics, L. longiclavatum differsfrom O. clavigerum in cultural appearance. The colonycolour of L. longiclavatum is olive (1E4), while thatof O. clavigerum is olive brown (4E4) on 2% DMEAat 7 d. The conidiophores of L. longiclavatum are moreabundant at the centre of the colony, while those ofO. clavigerum are spread throughout. L. longiclavatumappears fluffier, especially at the centre, and becomespigmented earlier than O. clavigerum. The sinuate col-ony margin of L. longiclavatum is distinct from thesmooth one of O. clavigerum.

The two species are also differentiated physiologi-cally. The optimal growth rate of L. longiclavatum is ap-proximately half that of O. clavigerum (14.3 mm dx1)on 2% OMEA at 25 x. Furthermore, in contrast toL. longiclavatum, O. clavigerum shows a high degree ofplasticity losing complex structures of anamorphs aftercontinuous subculturing (Tsuneda & Hiratsuka 1984).The phylogenetic analysis with rDNA, b-tubulin, actin,and GPD gene sequences also strongly supported theseparation of these two fungal species. In all thephylogenetic trees, L. longiclavatum and O. clavigerumconsistently formed distinct clades. In the highlyresolved combined tree, L. longiclavatum was placedat the base of the O. robustum, L. terebrantis, andO. clavigerum clades. All these phylogenetically close

species have a common anamorph: Leptographium.However, the shape and the size of their conidia, andthe length of their conidiophores differed (Table 2).In O. robustum and L. terebrantis, conidia are obovoidand oblong, with truncated ends and round apices, likethose of another related species, O. aureum. None ofthese species produce long clavate conidia, whichare typical of L. longiclavatum and sometimes ofO. clavigerum. Furthermore, L. longiclavatum conidio-phores are longer than those of the other species,particularly those of O. robustum, L. pyrinum andL. terebrantis. Four of the species have common hosts ;L. longiclavatum, O. clavigerum (Robinson-Jeffrey &Davidson 1968), and L. terebrantis (Harrington 1988,Hausner, Reid & Klassen 2000) have been foundon both lodgepole pine and ponderosa pine (Pinusponderosa) infested with D. ponderosae, whileO. robustum (Robinson-Jeffrey & Davidson 1968) hasbeen only isolated from ponderosa pine.

L. longiclavatum can therefore be readily dis-tinguished from the most similar Leptographium, theanamorph of O. clavigerum, by its longer conidio-phores, homogenous spore shapes, slower growth rate,b-tubulin RFLP profiles, and multigene phylogenies.In order to understand the role of L. longiclavatum inthe D. ponderosae-vectored infestation process, it willbe necessary to investigate its pathogenicity on maturelodgepole pines.

ACKNOWLEDGEMENTS

This work was supported by the Natural Sciences and Engineering

Research Council of Canada. We thank Lorraine Maclauchlan

(Southern Interior Forest Region, BC Ministry of Forests), Allan

Carrol (Pacific Forestry Centre, Natural Resources Canada) and Jeff

Alexander (Lignum Ltd., Williams Lake, BC) for providing infested

trees, and Diana L. Six (University of Montana) for sharing some

isolates from the mycangia of D. ponderosae. We are also grateful to

Mary Berbee (University of British Columbia) for reading our

manuscript and her valuable comments.

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Leptographium longiclavatum sp. nov., a new sapstaining fungus 1170