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Department of Wood Science, University of British Columbia, Vancouver B.C., V6T 1Z4 Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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An undescribed Leptographium species was isolated from the spruce-infesting bark beetle Ips perturbatus collected from felled spruce trees and logs in northern British Columbia and Yukon Territory. Morphologically, this fungus is similar to L. abietinum and L. hughesii but differed in a number of characteristics (e.g. the arrangement of its conidiophores). The fungus grew optimally at 25 C on 2% malt-extract agar and showed a high level of tolerance to cycloheximide. Comparison of rDNA and ß-tubulin gene sequences also confirmed that this Leptographium species represents an undescribed taxon. Thus we described it as a new species, Leptographium fruticetum sp. nov.
Key words: ß-tubulin, fruticetum, fungi, Ips, ITS2, Leptographium, LSU, ophiostomatoid, perturbatus, phylogeny, rDNA, spruce
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). This beetle frequently infests freshly harvested logs and stressed trees (Raffa et al 1993, Gara et al 1995, Holsten 2001). In spruce forests of Alaska, southwestern Yukon Territory and British Columbia major attacks of I. perturbatus also have been killing trees (Kondo and Taylor 1986, Holsten 2001, Garbutt 2003). Like many other bark beetles (Coleoptera: Curculionidae: Scolytinae), I. perturbatus carries some ophiostomatoid fungi that cause damage to trees, logs and lumber. The fungal associates of spruce-attacking Ips species, except for those of I. typographus (Linnaeus) in Eurasia, have not been examined thoroughly (Jacobs et al 2004). In North America the major focus has been given to the fungi associated with the spruce bark beetle, Dendroctonus rufipennis (Kirby), and almost no information is available on fungi carried by Ips beetles attacking spruce (Picea A. Dietrich) species.
Ophiostomatoid fungi, especially those of the genus Ophiostoma H.&P. Sydow, are the predominant associates of conifer-infesting bark beetles (Mathiesen-Käärik 1953, Wingfield and Gibbs 1991, Jacobs and Wingfield 2001). The genus Ophiostoma includes a number of different anamorphs that are classified in the genera Leptographium Lagerberg & Melin, Pesotum Crane & Schocknecht sensu Okada & Seifert, Hyalorhinocladiella Upadhyay & Kendrick and Sporothrix Haktoen & Perkins (Wingfield 1993, Okada et al 1998). Leptographium species are recognized by their tall, single conidiophores and their complex conidiogenous cells that produce slimy masses of hyaline, single-celled conidia (Kendrick 1962, Jacobs and Wingfield 2001). Teleomorphs have not been observed for many of these fungi, and only the morphology of their anamorphs has been described (Jacobs and Wingfield 2001). Leptographium species are economically important as tree pathogens (Cobb 1988, Harrington and Cobb 1988, Harrington 1993) agents of wood discoloration (blue-stain) (Solheim and La°ngström 1991; Seifert 1993; Solheim 1995a, b), and they are frequently involved in phytosanitary issues. These fungi depend on bark beetles for their dispersal. In nature they sporulate in their vector’s galleries in the phloem and sapwood of the infested trees. The association between the bark beetles and the fungi can be specific, as in the case of O. penicillatum (Grosmann) Siemaszko that is always associated with I. typographus on spruce trees (Solheim 1993, Krokene and Solheim 1996, Yamaoka et al 1997), or it can be casual, as with O. piceaperdum (Rumbold) C. Moreau, which has been found on many different bark beetles (Rumbold 1936, Krokene and Solheim 1996, Jacobs and Wingfield 2001, Yamaoka et al 2004).
During the first comprehensive survey of the ophiostomatoid fungi associated with the Ips beetles infesting felled spruce trees and logs in northern British Columbia (BC) and Yukon Territory, a Leptographium species consistently was isolated from I. perturbatus and its galleries. This fungus differed morphologically and genetically from other species described in the literature. In this study we compared the sequences of partial rDNA and ß-tubulin genes to establish whether this species is a member of Leptographium clade. We also determined the closest related taxa based on ecological, morphological and molecular characters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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A total of 87 beetles and 157 samples from beetle galleries were used for isolations. Small pieces of phloem and the surrounding sapwood were removed from different parts of Ips galleries and placed on 2% OMEA (33 g malt-extract agar "Oxoid CM59", 10 g agar "tech. No. 3" and 1L distilled water) amended with ampicillin at 50 µg/mL. Living beetles also were removed from their galleries and individually stored in a sterile 1.5 mL microtube. Tween 20TM (Sigma-Aldrich, Oakville, Canada) wash solution (0.01%) was added to each tube. The tubes were vortexed 3 min to dislodge fungal spores from the beetle bodies. The washed solution from the insect was serially diluted (1 : 5, 1 : 25 and 1 : 125), and then 100 µL of each dilution was spread onto plates of 1% OMEA. Petri dishes were incubated at room temperature. When mycelia overgrowths were observed, the individual growing tips of the separated colonies were subcultured on 2% OMEA plates to obtain pure cultures. From each insect-wash-dilution plate, 15 colonies were transferred randomly to fresh media. Pure cultures of each fungal isolate were obtained with a single conidia isolation technique (Uzunovic et al 2000). All isolates (TABLE I) in this study are maintained at the Breuil Culture Collection, University of British Columbia, Vancouver, Canada (BUBC). Representative isolates also have been deposited in the Canadian Collection of Fungal Cultures (CCFC/DAOM), Agriculture and Agri-Food Canada, Ottawa, Ontario.
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). The growth rates were determined at 4, 10, 15, 20, 25, 30, 35 and 37 C. Small plugs of agar (5 mm diam) were taken from the actively growing margins of the purified cultures and were placed at the center of Petri dishes containing 20 mL of 2% OMEA, with three replicate plates for each test temperature. The growth was calculated as a mean diameter increment per day. Average growth rates were calculated as millimeters per day (mm/d). Cycloheximide tolerance of isolates was assessed by measuring growth at 25 C on 2% OMEA amended with 0.05, 0.1, and 0.5% of cycloheximide.
Fungal structures produced on 2% DMEA (20 g malt-extract agar, Difco, 10 g agar, Difco, and 1L distilled water) and/or on gamma ray-sterilized spruce sapwood blocks were used for microscopic study. The fruiting structures from 1–4 wk old cultures, grown at room temperature, were mounted in water and observed with a Zeiss Axioplan compound light microscope. Fifty measurements were made for each taxonomically informative structure so that ranges and averages could be computed. For scanning electron microscopy (SEM), small wood blocks (5 x 2 x 5 mm) bearing fungal structures were fixed using the method described by Lee et al (2003). After fixation samples were dried with a Blazers CPD 020 critical point dryer. They were coated with gold palladium with a Nanotech Semprep II sputter coater and examined with a Hitachi S4700 scanning electron microscope.
DNA extraction, PCR amplification and sequencing.— –
Isolates of various Leptographium species were used in DNA sequence comparison (TABLE I
). DNA was extracted from mycelia grown on 2% OMEA plates overlaid with cellophane (gel dry grade, BioRad) following the method described by Kim et al (1999). The rDNA genes, including the ITS (internal transcribed spacers) and the partial LSU (large subunit 28S) were amplified using the primer set ITS1F/ITS4 and ITS3/LR3 (Vilgalys and Hester 1990, White et al 1990). A portion of the ß-tubulin gene was amplified using the primer set T10/BT12 (O’Donnell and Cigelnik 1997, Kim et al 2003). PCR amplification was performed as described by Kim et al (2004). PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Ontario, Canada). The purified products were sequenced with the same primer sets used for the PCR. Sequencing was performed on an ABI 3700 automated sequencer (Perkin-Elmer, Foster City, California) at the DNA synthesis and Sequencing Facility, Macrogen (Seoul, Korea).
Phylogenetic analysis.— –
Sequences of related taxa were retrieved from GenBank. The ITS2/LSU sequences of 47 and the ß-tubulin gene sequences of 21 ophiostomatoid fungi with Leptographium anamorphs were aligned with Clustal X version 1.81(Thompson et al 1997), on its default settings. The resulting alignments were corrected manually with PHYDIT version 3.2 (http://plasza.snu.ac.kr/jchun/phydit/). The localization of introns and exons in ß-tubulin gene fragments was approximated, first by performing BLAST searches of GenBank and second by comparing the homologous-characterized genes of species in GenBank to sequences of the Leptographium sp. considered in this study. The intron sequences of ß-tubulin gene were aligned ambiguously and, thus, were removed from the analysis. The aligned rDNA and ß-tubulin sequences were subjected to phylogenetic analyses performed by parsimony methods of PAUP*4.0b10 (Swofford 2002). Gaps were treated as missing data. Maximum parsimony trees (MPTs) were identified by heuristic searches with 100 random stepwise additions and tree-bisection-reconnection (TBR) branch-swapping options. The MAXTREE option was set to auto-increase. All characters were unordered and of equal weight. Statistical support for the phylogenetic groupings was assessed by bootstrap analyses using 1000 replicate datasets with the random addition of sequences during each heuristic search (Felsenstein 1985). Based on previous studies ( Jacobs et al 2001), L. elegans M.J. Wingfield, Crous & Tzean was assigned as the outgroup taxon.
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RESULTS |
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Morphology.— –
Leptographium sp. isolated from I. perturbatus exoskeleton and its gallery is typical of the genus Leptographium, having dark, long macronematous conidiophores and conidia accumulated in mucilaginous masses at the apex of the conidiogenous apparatus (FIGS. 1–4). The fungus has an optimal growth temperature of 25 C and can tolerate a high concentration (0.5%) of cycloheximide, an indication that it is an anamorph of the genus Ophiostoma. The most distinct characteristic of this fungus is in its conidiophore arrangement, which is composed of groups of greater than 30 conidiophores (FIGS. 1, 2). The grouped conidiophores often are produced on wood and on artificial media, particularly on 2% DMEA cultures of about 1–2 wk old. Comparisons with other Leptographium species revealed that this species is morphologically most similar to L. abietinum (Peck) M.J. Wingfield and L. hughesii K. Jacobs, M.J. Wingfield & Harrington (Davidson 1955, Kendrick 1962, Wingfield 1985, Jacobs et al 1988, Jacobs and Wingfield 2001). These species, however, could be distinguished from each other because of differences in their conidiophore arrangements, conidiophore lengths, conidial shapes and growth rates (TABLE II).
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). Alignments and trees also are accessible through TreeBASE (2005, http://www.treebase.org/treebase/submit.html) under reference number 25754.
Amplification of the ITS and LSU region for the Leptographium sp. resulted in fragments of 1179 base pairs (bp). The aligned ITS2/LSU dataset consisted of 621 characters, of which 429 were constant and 125 were parsimony informative. Fifteen MPTs were obtained with a length of 394 steps (CI = 0.73, RI = 0.9, HI = 0.26). Topological differences among these trees were due to branching order changes of a few taxa (i.e. L. lundbergii, L. alethinum, O. crassivaginatum and O. huntii); therefore one is presented (FIG. 13).
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), with a length of 73 steps (CI = 0.8, RI = 0.89, HI = 0.19), with essentially the same topology as the LSU/ITS2 tree.
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, 14). In contrast L. hughesii was placed in another clade composed of L. abieticolens K. Jacobs & M.J. Wingfield and L. bistatum J.-J. Kim & G.-H. Kim. The rDNA sequences of Leptographium sp. also showed the highest identity (1169/1176 = 99%) with that of O. abiocarpum. Although the branch in the rDNA MPTs separated the Leptographium sp. from the closely related species, it was not supported by the bootstrap analysis (FIGS. 13, 14).
Amplification of the partial ß-tubulin gene of the Leptographium sp. resulted in fragments of 719 bp. The ß-tubulin sequences shared only 97% (643/657) identity with that of the O. abiocarpum reference strain (MUCL 18351). The aligned ß-tubulin dataset consisted of 528 characters, of which 412 were constant and 94 were parsimony informative. Two identical MPTs were obtained with a length of 251 steps (CI = 0.63, RI = 0.8, HI = 0.36). The ß-tubulin gene tree had higher resolution than the rDNA tree (FIG. 15). The two isolates representing the unidentified Leptographium sp. formed a conspecific group, showing a sister relationship with O. abiocarpum. The clades of the unidentified Leptographium sp. and O. abiocarpum were supported with high bootstrap values: 98 and 92%, respectively. Regardless of the differences in the branch resolutions between the rDNA and ß-tubulin gene trees, the separation of Leptographium sp., as well as its monophyletic relationship with O. abiocarpum, L. abietinum and O. penicillatum was consistent.
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TAXONOMY |
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Leptographium fruticetum S. Massoumi Alamouti, J.-J. Kim & C. Breuil sp. nov. FIGS. 1–12
Crescit optime ad 25 C tum (8.2–) 8.6–9.2 mm per diem in 2% OMEA. Non crescit infra 4 C vel supra 37 C. In 2% OMEA cum alio 0.05, 0.1 et 0.5% cycloheximide, crescit ad 25 C alium 9.0, 8.0 et 7.4 mm per diem. Coloniae in OMEA plumbeus olivaces laete, exemplum stellate per iacio margins. Hypharum parietes laeves, olivaces, 1.5–3.0 µm lati, in substrato submersa. Conidiophorae singulae vel ad riginta aggregatae, mononematosa, macronematosa, (205–)400–645(–820) µm longi, structuris similes-rhizoideis praesentibus. Stipites brunnei, leves, cylindracei, simplices, 4–13-septati, (105–)320–630(–750) µm longi, ad basim 6.7–9.8 µm lati. Apparatus conidiogenus (35–)75–66(–100) µm longi, massa conidiali exclusa, 2–4(–5) seriebus ramorum cylindricorum, 2–3 metutae primariae sed plerumque duo inter se adjacentes, ramus centralis distinctus nullus, brunnei olivaceum, leves, cylindricae, aseptatae, 9.5–16 x 3.0–5.0 µm. Cellulae conidiogenae discretae, hyalinae, sursum attenuatae, 9.0–19(–25) x 1.0–2.5 µm. Evolutio conidii per aedificationem parietis supplementariae ontogenia holoblastica et proliferatione percurrenti cum secessione retardata, ut falso videtur per proliferationem sympodialem. Conidia hyalina, aseptata, oblonga, aliquando exigue curvata, apicibus rotundatis, basibus distinctis truncatis (3.0–) 4.7–5.2(–7.5) x 1.0–2.5 µm.
The optimal growth temperature for the Leptographium sp. was 25 C with a growth rate of (8.2–)8.6–9.2(x = 8.8 ± 0.28) mm/d diam on 2%OMEA. No growth was found below 4 C and above 37 C. On 2% OMEA amended with 0.05, 0.1 and 0.5%cycloheximide, growth at 25 C were 9.0, 8.0 and 7.4 mm/d diam, respectively. Colonies dull olive gray (2F2) at first, becoming a dull dark gray (1F1) with age, aroma present in both fresh and old cultures. Colony pattern was stellate with diffuse margins. Hyphae smooth-walled, olive (3F5), not constricted at the septa, 1.5–3.0 µm wide, mostly submerged in the agar with little aerial mycelia. Conidiophores single or in groups of greater than thirty, erect, mononematous, macronematous, (205–)400–645 (–820) (x = 461 ± 153) µm long with rhizoid-like structures present at the base, sometimes produced on aerial hyphae (FIGS. 1–3). Stipes light brown (6D8), smooth, cylindrical, simple, 4–13-septate, (105–)320–630 (–750)(x = 384 ± 134) µm long, 3.5–6.0 µm wide below primary branches, apical cell not swollen, basal cell 6.7–9.8 µm wide and swollen (FIGS. 3, 6, 7, 8). Conidiogenous apparatus (35–)75–66(–100)(x = 66.7 ± 13) µm in length (excluding the conidial mass) consisting of 2–4(–5) series of cylindrical branches, 2–3 primary branches but mostly two adjacent to each other without a distinct central branch, olive brown (4D4), smooth, cylindrical, aseptate, 9.5–16(x = 11 ± 1.5) x 3.0–5.0 µm. Arrangement of the primary branches on stipe-type B (Jacobs and Wingfield 2001). Conidiogenous cells discrete, hyaline, 2–3 per branch, cylindrical, tapering slightly from the base to the apex, 9.0–19 (–25)(x = 15 ± 4) x 1.0–2.5 µm. Conidium development annellidic (FIGS. 9–11), occurring through replacement wall building with holoblastic ontogeny and percurrent proliferation and delayed secession, giving a false appearance of sympodial proliferation (Wingfield 1993). Conidia hyaline, aseptate, oblong, sometimes clavate, with rounded apices and truncate bases, (3.0–) 4.7–5.2(–7.5)(x = 5.1 ± 1) x 1.0–2.5 µm (FIGS. 11, 12).
Species examined. – CANADA. BRITISH COLUMBIA: Prince George, Aleza Lake Research Forest. Ips perturbatus young adults on Picea engelmannii x glauca, 5 Jul 2002, S. Massoumi Alamouti 2PG6P-L1 (culture DAOM234389) (HOLOTYPE). CANADA. YUKON TERRITORY: White-horse. Ips perturbatus gallery on Picea glauca, 2 Aug 2003, S. Massoumi Alamouti 3YT1P-L1 (culture DAOM234390). CANADA. BRITISH COLUMBIA: Prince George. Stained sapwood of freshly cut logs of Picea engelmannii x glauca, Sep 1997, A. Uzunovic AU157-253 (culture DAOM234391).
Etymology. – The Latin fruticetum refers to the bouquet-like arrangements of conidiophores.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Species of the genus Ophiostoma, in particular those with Leptographium anamorphs, are the most frequent fungal associates of bark beetles (Solheim 1986; Krokene and Solheim 1996; Yamaoka et al 1997, 1998; Jacobs and Wingfield 2001; Yamaoka et al 2004). Many of these fungi, such as O. penicillatum, L. pyrinum R.W. Davidson, L. euphyes, L. yunnanense X.D. Zhou, K. Jacobs, M.J. Wingfield & M. Morelet, O. americanum and O. dryocoetidis W.B. Kendrick & Molnar, are known to be restricted to one beetle species (Kendrick and Molnar 1965; Harrington 1988; Jacobs et al 1997, 2000; Zhou et al 2000). In our survey L. fruticetum was the most common species isolated from I. perturbatus adults and their galleries at all the sampling sites.
L. fruticetum seems to have a specific relationship with I. perturbatus adults. First, in this survey spruce trees infested by I. perturbatus often were colonized by other bark beetle species at the same time, in particular Dryocoetes affaber Mannerheim and Polygraphus rufipennis Kirby. Although these bark beetles shared the same habitat, L. fruticetum was not isolated from either D. affaber or P. rufipennis (Massoumi Alamouti 2005). Second, previous studies on fungal associates of Dendroctonus rufipennis and other spruce-colonizing bark beetles in North America, as well as those of I. typographus in Eurasia, have not reported a similar fungus (Safranyik et al 1983; Solheim 1993, 1995b; Krokene and Solheim 1996; Yamaoka et al 1997; Ohsawa et al 2000; Haberkern et al 2002; Six and Bentz 2003; Viiri and Lieutier 2004).
L. fruticetum is morphologically most similar to L. abietinum and L. hughesii (Kendrick 1962, Davidson 1955, Jacobs et al 1998, Jacobs and Wingfield 2001). These fungi do not produce sexual structures, have olivaceous colonies, similar conidiogenous apparatus and tall dark conidiophores that are produced abundantly on artificial media. L. abietinum is the most common fungus isolated from spruce trees infested with D. rufipennis in North America (Kendrick 1962, Harrington 1988). L. hughesii has a different host and geographic distribution. This fungus is associated with tropical hardwoods, whereas L. fruticetum and L. abietinum, such as many other Leptographium spp., occurs on coniferous hosts ( Jacobs et al 1998, Jacobs and Wingfield 2001). L. hughesii has been reported only in southeastern Asia and its insect vectors have yet to be identified ( Jacobs et al 1998). L. fruticetum, L. abietinum and L. hughesii also can be distinguished morphologically. The most distinct morphological character of L. fruticetum is the unique arrangement of its conidiophores, which arise in groups of greater than 30. In contrast no other fungus that has a Leptographium anamorph, including L. abietinum and L. hughesii, has been reported as having more than 10 conidiophores in a group (Jacobs et al 1998, Jacobs and Wingfield 2001). L. fruticetum and L. hughesii can be distinguished from L. abietinum by their longer conidiophores and the presence of rhizoid-like structures at the bases of conidiophores. Kendrick (1962) recorded similar structures for L. abietinum. However Jacobs et al (1998) re-examined the cultures used by Kendrick and found that they include both L. abietinum and L. hughesii. It has been indicated that L. hughesii produces rhizoids whereas these structures are absent or very rarely found in isolates of L. abietinum (Jacobs et al 1998, Jacobs and Wingfield 2001). A further difference among these three fungi is conidial shape. Although they all have conidia of similar lengths, conidia of L. fruticetum are oblong and occasionally curved. In contrast L. abietinum has distinctively narrow and prominently curved conidia while L. hughesii has ellipsoid to obovoid conidia that can be slightly curved (Jacobs et al 1998, Jacobs and Wingfield 2001). In addition to these microscopic characteristics, L. fruticetum differs from L. abietinum and L. hughesii by its ability to grow at 35 C. Further L. fruticetum has fewer aerial mycelia and becomes pigmented earlier than L. abietinum. It grows more slowly and has a prominent star-shaped colony pattern, which differs from that of L. abietinum. In contrast L. hughesii produces abundant aerial mycelia and grows 8–10 times slower than the two other fungal species.
Although L. fruticetum is morphologically similar to L. hughesii, the rDNA phylogenies showed that these two species are related distantly. In addition phylogenetic analyses of the rDNA and ß-tubulin gene sequences showed that while L. abietinum and L. fruticetum are closely related they can be distinguished if appropriate gene regions are selected. In the LSU and ß-tubulin phylogenetic trees, L. fruticetum formed a clade distinct from that containing L. abietinum.
L. fruticetum and O. abiocarpum share similar ecological niches; both are isolated from felled white spruce and logs infested by Ips species (Davidson 1966, Olchowecki and Reid 1974). However our results suggested that they might be carried by different beetle species. In our survey some felled spruce trees were infested by both I. perturbatus and Ips tridens Mannerheim. Results showed that the most common fungal associates of I. tridens were those that both morphologically and genetically matched the O. abiocarpum reference strain (Massoumi Alamouti 2005). O. abiocarpum was not isolated from I. perturbatus (Massoumi Alamouti 2005). L. fruticetum and O. abiocarpum also can be distinguished by their morphological characteristics (TABLE II). L. fruticetum produces numerous conidiophores spread throughout its colony while O. abiocarpum rarely produces asexual structures. Upadhyay (1981) described a Leptographium state for O. abiocarpum but Davidson (1966) could only observe and describe the teleomorph of this fungus. Results of the present phylogenetic analyses confirm the relationship of O. abiocarpum to Leptographium, as suggested by the morphological study of Upadhyay (1981). O. abiocarpum produces perithecia on artificial media and wood, whereas we were unable to find evidence of a teleomorph for L. fruticetum in the galleries of I. perturbatus or on artificial media. We also failed to produce perithecia by crossing L. fruticetum isolates. However the phylogenetic position and the cycloheximide tolerance of L. fruticetum confirm its relationship to Ophiostoma species.
In both rDNA and ß-tubulin trees L. fruticetum formed a monophyletic clade not only with L. abietinum and O. abiocarpum but also with O. penicillatum. All four species have a Leptographium anamorph, are closely associated with a bark beetle and commonly occur on spruce species. O. penicillatum is one of the most common associates of the destructive spruce-colonizing bark beetle I. typographus in Eurasia (Krokene and Solheim 1996, Jàcobs et al 1997, Yamaoka et al 1997). O. penicillatum is distinguished from L. fruticetum by its large allantoid conidia, fast growth, host and vector species, as well as its geographical distribution (TABLE II) (Grosmann 1931, Upadhyay 1981, Jacobs et al 1997, Yamaoka et al 1997, Jacobs and Wingfield 2001).
In conclusion the unknown Leptographium sp. described here is a new taxon that was named L. fruticetum because of its grouped-conidiophore arrangement. This fungus is distinguished readily from the morphologically close species by its abundant asexual reproduction, unique conidiophore arrangement, conidial characteristics and growth rate, as well as by the gene-selected phylogenies. L. fruticetum was isolated from I. perturbatus adults and their galleries, and from the stained sapwood of felled spruce trees and logs. L. fruticetum was not isolated from other bark beetles infesting spruce and seems to have a specific relationship with I. perturbatus. The morphological and isolation results were supported by the phylogenetic analyses, showing that L. fruticetum is closely related to the fungal species (i.e. O. abiocarpum, O. penicillatum and L. abietinum) that also have Leptographium anamorphs and are closely associated with other spruce-attacking bark beetle species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: alamouti{at}interchange.ubc.ca
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LITERATURE CITED |
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———, ———, Crous PW, Harrington TC. 1998. Leptographium engelmannii, a synonym of Leptographium abietinum, and description of Leptographium hughesii sp. nov. Can J Bot 76:1660–1667.[CrossRef]
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