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Leptographium pruni, sp. nov. from bark beetle-infested Prunus jamasakura in Japan

     JST domestic fellow, Tohoku Research Center of Forestry and Forest Products Research Institute, Nabeyashiki 92-25, Shimo-Kuriyagawa, Morioka, Iwate 020-0123, Japan

M. J. Wingfield

     Forestry and Agricultural Biotechnology Institute (FABI), Pretoria 0002, Republic of South Africa

T. Kubono
Y. Ichihara

     Tohoku Research Center of Forestry and Forest Products Research Institute, Nabeyashiki 92-25, Shimo-Kuriyagawa, Morioka, Iwate 020-0123, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Leptographium species are anamorphs of Ophiostoma, commonly isolated from conifer. There are, however, a small number of these fungi that have been collected from angiosperm hosts. In this study, we describe Leptographium pruni, sp. nov. isolated from the bark of Prunus jamasakura infested by the bark beetle Polygraphus ssiori. This new species is unusual in having a distinct Sporothrix synanamorph with ramoconidia. No evidence of a teleomorph was found, but a high level of tolerance to the antibiotic cycloheximide and the presence of a Sporothrix synanamorph suggest that L. pruni is an Ophiostoma anamorph. Analysis of sequence data for the domain 1 region of the LSUrDNA operon also supports the phylogenetic relationship of L. pruni with Ophiostoma. In addition, sequence data suggest that L. pruni is related to other species of Leptographium rather than Pesotum species with distinct Sporothrix synanamorphs.

Key words: Leptographium, Prunus, Sporothrix synanamorph, ITS, LSUrDNA, Ophiostomatales

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Species of Leptographium Largeberg & Melin are characterized by dark mononematous conidiophores terminating in a conidiogenous apparatus made up of a series of branches and conidiogenous cells that produce hyaline 1-celled conidia in mucilaginous masses (Kendrick 1962, Wingfield 1993). This morphology is adapted for dispersal by insects, especially bark beetles (Coleoptera:Scolytidae; Lagerberg et al 1927, Upadhyay 1981, Harrington 1988, Jacobs and Wingfield 2001). Most Leptographium species are thought to be anamorphs of Ophiostoma H. & P. Sydow (Harrington 1988, Wingfield 1993, Jacobs and Wingfield 2001), although teleomorphs are known only for a small number of the species.

Leptographium includes economically important species that cause blue stain in lumber (Lagerberg et al 1927, Harrington 1988, Gibbs 1993, Seifert 1993, Jacobs and Wingfield 2001). A few species, such as L. wageneri (W.B. Kendrick) Wingfield, are important root pathogens of conifers (e.g., Wagener and Mielke 1961, Leaphart 1960, Smith 1967). Most species of Leptographium occur on conifers, and only a few occur on angiosperms ( Jacobs and Wingfield 2001). This might reflect the relatively few studies on Ophiostoma spp. or their anamorphs on hardwoods, or the absence of blue-stain problems in this wood type.

During a survey of ophiostomatoid fungi in Japan, a species of Leptographium was isolated from Prunus infested with the bark beetle Polygraphus ssiori Nijima. This fungus was unusual because of a distinct Sporothrix Hektoen & Perkins synanamorph, particularly, with ramoconidia. The well developed Sporothrix-like synanamorph and ramoconidia are unknown in other Leptographium species or unnamed Leptographium anamorphs of Ophiostoma ( Jacobs and Wingfield 2001). The aim of this study was to describe this species. In addition, its taxonomic position was confirmed based on partial rDNA sequence data.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Fungus isolations. – Isolations were made from the bark beetle P. ssiori and from the walls of its galleries in the bark of dead trees of a Prunus jamasakura Seib. The fungus was collected on the same tree species at two localities in northern Japan (Higashidohri Village, Aomori Prefecture, and Morioka City, Iwate Prefecture) in May 2000. A total of 162 beetles and 100 pieces of bark from galleries of the insects were used for isolations.

Adult beetles or two small pieces of bark were placed on the surface of 1% malt-extract agar (Difco malt extract, 10 g; agar, 15 g; distilled water, 1000 mL) without surface sterilization, and the plates were incubated at 15 C in the dark. After 2 mo, fungi that had grown on the plates were isolated by transferring hyphal tips or by lifting conidial masses, using a sterilized tungsten needle, to Petri dishes containing 2% MEA (Difco malt extract, 20 g; agar, 15 g; distilled water, 1000 mL). These dishes were incubated at 15 C in the dark for an additional 2 wk.

Morphology. – The isolates were incubated at 15 C in the dark on 2% MA, and after 2 wk small pieces of sterilized twigs or bark of Prunus sp. were added to the plates to stimulate sporulation. Conidiophores and conidia that were produced in cultures were mounted on glass slides in 1% lacto-fuchsin and observed and measured with a light microscope. Fifty measurements were made of each structure, and the ranges, averages and standard deviations were computed.

In preparation for scanning electron microscopy (SEM), agar disks 8 mm diam were cut from the colonies and fixed in 2.5% glutaraldehyde overnight and 1% osmium tetraoxide for 1 h. Specimens were dehydrated in a graded ethanol series, passed through ethanol-isoamylacetate and dried with a Hitachi critical-point dryer. The specimens were mounted and coated with gold palladium alloy and examined with a JOEL JSM5310LV scanning electron microscope.

The growth rates of isolates were determined at 4, 10, 15, 20, 25 and 30 C. Agar disks 5 mm diam were cut from actively growing margins of colonies of each isolate to be tested and placed at the center of plates containing 2% MEA. Three replicate plates were prepared for each isolate. In addition, cycloheximide tolerance of isolates was tested at a range (0, 0.05, 0.1, 0.5, 1.0, 2.5 and 5.0 g/L) of concentration. Colony diameter on each plate was measured after 1 wk of incubation at 20 C, and growth rates were calculated as mm/d.

DNA sequence comparisons. – Cultures were incubated on 2% MA plates for 4 wk. DNA was amplified directly from the mycelium, using the polymerase chain reaction (PCR) in a GeneAmp 9600 thermal cycler (Perkin-Elmer). The method used was that of Suyama et al (1996) with slight modification. A small amount of aerial mycelium was removed from cultures and crushed in a 100 µm PCR tube with a pipette tip, under a dissection microscope. Fifty µL of reaction mixture containing 5 µL 10x buffer, 6 µL of 25 mM MgCl2, 10 mM of dNTPs, 20 pmol of each primer, ITS1 and ITS4 for ITS1-5.8s-ITS2 region (White et al 1990), NL1 and NL4 for 5' terminal end of the LSU rDNA gene containing D1/D2 regions (O’Donnell 1993), 10 µL 5x CG-RICH solution (with FastStart Taq DNA polymerase, Roche Molecular Biochemicals), 2.5 unit of Fast Start Taq DNA polymerase (Roche Molecular Biochemicals) were added to the template. The PCR conditions were: initial denaturation at 95 C for 4 min, followed by 40 cycles of denaturation at 94 C for 20 s and annealing at 56 C for 1 min. Extension was done during the change of temperature from annealing and denaturing. Final elongation reaction was done at 72 C for 10 min. The PCR products were purified with Microcon-30 Microconcentrators (Amicon Inc., U.S.A.) and used for sequencing using Big Dye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 310 genetic analyzer (Perkin Elmer Applied Biosystems). Sequences of three isolates were determined, but all of them were identical. Sequences for the isolate MAFF410951 were deposited in GenBank (AB09121' for ITS and AB104900 for LSrDNA D1/D2 regions).

Sequence data for Leptographium species are relatively limited, but data for the LSUrDNA D1 and ITS2 regions were available for many species of this genus. Thus sequence data were analyzed together with previously reported sequences for the domain 1 regions of the large-subunit ribosomal DNA operon (Hausner et al 2000) and for the ITS2 region ( Jacobs et al 2001). Overall, the dataset for LSrDNA D1 region included 36 sequences, including sequence data produced in this study. The dataset for the ITS2 regions included the 41 sequences for species of Leptographium derived from Jacobs et al (2001) as well as the sequence data obtained in this study. Species included in this study and their GenBank accession numbers were those provided in Hausner et al (2000) and Jacobs et al (2001).

Sequences were aligned using ClustalX version 1.81 (Thompson et al 1997). Alignments were adjusted manually using the program BioEdit version 5.0.' (Hall 1999). In addition, alignment of LSU rDNA domain 1 region was adjusted based on the alignment of Hausner et al (2000). The alignment of ITS2 sequences included many sites that were difficult to align. These sites in the alignment of ITS2 region were decided using SOAP version 1.2 (Löytynoja and Milinkovitch 2001) for comparing alignments obtained in 20 trials, each with a different gap penalty (2–22), and removed from the alignment. As a result, 250 bp and 75 bp of sequences, including gaps, for each of the LSU rDNA D1 regions and ITS2 region, respectively, were used in the phylogenetic analyses. Aligned dataset was analyzed using the programs PAUP*4.0 beta (Swofford 1999). A parsimony analysis was carried out using heuristic search with random stepwise addition and tree-bisection reconnection (TBR) option of the program. Gaps were treated as missing data. All characters were equally weighted. The MAXTREE option was set to auto-increase. Bootstrap and jackknife values (each 1000 replicates) also were calculated with MAXTREE option set to 1000.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Fungus isolations. – A species of Leptographium was isolated from beetles and their galleries but at a relatively low frequency (<5%). The fungus, therefore, does not appear to be a primary associate of P. ssiori.

Morphology. – The Leptographium species is characterized by dark mononematous, macronematous conidiophores, and 2–3 primary branches, and hyaline 1-celled, oblong to ellipsoidal conidia (FIGS. 1, 4 and 5). These characteristics are typical of the genus Leptographium, but the fungus is unusual in having an obvious synanamorph with micronematous conidiophores and conidia arising from sympodialy developing denticles. This synanamorph is typical of species of Sporothrix Hektoen & Perkins, with conidia produced on obvious denticles (FIGS. 6, 7, 14 and 16). In addition, these micronematous conidiophores often produced ramoconidia giving rise to secondary conidia (FIGS. 8, 9, 13 and 15). These conidiophores were particularly common in young cultures (about 2 wk old).

View larger version (123K): [in this window] [in a new window]   FIGS. 1–5. Leptographium pruni. 1. Macronematous conidiophore. 2. Colony characteristics on MEA after 1 mo at 20 C in the dark. 3. Base of a stipe. 4. Conidiogenous apparatus. 5. Conidia. Scale bars: 1 = 50 µm, 3, 4 = 20 µm, 5 = 5 µm.

View larger version (138K): [in this window] [in a new window]   FIGS. 6–9. Sporothrix synanamorph of Leptographium pruni. 6. Micronematous conidiophore (Sporothrix-type) forming from the base of macronematous conidiophore, 7. Micronematous conidiophore (Sporothrix-type) with distinct denticles. 8. Micronematous conidiophore (Hyalodendron-type) with catenulate conidia. 9. Ramoconidia (arrow) and conidia produced by micronematous conidiophores. Scale bars: 6–8 = 20 µm, ' = 5 µm.

View larger version (144K): [in this window] [in a new window]   FIGS. 10–16. Leptographium pruni (SEM) 10. Conidiogenous apparatus of macronematous conidiophore. 11. Conidia from macronematous conidiophores. 12. Conidiogenous cells showing percurrent proliferation. 13. Catenulate conidiophore and ramoconidia with distinct denticles. 14. Developed denticles of micronematous conidiophore. 15. Ramoconidia with distinct denticles. 16. Conidiogenous cells showing sympodial ontogeny. Scale bars: 10 = 20 µm, 11, 15, 16 = 5 µm, 12–14 = mm.

Phylogenetic analysis of LSU rDNA D1 region showed that the Leptographium sp. from Prunus jamasakura is related to Ophiostoma, particularly those species with Leptographium states. The fungus was not related specifically to species of Ophiostoma that are characterized by Pesotum states and having distinct Sporothrix synanamorphs. This relationship was supported by high bootstrap value (FIG. 18). In the phylogenetic analysis of LSU rDNA D1 region, from a total of 250 characters, 154 characters were constant, 35 variable characters were parsimony uninformative and 61 were informative.

View larger version (47K): [in this window] [in a new window]   FIG. 18. One of 1651 most-parsimonious trees of Ophiostoma, Leptographium and other Ascomycetous fungi based on 250 characters, including gaps, of D1 region of large subunit rDNA operon. The tree is unrooted. One thousand replicates of bootstrap values >50% and jackknife values >50% indicated above the branches (bootstrap value/jackknife value). Tree length = 205, consistency index (CI) = 0.6585, homoplasy index (HI) = 0.3415, retention index (RI) = 0.8006.

View larger version (48K): [in this window] [in a new window]   FIG. 19. One of the most-parsimonious trees of Leptographium species based on 75 characters, including gaps, of ITS-2 regions of rDNA operon. The tree is unrooted. One thousand replicates of bootstrap values >50% and jackknife values >50% indicated above the branches (bootstrap value/jackknife value). Arrows show the Leptographium species reported to have Sporothrix synanamorphs. Tree length = 41, Consistency index (CI) = 0.8049, Homoplasy index (HI) = 0.1951, Retention index (RI) = 0.9286.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

Leptographium pruni, sp. nov. H. Masuya & M. J. Wingfield. FIGS. 1–17

View larger version (26K): [in this window] [in a new window]   FIG. 17. Leptographium pruni. a. Macronematous conidiophore. b. Conidia (b1. Conidia developing from macronematous conidiophore. b2. Conidia from micronematous conidiophore and ramoconidia. b3. Secondary conidia). c. Micronematous (Sporothrix-type) conidiophore. d and e. Micronematous (Hyalodendron-type) conidiophore. f. Laterally formed secondary conidia on undifferentiated hyphae. Scale bars: a = 30 µm, b–f = 5 µm.

Colonies on 2% MEA hyaline at first becoming smoke gray (21""d, Rayner 1970), aerial hyphae abundant. Mycelium straight or curved, spreading radially on the medium and immersed in the medium, hyaline to pale brown, sometimes brown, verrucose, 1.5–12 µm diam, occurring singly or aggregated in strands of 2–5 hyphae. Aerial hyphae hyaline to pale brown, sometimes verrucose. Conidiophores mononematous and both macronematous and micronematous forms present.

Macronematous conidiophores arising laterally from hyphae, single but often also in loosely arranged groups on the hyphae, without rhizoidal hyphae at the bases. Stipes erect, pale brown to dark brown, 1–6-septate, tapered, 32–190(–324) (x = 86.3) µm long and 3–10.5 (x = 6.2) µm wide at base. Conidiogenous apparatus 22–80 (x = 46.5) µm long (excluding conidial mass) consisting of 3–5 but mostly three or four series of branches. Up to four primary branches present but mostly two adjacent to each other without a distinct central branch 6–24 x 2–6.5 (x = 13 x 4) µm. Arrangement of the primary branches on the stipe-type B ( Jacobs and Wingfield 2001). Conidiogenous cells discrete, hyaline, tapering from base to apex, 5–13(–16) x 1–2.5 (x = 10 x 1.5) µm. Conidium development replacement wall building with holoblastic, percurrent proliferation but with delayed secession, giving a false appearance of sympodial proliferation. Conidia hyaline, oblong to ellipsoid, sometimes clavate, with rounded apices and rounded to subtruncate bases, 2.5–8.5(–13) x 1–3 (x = 5 x 2) µm, accumulating around the conidiogenous apparatus in a hyaline mucilaginous mass.

Micronematous conidiophores, arising orthotropically from undifferentiated hyphae, often in terminal position or integrated in short side branches, sometimes developed on aerial hyphae, unbranched. Conidiogenous cells, subcylindrical, variable in shape and size, usually widest at the basal part and slightly tapering, bearing distinct denticles at their tip, 1–40 (x = 16.5) µm long, 0.75–2.5 (x = 1.25) µm width at the base. Conidia hyaline, 1-celled, oblong to ellipsoidal, 2–5 x 0.5–4 (4 x 2.5) µm width, sometimes developing into ramoconidia, which are larger than normal conidia, producing distinct denticles and secondary conidia. Ramoconidia hyaline, 1-celled, sometimes septate, 4–11.5 x 1.5–2.5 (x = 7 x 2) µm. Secondary conidia, hyaline, 1-celled, subglobe to ellipsoidal, 1.5–5 x 0.5–2 (x = 3.5 x 1.5) µm.

The growth rate of colonies on 2% MEA 5.5–6.5 (x = 5.' ±0.36) mm/d at 20 C. Growth reduced at temperatures below 20 C and above 30 C (TABLE I). The fungus is tolerant to cycloheximide, with growth at 20 C reduced by approximately 60% on 2% MEA containing 5.0 g/L cycloheximide (TABLE II).

Etymology. – pruni referring to Prunus, the host on which this fungus was found.

	DISCUSSION

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Macronematous conidiophores of L. pruni are similar to those of Leptographium grandifoliae Wingfield, L. sibiricum Jacobs & Wingfield and L. elegans Wingfield et al. However, unlike L. grandifoliae, the Leptographium state of the fungus has no rhizoids at the bases of conidiophores, which are typical in the former species. Leptograhium pruni morphologically is similar to L. sibiricum and L. elegans, and the three species have overlapping shapes and sizes of conidiophores and conidia. However, L. sibiricum lacks the typical synanamorph found in the L. pruni and L. elegans has an indistinct and rarely produced Sporothrix synanamorph (Wingfield et al 1994). L. sibiricum and L. elegans also occur on conifers, and there is no precedence for species of Leptographium on conifers being found on hardwoods ( Jacobs and Wingfield 2001).

Four Leptographium species have sympodial synanamorphs that might be considered Sporothrix-like and thus potentially confused with the L. pruni. These are L. aureum Wingfield, L. elegans Wingfield et al, L. franckegrosmanniae Jacobs & Wingfield and the unnamed anamorph of O. valdiviana Butin. Of these, L. aureum and L. franckegrosmanniae rarely produce a synanamorph and, when these structures are formed, conidia are produced directly on a superficial mycelium and in the absence of distinct conidiophores (Upadhyay 1981, Mouton et al 1992). In addition, their mononematous Leptographium morphs are quite different and easily distinguished from L. pruni. Ophiostoma valdiviana was described as having a reasonably well-developed Sporothrix synanamorph, but the Leptographium morph has stipes that are sometimes synnematous and that vary in length (Butin and Aquilar 1984) when compared to those of L. pruni.

Leptographium elegans is the species most similar to the L. pruni. Although L. elegans has a Sporothrix synanamorph, its conidia are similar to those produced by the Leptographium morph (Wingfield et al 1994, Jacobs and Wingfield 2001). This is in contrast to L. pruni, in which conidia produced by the Sporothrix morph are rounded at both ends and morphologically different from those of the Leptographium morph, which have rounded apices and truncate bases. In addition, the mycelium of L. elegans sometimes aggregates in strands of 4–13 hyphae, distinct from aggregations of 2–5 hyphae in L. pruni. To the best of our knowledge, there are no other species of Leptographium that have synanamorphs that produce ramoconidia.

Leptographium pruni originates from a host not previously associated with this group of fungi. The Prunus host is different from other Leptographium species that might be considered similar to it. L. grandifoliae was isolated from Fagus grandifolia in the United States (Davidson 1976), L. sibiricum from Abies sibirica in Russia ( Jacobs et al 2000), L. aureum from Pinus spp. in the United States (Harrington 1988), L. franckegrosmanniae from Quercus sp. in Germany (Davidson 1971), O. valdiviana from bark and wood of Nothofagus alpina (Poepp. & Endl.) Oerst. and N. dombeyi (Mirb.) Oerst. in Chile (Butin and Aquilar 1984) and L. elegans from Chamaecyparis formosensis Matsum. in Taiwan (Wingfield et al 1994). In general, Leptographium spp. are typified by having distinct hosts and the bark beetles that carry them tend to be relatively host specific ( Jacobs and Wingfield 2001). Thus, in addition to robust morphological differences, distinct host differences provide us with convincing evidence that L. pruni represents a distinct taxon.

We were unable to find evidence of a teleomorph for L. pruni. If it occurs frequently, we would have expected it to be present in the galleries of P. ssiori. We also failed to make successful crosses between our isolates of L. pruni isolate. However, the fact that the fungus is highly tolerant to cycloheximide in culture provides good evidence to suggest that it is an anamorph of Ophiostoma (Harrington 1981). Our DNA sequence data also have shown that L. pruni is related to other species in the Ophiostomataceae and support the views of previous authors (Zambino and Harrington 1992, Hausner et al 2000, Jacobs et al 2000) that the genus Leptographium should be reserved for anamorphs of Ophiostoma.

The most distinct characteristic of L. pruni is its Sporothrix synanamorph. The presence of acropetal chains of ramoconidia particularly is characteristic of this species. This synanamorph is more strongly developed than in the reduced Sporothrix states in other Leptographium spp. and is more similar to those observed in some Ophiostoma species with Pesotum anamorphs. Phylogenetic analyses in this study have shown that L. pruni is closely related to Ophiostoma with Leptographium anamorphs. Thus, despite its distinct Sporothrix state and ramoconidia, it is specifically not related to Ophiostoma species having similar Sporothrix anamorphs. This suggests that the presence of a distinct Sporothrix anamorph can occur widely among the Ophiostomataceae and supports the view of Hausner et al (1993), who suggested a polyphyletic origin for the Sporothrix-like morphology in Ophiostoma. Mouton et al (1992) also suggested that Sporothrix synanamorphs increasingly might be found as new Leptographium spp are discovered. Our discovery of the first Leptographium sp. having a distinct Sporothrix synanamorph with catenulate conidia, therefore, might reflect a poorly collected genus, rather than an unusual characteristic.

Recent DNA sequence-based studies have suggested that separation of the synnematous Pesotum and the mononematous Leptographium do not reflect phylogenetic relationships in Ophiostoma (Okada et al 1998, Hausner et al 2000). Harrington et al (2001) suggested that Pesotum should be restricted to the Ophiostoma species with synnematous anamorphs and Sporothrix synanamorphs. This suggestion was based on the fact that Pesotum-like species with Sporothrix synanamorphs have a monophyletic origin and reside in the Ophiostoma piceae (Münch) H. & P. Sydow complex, separate from other Pesotum-like anamorphs without Sporothrix synanamorphs. Thus, the Sporothrix synanamorph appears to be an important characteristic in the delimitation of Pesotum species from other synnematous anamorphs of Ophiostoma. In contrast, results of our study suggest that the Sporothrix synanamorph in Leptographium may have less value for generic treatment, than has been found in Pesotum. Our study also suggests that Sporothrix synanamorphs may not be unusual in members of Leptographium and that its generic concept might be modified to include this view.

Leptographium recently has been subjected to a monographic study by Jacobs and Wingfield (2001). DNA-based comparisons also have been undertaken (Hausner et al 2000, Jacobs et al 2001), and they already are proving to be useful for the identification of known species. However, little evidence has emerged for phylogenetic relationships linked to morphological characters (Hausner et al 2000, Jacobs et al 2001). This might be due to the fact that data for variable regions of the genome for this group of fungi are relatively limited and sequences for additional genes are required. Sequence data also are not available for all species of Leptographium, and the addition of taxa could greatly improve the value of phylogenetic trees for this group. Thus surveys and collection of additional species of Leptographium from new environments and particularly from Angiosperms should add valuable insight into the taxonomic concepts and evolution of Leptographium species.

	FOOTNOTES

 
Accepted for publication November 22, 2003.

¹ Corresponding author. E-mail: H_masu{at}hotmail.com

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