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Faculté de foresterie et de géomatique, Université Laval, Québec City, Québec, G1K 7P4
Jean A. Bérubé
Natural Resources Canada, Canadian Forest Service, 1055 du PEPS, P.O. Box 3800, Sainte-Foy, Québec City, Québec, G1V 4C7
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ABSTRACT |
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The new species Lophodermium macci is described. It is similar in its morphology, habitat, geographic range and ecology to L. pini-excelsae, L. staleyi and L. nitens and often is misidentified as L. pinastri on Pinus strobus in herbaria. A modified technique was used to extract DNA from minute ascomata on herbarium specimens, and new primers were made to amplify the damaged DNA from these specimens. It provides added evidence to separate L. macci from L. pini-excelsae, its closest morphological taxon.
Key words: DNA, herbarium specimens, ITS, Lophodermium, L. pinastri, L. pini-excelsae, Pinus strobus, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It has been studied intensively in herbaceous ( Johnston 2001) and coniferous needles (Minter 1981, Cannon and Minter 1986).
Our work is focused on describing the fungal endophyte biodiversity inside conifer foliage. Endophytes are organisms living inside a plant without showing any symptoms of their presence (Carroll 1995). It is possible to detect many of these organisms by plating a small part of the plant on nutrient agar, usually yielding the asexual stages of the endophytes. While collecting the fruiting stages of fungal needle endophytes of eastern white pine (Pinus strobus L.) to match with our collection of asexual endophyte stages, we came upon a species new to science, which is described below as Lophodermium macci. We provide a description of this species, its habitat, ecology, geographic range and morphological and evolutionary relatedness to other species.
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MATERIALS AND METHODS |
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).
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) are kept at QFB (Laurentian Forestry Centre, Canadian Forest Service, Québec City, Québec), ATCC (American Type Culture Collection) and CBS (Centraalbureau voor Schimmelcultures). Cultures were grown at room temperature under diffused natural light and cool white fluorescent light.
DNA extraction from fresh cultures. –
Colonies were grown on malt-extract broth (Difco), the mycelium was frozen and lyophilized, then extracted using a 2x CTAB procedure modified from Zolan and Pukkila (1986). DNA was extracted with CTAB, purified with chloroform : isoamyl alcohol : phenol precipitated with isopropanol and resuspended in TE-8 buffer.
DNA extraction from herbarium specimens (Mixer Mill protocol). – One ascoma was removed carefully from the needle and immediately submitted to the DNA extraction protocol, using the reagents and material of the DNeasy Plant mini-kit (Qiagen, Germany, 2000), with modifications of the original protocol. The extraction process was: Clean thoroughly one small tungsten ball per 2.5 mL tube with ethanol then HCl, and autoclave; add 500 µL AP1 buffer, 1 µL RNase A (stock solution 100 mg/ml), 1 µl DX reagent; heat 10 min at 80 C; place the tubes in the Mixer Mill (MM300); shake twice for 2 min at 30 vibrations/s; heat the tubes at 100 C for 5 min; centrifuge 10 s at 13 000 rpm; add 150 µL AP2 per tube; shake twice in the Mixer Mill for 30 s at 30 vibrations/s; centrifuge a few seconds at 13 000 rpm, then cool at –20 C for 10 min; centrifuge again at 13 000 rpm for 2 min; recuperate the supernatant and add it to 850 µL of AP3 in a new tube; mix by inverting; centrifuge a few seconds at 13 000 rpm; place half of the solution on a DNeasy minispin column sitting on a collection tube; centrifuge 1 min at 8000 rpm; discard flow-through; repeat with remaining sample; discard flow-through and collection tube; add 500 µL Buffer AW (wash buffer) on the column, centrifuge 1 min at 8000 rpm; discard flow-through; repeat with the Buffer AW; discard flow-through; centrifuge again 1 min; place the column on a new collection tube; add 50 µL Buffer AE (elution buffer) heated at 80 C and incubate 5 min; centrifuge at 13 000 rpm 2 min; add 50 µL AE and centrifuge again; final volume is 100 µL; store at –20 C.
PCR (ITS and 200 bp sections of ITS). –
The internal transcribed spacer region (ITS) of the nuclear ribosomal gene was amplified using ITS-1F and ITS-4 primers (White et al 1990) on a MJ Research PTC-100 thermocycler with a program described by Abeln et al (2000). The ITS region also was amplified in small sections of 200 bp using eight primers based on the sequence of L. macci. These primers (TABLE III, FIG. 3) were designed using PrimerPremier 2002 (Biosoft International, California).
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), 1 unit TAQ polymerase (Invitrogen), and 3 µL of 1:10 dilution of ca. 10 ng/µL genomic DNA (undiluted 3 µl DNA from the Mixer Mill protocol). PCR: 3 min at 94 C, 40 cycles at 92 C for 1 min, 56 C (52 C for the small sections) for 1 min, 72 C for 1.5 min with a last cycle of 10 min at 72 C. The amplified DNA then was purified using QIAquick PCR Purification Kit (Qiagen, Maryland) and sequenced on a Genetic Analyzer 3100 (Applied Biosystems) sequencer. The ITS sequences of L. macci are deposited in GenBank (TABLE I).
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) and phylogenetic relationships were investigated using PAUP 4.8b10 (Swofford 2001) and MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001). Support for the branching topologies was evaluated using bootstrap analysis (Felsenstein 1985). Parsimony analysis was performed using a heuristic search with these parameters: Characters were unordered and had equal weight, the maximum number of trees was set at 1000, the swapping algorithm was tree bisection-reconnection (TBR), the steepest descent option was not in effect. Bootstrap analysis was performed using 1000 replicates and with MAXtrees set at 1000. MrBayes analysis consists of 1 000 000 replicates. Cyclaneusma minus (U92309
[GenBank]
) and Try-blidiopsis pinastri (U92307
[GenBank]
) were selected as outgroups.
Microscopic observations. –
Needles with ascomata were mounted in EPON resin (Spurr 1969) and stained with to-luidine blue for thin sectioning (6–8 µm) and microscopic observation.
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RESULTS |
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). Because no complete ITS sequences of L. pini-excelsae are deposited in GenBank, this species could not be included in the phylogenetic analysis. An attempt was made to amplify the complete ITS of herbarium specimens using ITS1F and ITS4, but it failed. However, the amplification of small sections of 200 bp using appropriate primers was successful. One of these amplified fragments is the 5.8s region (primers Lm.up and Lm.down) and was used to confirm that the amplified DNA was that of a species of Lophodermium. The other fragment is the variable portion of ITS2 of Lophodermium, and sequence comparison distinguished L. macci from L. pini-excelsae (IMI 87030a, holotype; IMI 249596; IMI 139876) (FIG. 5). However, because the sequence of L. pini-excelsae is partial, it cannot be used in phylogenetic analysis of the genus.
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).
TAXONOMY –
Lophodermium macci Sokolski & Bérubé, sp. nov. FIGS. 1, 2
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Ascomata forming on the abaxial side of secondary needles, shiny and black in the center, with a gray margin and a black perimeter line making a clearly marked outline, usually elliptic, sometimes round, raising the surface of the needle, partly subepidermal (not more than 4, usually 2–3 host epidermal cells extending on clypeus), perimeter line 500–900 x 300–500 µm, clypeus 400–650 x 150–300 µm in extent, opening by a single longitudinal split (TABLE II). Fewer than 5% of needles exhibiting one or two ascomata on the edge of the stomatal side. Lips inconspicuous under dissecting microscope at low magnification but visible in thin sections under compound microscope at higher magnification; in midpoint vertical section partly subepidermal, more than eight epidermal cells being displaced and lying in a group on the basal wall, the clypeus with thin-walled staining cells in the region of the split, the rest of the clypeus becoming paler on either side and not extending as far as the basal wall, which is developed poorly. Paraphyses rare (seen only once), with hooked tips, septate, 70–110 x 2 µm. Asci 70–110 x 7–8 µm. Ascospores 55–80 x 2 µm, filiform, arranged in fascicles, sometimes helically coiled, slightly tapering toward the base, hyaline, aseptate; discharged ascospores covered with a gelatinous sheath 2–3 µm thick. Conidiomata not observed.
Cultures.. Isolated, single ascospores not germinating (21 C, daylight), germination obtained from groups of ascospores; isolates on MA slow growing (less than 3 mm/d), never reaching the edge of the 9 cm diam Petri dish, usually cream with some small brown or black spots (sclerotium 25–50 µm), irregular, farinaceous, thin, no black lines (S164, S171, S172).
Ecology.. Lophodermium macci has been observed only in collections from Canada and the United States from secondary needles of haploxylon (five-needle) pines, sporulating on fallen senesced needles that have overwintered in the litter. Lophodermium macci never has been observed on needles attached to the tree or has it been recovered as a foliar endophyte.
Hosts.. Haploxylon pines (Pinus strobus, P. monticola Douglas ex Don, P. koraiensis Siebold and Zucc).
Known distribution.. Canada (Québec, Ontario), USA (New York).
HOLOTY PE: CANADA. QUÉBEC: Valcartier, on senesced needle of Pinus strobus, 18 Dec 2001, Sokolski & Bérubé (QFB 14690).
Paratype specimens of L. macci. CANADA. QUÉBEC: Valcartier, on senesced needle of Pinus strobus, 18 Dec 2001, Sokolski & Bérubé (four specimens: QFB 14691, QFB 14692, DAOM 231108, IMI 390862).
Specimens of L. macci misidentified as L. pinastri in herbaria. QFB 7023 (P. monticola), QFB 1478 (P. strobus), QFB 7419 (P. strobus), DAOM 2095 (P. koraiensis).
Specimens of L. pinastri examined. DAOM 92984 (P. banksiana), IMI 11840 (P. sylvestris), IMI 234613 (P. sylvestris), IMI 243685 (P. mugo), IMI 243694 (P. mugo), IMI 247600 (P. ponderosa). All other specimens reported in Minter’s monograph (1981) are on 2- or 3-needled pines; these were not studied by us.
Specimens of L. pini-excelsae examined. DAOM 191554 (P. strobus, Canada), IMI 87030a (holotype, P. excelsa), IMI 139876 (P. excelsa), IMI 231775 (P. pumila), IMI 247583 (P. patula), IMI 247596 (P. strobus, Japan).
Specimens of L. staleyi examined. IMI 225067 (P. sylvestris).
Specimens of L. nitens examined. DAOM 182074, DAOM 166823, DAOM 182068, IMI 23092, IMI 23111, IMI 216495, IMI 337850, IMI 365033, IMI 365046.
Specimens of L. conigenum misidentified as L. pinastri. QFB 3484 (P. contorta), QFB 19633 (P. sylvestris), QFB 1481 (P. contorta), QFB 3445 (P. sylvestris), DAOM 115201 (P. banksiana), DAOM 86491 (P. banksiana).
Comments..
Lophodermium macci is characterized by the black ascomata that have a gray margin and a black perimeter line, partly subepidermal, lips inconspicuous, abaxial only; conidiomata not observed in nature; black zone lines present only when adjacent to another Lophodermium species; on haploxylon pines. The clypeus with thin-walled staining cells in the region of the split is similar to lip cells of L. pinastri (Schrad. ex Hook.) Chev. and L. pini-excelsae Ahmad. Cultures of L. macci are distinct from those of L. pinastri and L. pini-excelsae (as it was described by Minter 1981), neither of which has a farinaceous colony. Colony characters are not available for other species of Lophodermium. Lophodermium macci and L. nitens Darker have similar ecologies.
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DISCUSSION |
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, show similarities: the black clypeus with gray margin, a black perimeter line and the subepidermal nature. However L. macci can be differentiated by the abaxial position of the ascocarps, the inconspicuous lips at the macroscopic level, the poorly developed basal wall and the absence of conidiomata (TABLE II). The most useful characteristic for distinguishing L. macci from these species (L. pini-excelsae in particular) is the manner in which the ascocarp is embedded in the needle. This is the principal criterion separating L. pinastri from L. pini-excelsae used by Minter et al (1978). In this case the differences are the depth of embedding of the ascocarps. Lophodermium macci displaces two or three epidermal cells (rarely four) of the host on each side of the clypeus, while L. pini-excelsae displaces more than four, usually five. The number of epidermal cells left on the bottom of the ascocarp is five and more for L. pini-excelsae and eight and more for L. macci.
All the herbarium specimens of L. pinastri on P. strobus, P. koraiensis and P. monticola originating from Canada and the United States (QFB and DAOM herbaria) that we have examined so far were L. macci. Confusion in identification made before 1981 was due to the broad description found in the literature (Darker 1932). Minter (1981) clearly delineated L. pinastri, and none of the L. pinastri specimens he listed were on P. strobus or any other five-needle pine.
We observed that L. nitens, followed by L. macci, are the commonest Lophodermium species sporulating on senesced P. strobus needles in the litter in eastern Canada. The goal of this study was to link needle fungal endophytes with ascomata collections on senesced needles. L. nitens has been found recently as a needle endophyte on P. strobus (Bérubé unpubl, Deckert et al 2002), but despite numerous isolations, L. macci never has been isolated as an endophyte. Because ascocarps of L. macci are somewhat common on senesced needles, they either colonize the needles during or after senescence or may not grow readily from healthy green needles used to isolate endophytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: jberube{at}nrcan.gc.ca
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LITERATURE CITED |
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Cannon PF, Minter DW. 1986. The Rhytismataceae of the Indian subcontinent. Mycol Pap 155:i–vii, 1–123.
Carroll G. 1995. Forest endophytes: pattern and process. Can J Bot 73 (suppl. 1):S1316–S1324.
Darker GD. 1932. The Hypodermataceae of conifers. Contr Arnold Arbor 1:1–131.
Deckert RJ, Hsiang T, Peterson RL. 2002. Genetic relationships of endophytic Lophodermium nitens isolates from needles of Pinus strobus. Mycol Res 106:305–313.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791.
Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics Appl Notes 17:754, 755. (Software available at http://morphbank.ebc.uu.se/mrbayes/)
Johnston PR. 2001. Monograph of the monocotyledon-inhabiting species of Lophodermium. Mycol Pap 176:i–vii, 1–239.
Minter DW. 1981. Lophodermium on pines. Mycol Pap 147: 1–54 + 65 figures.
———, Staley JM, Millar CS. 1978. Four species of Lophodermium on Pinus sylvestris. Trans Br Mycol Soc 71:295–301.
Petrak F, Ahmad S. 1954. Beitrage zur Pilzflora Pakistanis. Sydowia 8:162–185.
Rambaut A. 1996. Se-Al: Sequence Alignment Editor. Available at http://evolve.zoo.ox.ac.uk
Spurr AR. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastr 26:31–43.
Swofford DL. 2001. PAUP: 4.8b10 test version 2001. Sun-derland, Massachusetts: Sinauer Associates.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press. p 315–322.
Zolan ME, Pukkila PJ. 1986. Inheritance of DNA methylation in Coprinus cinereus. Mol Cell Biol 6:195–200.
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