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Division of Applied Bioscience and Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Buk-gu, Gwangju 500-757, Korea
Jae Young Park
Hack Sung Jung 1
Department of Biological Sciences, College of Natural Sciences, Seoul National University, Kwanak-gu, Seoul 151-747, Korea
Richard C. Summerbell
Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands
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ABSTRACT |
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Two new epiphytes of anamorphic ascomycetes, Phaeomoniella zymoides sp. nov. and Phaeomoniella pinifoliorum sp. nov., were isolated from the needle surface of Pinus densiflora in Korea. The new taxa were characterized by acid-tolerant, slow, partially yeast-like growth and extensive production of emerging cells on convex wrinkled mycelial colonies. Phaeomoniella zymoides produced mycelium with large numbers of intercalary and lateral or terminal vesicles or swollen cells. Large conidiogenous cells had a swollen base and appeared to be phialidic, and many phialoconidia also were produced from lateral hyphal apertures. Maturing colonies of Ph. zymoides were made up of dark green to blackish areas and produced a Phoma-like synanamorph. Primary conidia became elongate mother cells giving rise to polar or lateral secondary conidia. Phaeomoniella pinifoliorum was characterized by reduced, swollen, phialide-like cells, lateral production of conidia from hyphae and terminal or subterminal, or less commonly lateral, secondary production of conidia from yeast-like primary conidia. When ITS and 28S rDNA sequences were compared and analyzed with those of best matching GenBank taxa, the Phaeomoniella group consisted of three lineages, "zymoides," "pinifoliorum" and "chlamydospora" clades, which again showed a complete sister relationship to Moristroma quercinum ined.
Key words: ITS, Moristroma, Phaeomoniella chlamydospora, Pinus densiflora, 28S rDNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant leaf surfaces are inhabited by diverse microorganisms, such as filamentous fungi, yeasts and bacteria (Preece and Dickinson 1971
, Goulder and Baker 1991, Andrews and Harris 2000, Lindow and Brandle 2003). Some microorganisms are beneficial to their hosts, while others are pathogenic. The leaf surface habitat, the phyllosphere, is colonized by these microbes, which are referred to as epiphytes (Leben 1965, Stockwell et al 1999, Lindow and Brandle 2003), while organisms growing within healthy leaves are referred to as endophytes (Bayman et al 1997, Azevedo et al 2000, Strobel and Daisy 2003). Farr et al (1989) listed 77 830 different epiphytic and endophytic plant-fungus combinations in the United States. Lindow et al (2002) reviewed the biology of phyllosphere fungi, addressing current topics of interest such as mutual interactions of leaf surface fungi, characteristics of endophytic and epiphytic colonization and the mechanism by which yeasts adhere to leaf surfaces.
Fungi associated with leaves are a topic of increasing interest in bio-exploitation and biodiversity studies. Among epiphytic microorganisms, many species produce biotic substances such as vitamins, auxin, folic acid, thiamine, riboflavin and other compounds, as well as antibiotic substances with strong antimicrobial properties (Krasil’nikov 1958). Some endophytes also produce bioactive substances that are involved in host-microbe relationships. For example Seimatoantlerium tepuiense Strobel, E.J. Ford, J. Yi Li, J. Sears, Sidhu & W.M. Hess (a coelomycetous anamorph related to the xylarialean genera Broomella and Pestalosphaeria) from the Guayana region of Venezuela is a unique epiphytic fungus reported to produce the anticancer agent taxol (Strobel et al 1999).
For many years extensive studies have been conducted on endophytic and epiphytic fungi associated with the leaves of conifers, including those of the Pinaceae, especially the species of Pinus L. (Millar 1980, Carroll 1986, Miller 1986, Bills 1996, Suryanarayanan et al 2002, Ganley et al 2004). Endophytes, microorganisms that reside in the tissues of living plants, are relatively unstudied and are potential sources of novel natural products for the exploitation in medicine, agriculture and industry (Strobel and Daisy 2003). The role of the specific fungi in keeping the host plants healthy has been described as mutualistic symbiosis (Millar 1980, Carroll 1986, Miller 1986, Ganley et al 2004). However in Korea epiphytes on the Pinaceae or of their interactions with pine pathogens have been little studied. In the course of investigating the occurrence of epiphytic fungi on various pine plants examined for quarantine purposes we have found a high occurrence of undescribed organisms, including two new species related to Phaeomoniella Crous & W. Gams (previously Phaeoacremonium W. Gams, Crous & M.J. Wingfield pro parte). Phaeomoniella is a genus recently established for Ph. chlamydospora (W. Gams, Crous, M.J. Wingfield & Mugnai) Crous & W. Gams that is one of the agents involved in causing Petri vine decline (PVD) in young grapevines (Crous et al 1996, Crous and Gams 2000, Groenewald et al 2000, Whiting et al 2001). The aim of the present study was to characterize these new epiphytes, all of which were isolated from healthy-looking needles of Pi. densiflora in Korea. Both phenotypic and ribosomal sequence characters were studied and analyzed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. In addition pH of broth medium was aseptically adjusted from 2 to 10 with 1 N HCl and NaOH solution after autoclaving. The solutions were filtered with a disposable filter (MFS 25 disposable syringe unit, Advantec MFS Inc., Japan).
Isolates.— –
Ten representative isolates were collected from 20 samples of 20 Pi. densiflora trees. They were stored in the collections of both SFC (Seoul National University Fungus Collection, Seoul, Korea) Herbarium and CBS (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) Fungal Biodiversity Centre as SFC AW203 (= CBS 114905), SFC AW304 (= CBS 114904), SFC BW301 (= CBS 114907), SFC BC208 (= CBS 114906) and SFC CW202 (= CBS 114903). Additional taxa from GenBank used in sequence comparisons are listed (TABLE I).
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) and water agar (WA, agar 20 g l–1 in distilled water). The strains were stored in a lyophilized condition as well as under liquid nitrogen. For descriptive and molecular studies the strains were maintained at 2–3 C before subculture. Detailed morphological studies were done in Riddell slide culture, as outlined by Gams et al (1998), on MEA and OA. For the studies on pH optima and tolerances strains were precultured in yeast-malt (YM) extract broth (same composition as YMA) on a rotary shaker (150 rpm) at 28 C 7–10 d. They were inoculated to liquid potato-dextrose (PD) broth adjusted at unitary intervals at pH 2–10. In a separate experiment temperature responses were tested on PDA at 5 C increments at 2–33 C. Mycelial growth of the isolates was recorded periodically by measuring diameters of the colony at right angles after they were incubated at given temperatures in the dark for up to 5 wk. All the experiments were carried out with two separate plates per treatment. Microscopy.— – In light microscopic observations of micro-morphological features a Nikon Labophot 2 Microscope (Nikon, Tokyo, Japan) and an Olympus BX50 microscope (Olympus, Tokyo, Japan) were used and both were equipped with differential interference contrast. The slide culture preparations for the fungal isolates were made from 10 d old growth on MEA and OA at 22 C. For scanning electron microscopy (SEM) samples were fixed in 2.5% paraformaldehyde-glutaraldehyde mixture buffer with 0.1 M phosphate (pH 7.2) for 2 h, post-fixed in 1% osmium tetroxide in the same buffer for 1 hr, dehydrated in graded ethanol and substituted by isoamyl acetate. They were dried at the critical point in CO2. Finally the sample was covered in gold in a sputter coater (SC502, Polaron, West Sussex, UK) and observed with a SEM515 scanning electron microscope (Phillips, Eindhoven, the Netherlands) and a S-3500 N low vacuum scanning microscope (Hitachi, Tokyo, Japan).
DNA extraction, amplification and sequencing.— –
Strains maintained at 2–3 C were inoculated into YM broth in 5 mL test tube and incubated at 25 C 4–5 d. The broth-cultured cells were transferred to 1.5 mL Eppendorf tube (Westbury, New York) and centrifuged at 5000 g 3 min. Distilled water (1.5 mL) was added, the pellet was resuspended by vortexing to mix until precipitant dissolves and the suspension was centrifuged again. The supernatant was removed carefully with a pipette and the pellet was harvested. Total genomic DNAs were extracted with AccuPrep® Genomic DNA Extraction Kit (Bioneer Corp., Daejeon, Korea). From extracted genomic DNA the 28S and internal transcribed spacer (ITS) 1 and 2 regions inclusive of the 5.8S rDNA were amplified with ITS1 and LR5 primers (White et al 1990) using Quick PCR Premix (GENENMED, Daejeon, Korea) containing Taq DNA polymerase, dNTPs, reaction buffer and tracking dye (GENENMED, Daejeon, Korea). PCR reactions were conducted with 30 thermal cycles according to these conditions: 1 min at 95 C for denaturation, 1 min at 52 C for annealing, 1 min at 72 C for extension and 10 min at 72 C for terminal extension. Amplified PCR products were detected on 0.75% agarose gel through electrophoresis. Checked amplicons were purified with AccuPrep® PCR Purification Kit (Bioneer Corp., Daejeon, Korea). The purified PCR products were sequenced with an ABI 3700 automated DNA sequencer (Applied Biosystems Inc., Foster City, California). For sequencing ITS and 28S regions, primer pairs ITS4 and LR3 (White et al 1990) were used.
Phylogenetic analyses.— –
Sequences generated for the 10 representative isolates of new taxa were aligned with sequences obtained from GenBank using Clustal X version 1.83 (Thompson et al 1997) (gap opening penalty = 10.0, gap extension penalty = 0.02). Using PHYDIT version 3.2 (Chun 1995), ambiguous and uninformative variable sites were excluded and a sequence dataset was submitted to subsequent phylogenetic analyses. Phylogenetic analyses were performed based on the parsimony analysis of PAUP 4.0b10 (Swofford 2002) using tree bisection reconnection (TBR) branch swapping with MAXTREES unrestricted. All gaps were treated as missing data. Degrees of similarity between the sequences were analyzed with the similarity comparison feature of PHYDIT. The aligned sequences were deposited at TreeBase.
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RESULTS |
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–2, FIGS. 3–11
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Colonies attaining a diameter of 18 mm at 22 C in the dark after 3 wk on PDA medium, deeply folded and wrinkled with radial striations extending to the lobate margin; surface coloration featuring extensive patches of greenish to black intermixed with pale patches; surface texture moist to mucoid (FIG. 1
). Hyphae beginning hyaline, mostly becoming melanized greenish to black in later development, frequently producing vesicular cells 5.7–7.2 µm wide singly or in relatively widely spaced chains. Little or no aerial mycelium formed except for occasional occurrence in older cultures (FIG. 1). Phialides formed mainly in areas of toruloid mycelium as swollen intercalary cells of 2.5–8.0 x 2.5–4.0 µm with one medially or subterminally situated, slightly protuberant fertile aperture or rarely two subterminal apertures, less commonly formed as intercalary cells in otherwise undifferentiated hyphae, or as discrete, hyaline side branches or terminal cells, frequently wholly swollen or with a swollen base, 2.5–8.8 x 2.5–3.5 µm (FIGS. 3–11). Larger conidiogenous cells with a swollen base appearing to be phialidic and many phialoconidia produced also from lateral hyphal apertures (FIG. 8). Conidia unicellular, hyaline, slender, rod–shaped to narrowly ellipsoidal, sometimes flexuose to allantoid, (2.6–) 3.5–6.0 (–7) x 0.8–1.9 µm. Primary conidia expanding after detachment, mostly becoming broadly cylindrical to long-ellipsoidal, 5.5–8.5 x 1.2–2.5 µm, sometimes 1- or 2-septate and extending up to 16.4 µm long, transforming into yeast mother cells giving rise to polar or lateral secondary conidia identical to primary conidia in form. Secondary conidiation process producing a yeast-like layer of detached cells held in copious slime. Vesicular chlamydospores abundant, hyaline to brownish, globose to subglobose, occurring singly or in chains, often found in interwoven clumps (FIGS. 8–11). Optimal growth at 17–20 C, strongly inhibited but not eliminated at >30 C, and moderate at 4 C. In acidified medium growth occurring at pH 3 but not at lower pH (TABLE II).
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Habitat. – Phylloplane of pine leaves.
Distribution. – Known only from the type locality.
Specimens examined. – KOREA: Mount Juwang, Gyungsangbug-do, from the needles of Pi. densiflora, 24 Jan 2004, H.B. Lee (HOLOTYPE: SFC P00130 [GenBank] ); ex-type culture CBS 114904 (= SFC AW304); additional specimens from the same site and substrate: SFC AW203 (= CBS 114905), SFC AW304 (= CBS 114904), SFC BW301 (= CBS 114907), SFC BC208 (= CBS 114906).
Commentary. –
After 21 d in culture Ph. zymoides isolates yielded a synanamorph suggestive of Phoma Sacc. on OA. Some days later such structures also were seen on MEA. Pycnidia were single or in a clump of up to three appressed individuals, subglobose, obscurely papillate, dark, 130–240 x 130–220 µm, with a moderately thin wall of textura epidermoidea externally ornamented with a few stiff, outwardly directed, hyaline, irregular, thick-walled, blunt-ended, unbranched or once-branched sterile hairs up to 35 µm long; ostioles roundish, surrounded by a ring of relatively heavily melanized cells; phialides hyaline, subglobose or coniform, 2.5–5 x 1.5–3 µm, giving rise to conidia of 2.5–4.5 (–6) x 1.0–1.7 µm, consistent with the mycelial conidia in shape but with a relatively pronounced tendency to be slightly curved. A Phoma-like synanamorph as above also has been described from Ph. chlamydospora in growth on carnation leaf agar (Crous and Gams 2000).
The extensive patches of greenish to black mycelium of Ph. zymoides were suggestive of the so-called "black yeasts" (de Hoog et al 2000), particularly of genera such as Aureobasidium Viala & G. Boyer in which melanized and whitish patches may co-occur. Colony colors varied according to the incubation time and type of used medium. Like Aureobasidium our Phaeomoniella isolates also showed a patchy appearance at the yeasty or slimy areas of the colony. Phaeomoniella zymoides is readily distinguished from Aureobasidium in that it has phialidic conidiogenesis while the latter produces synchronous blastoconidia. The most morphologically similar fungi belong to four phylogenetically distant groups, namely Hyphozyma de Hoog & M.T. Smith, Lecythophora Nannfeldt, the members of the Phialemonium curvatum W. Gams & W.B. Cooke complex (Proia et al 2004), and the unnamed anamorph of the discomycete Tromeropsis microtheca (P. Karsten) Sherwood (Weber 2002). The first of these is morphologically distinct in producing colonies with orange to pinkish primary growth and blastoconidia from lateral openings on otherwise undifferentiated hyphal cells. Lecythophora spp. and Ph. curvatum complex members are distinguished mostly by their regular production of more or less uniformly curved mycelial conidia from adelophialides integrated into nontoruloid hyphae and occasionally or frequently from relatively elongated discrete phialides. Many species have one or more special features that are not found in Ph. zymoides, such as orange colony coloration (L. hoffmannii [J.F.H. Beyma] W. Gams & McGinnis), small, dark chlamydospores (L. mutabilis [J.F.H. Beyma] W. Gams & McGinnis), or a sporodochial synanamorph (Ph. curvatum sensu lato pro parte) (Proia et al 2004). The Ph. curvatum complex lacks the yeast-like secondary conidiation. The Tromeropsis (P. Karsten) Sherwood anamorph produces distinctly curved blastoconidia from otherwise undifferentiated hyphal cells.
Phaeomoniella zymoides shows a degree of similarity with the closely related Ph. chlamydospora (previously Phaeoacremonium chlamydosporum W. Gams, Crous, M.J. Wing. & Mug.). It looks similar in its overall cultural growth on PDA and OA, especially in its production of greenish to dark colonies in extended growth. The white, yeast-like colonies of Ph. zymoides on YMA and YpSs media, however, have no equivalents in Ph. chlamydospora. Phaeomoniella zymoides has lightly melanized, 1–2-celled vesicular chlamydospores, which are different from the dark chlamydospores occurring sparsely to commonly in Ph. chlamydospora. The occurrence of chlamydospores, the sharp delimitation in pigmentation of conidiophore stipes against the hyaline phialides, and consistently straight conidia were considered as significant characters supporting the distinction of Phaeomoniella from Phaeoacremonium, after molecular studies had shown that these fungi were not closely interrelated (Dupont et al 1998, Groenewald et al 2001). The character of chlamydospore formation observed in Ph. chlamydospora was shared with Ph. zymoides. However the mostly integrated or, when discrete, small, broad-based phialides of Ph. zymoides markedly differed from the elongate-ampulliform to subcylindrical phialides of Ph. chlamydospora. Also Ph. zymoides formed no structure reminiscent of the contrastingly melanized conidiophores of Ph. chlamydospora. As with Ph. chlamydospora Ph. zymoides has no known teleomorph but the production of a Phoma synanamorph is common to both species.
Two conidial forms were observed in association with the mycelium of Ph. zymoides colonies: relatively small, newly formed oblong to narrow-ellipsoidal conidia, which were the predominant type, and larger, somewhat broadly cylindrical to ellipsoidal conidia that were in the process of transformation to become mother cells of secondary conidia after detachment. The conidia of Ph. chlamydospora, measuring (1.5–)3.0–4.0(–4.5) x 1.0–1.5(–2.0) µm, are slightly smaller than the primary conidia, mostly 3.5–6.0 x 0.8–1.9 µm, of Ph. zymoides, and the mixture of the conidia that measured 5.5–8.5 x 1.2–2.5 µm and differentiated as yeast cells in Ph. zymoides is not seen in Ph. chlamydospora.
Phaeomoniella zymoides grew well at rather low temperatures of 12–18 C as well as at relatively strongly acidic pH 3–4 and thus can be characterized as acid-tolerant (FIG. 2, TABLE II). Preliminary studies showed that the organism grew well on all of the various growth media in this study. Phaeomoniella zymoides has been isolated frequently from pine tree needles with acidic YM medium and dilution plating. When the growth in YM broth was tested at pH 2–10, Ph. zymoides was shown to have a broad spectrum of tolerance, with optimal growth at pH 5–7 but also with extended growth at an acidic pH 3. The fungus, however, barely grew at more than pH 8 (TABLE II).
Phaeomoniella pinifoliorum H.B. Lee, J.Y. Park, R.C. Summerbell & H.S. Jung, sp. nov. FIGS. 1–2, FIGS. 12–17
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Colonies slowly growing, reaching 16 mm diam on PDA medium at 22 C in the dark after 3 wk; surface consisting of flat, whitish patches of mucoid, yeasty material intermingled with patches, developing convex wrinkled growth, and with sparse patches of aerial mycelium at the center in mature colonies; margins radially striate on the surface, lobate at the edge. Mycelium densely interwoven, forming abundant, globular to ovoid, intercalary or terminal vesicles. Phialides commonly simple, inflated, forming slightly papillate lateral branches, 2.5–9.0 x 2.5–3.5 µm but also integrated as swollen cells into toruloid hyphal segments, and less commonly as uninflated hyphal cells with a fertile aperture; fertile apertures occurring terminally, subterminally or laterally on discrete phialides and medially or subterminally on integrated hyphal cells, often without an obvious collaret but possessing a discernible small collaret in some cases. Conidia hyaline, cylindrical to ovoid, often slightly curved to allantoid, (2.5–) 3–4.5 x 1.7–2.3 µm when newly mature, expanding to become potentially reproductive yeast-like cells after dehiscence, usually remaining unicellular but sometimes becoming 1–2-septate, measuring 5.7–15 x 1.6–2.1 µm. Secondary conidiation common, with the yeast-like conidia arising mostly from the obscure phialidic apertures at the ends of primary conidia. Vesicular chlamydospores abundant, hyaline or subhyaline, globose to subglobose, formed singly or in linear or branched chains of up to seven pieces. Optimal growth at 20–22 C, almost inhibited in growth at >33 C. Growth occurring at 4 C and down to an acidic pH 3 (TABLE II).
Etymology. – The specific name "pinifoliorum" indicates "from the needles of pine trees".
Habitat. – Phylloplane of pine leaves.
Distribution. – Known from the type locality as yet.
Specimens examined. – KOREA: Mount Juwang, Gyungsangbug-do, from the needles of Pi. densiflora. 20 Jan 2004, H.B. Lee (HOLOTYPE: SFC P00327 [GenBank] ); ex-type culture CBS 114903 (= SFC CW202).
Commentary. –
Dark, rounded structures suggestive of the development of a pycnidial synanamorph were observed in CBS 114903 after the revival from lyophilization and growth for 30 d on OA. Maturation of structures as functional pycnidia has not been observed. Small areas of darkened mycelium were seen in areas where pycnidium-like structures were formed. The original isolation of Ph. pinifoliorum was on acidic YM medium, and the species grew particularly well on this medium that contains acidic carbon sources including succinic and aspartic acids. Like Ph. zymoides Ph. pinifoliorum grew favorably at low temperatures of 12 to 18 C and at relatively strongly acidic conditions at pH 3–4 (FIG. 2, TABLE II). Unlike the two isolates of Ph. chlamydospora tested in this study, which have optimal growth at 25 C, Ph. pinifoliorum showed an optimal growth at ca. 21 C. Phaeomoniella pinifoliorum showed similarity with Ph. chlamydospora in slow cultural growth, predominantly small, oblong to ellipsoidal conidia, the tendency for discrete conidiogenous cells to be basally inflated and the differentiation of vesicular chlamydospores. However Ph. chlamydospora lacks the tendency for conidia to expand after dehiscence and become elongated, sometimes allantoid, yeast cells. Whereas Ph. chlamydospora forms aerial mycelium abundantly Ph. pinifoliorum produced little or no aerial mycelium in young colonies even under near-UV light. Phaeomoniella pinifoliorum, nevertheless, produced a modest amount of aerial mycelium in later growth at near-UV wavelength.
In comparison to the morphologically somewhat similar Ph. zymoides, Ph. pinifoliorum showed several differences. The most striking difference was the degree of colony melanization and Ph. pinifoliorum formed no dark patches, except in small areas where pycnidium-like initials were formed, thus remaining pale on most media. The conidia, conidiogenous structures and vesicular chlamydospores formed by both species were relatively similar in size and shape. However Ph. pinifoliorum grew more slowly than Ph. zymoides, which was reflected in a slight difference in colony diameters as noted in descriptions (18 mm for Ph. zymoides and 16 mm for Ph. pinifoliorum in 3 wk). Like Ph. zymoides, Ph. pinifoliorum had a broad growth spectrum of pH 3–7 and was able to grow at pH 3 but was hardly able to grow at pH 8 or above (TABLE II).
Sequence analyses.— –
The alignment of ITS1, ITS2 and 5.8S rDNA sequences included 570 nucleotide positions. Maximum parsimony analyses resulted in two most parsimonious trees (tree length = 1315, CI = 0.575, RI = 0.811, RC = 0.466, HI = 0.425) of which one is shown (FIG. 18). In addition maximum parsimony analysis of 523 nucleotides from the alignment of 28S rDNA sequences resulted in a single parsimonious tree (tree length = 456, CI = 0.612, RI = 0.688, RC = 0.412, HI = 0.388) (FIG. 19). The results of these analyses, along with our observations on phenotypic characters of the isolates, indicated that all isolates listed under the name of Ph. zymoides were closely related to one another, forming a clade labeled "Clade zymoides" that was fully supported by a 100% bootstrap value in both trees. They were well distinguished from other Phaeomoniella species as a separate clade and also were different from Ph. chlamydospora by 83.5–83.7% and 86.0% respectively in ITS and 28S sequence similarities.
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DISCUSSION |
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The abundance of these Phaeomoniella isolates may vary depending on locality, leaf age and environmental conditions such as pH, temperature and water potential. Little information is available on the pH of the foliage surfaces for most conifers, but it is well known that the wood of Picea has low pH values and this also seems to be the case for Pinus (Gourbière 1975). In general it is known that the numbers of both phylloplane and endophytic fungi of Pinus species increase as the needles age (Mishra and Das 1981). Woo et al (1998) reported that, when the leaves of a Korean pine species, Pi. koraiensis, were immersed in an aqueous solution at pH 3.0–6.0, the equilibrium pH at 48 h was 3.5–4.5 and was significantly lower than pH 5.0–6.0 observed for Zelkova serrata, Robinia pseudoacacia, Quercus acutissima and Prunus serrulata. Nowadays the abundance of acid-tolerant species might have been affected by low pH due to acid rain. In this study the average percentage of incidence of Ph. zymoides was approximately 4.5%, but the incidence increased to 25% in January, showing that the fungus could be potentially psychro-tolerant. Phaeomoniella pinifoliorum was found only once in this study and this isolation also occurred in January.
Phaeomoniella chlamydospora originally was described as a species of Phaeoacremonium, but after Dupont et al (1998) reported that the species was phylogenetically unrelated to other Phaeoacremonium species, a new monotypic genus Phaeomoniella was introduced by Crous and Gams (2000) to accommodate the species. Phaeomoniella was confirmed as distinct from Phaeoacremonium based on ITS and ß-tubulin sequence data and classified in the Chaetothyriales (Groenewald et al 2001). The distinction was reinforced further after Phaeoacremonium was discovered to be connected to the time-honored teleomorphic genus Togninia Berlese that is considered to be related to the order Calosphaeriales (Scheck et al 1998, Tegli 2000, Tegli et al 2000a, Tegli et al 2000b, Mostert et al 2003). However Réblová et al (2004) have shown that Togninia belongs to the Diaporthales rather than to the Calosphaeriales and described a new family Togniniaceae to accommodate it. To date no teleomorph directly connected to Phaeomoniella is known yet, and the taxonomic relationship of the genus requires further investigation.
The relative phylogenetic proximity of our new taxa to Ph. chlamydospora is poorly reflected in morphological features. In particular the elongate and particolored conidiophores of Ph. chlamydospora, despite the basal swelling seen in the phialides of this organism, do not convincingly resemble the highly reduced, vesicular phialides and minimally visible intercalary conidiogenous openings of Ph. zymoides and Ph. pinifoliorum. Our phylogenetic analysis of ITS sequences groups the single available isolate of Ph. pinifoliorum somewhat closer to Ph. chlamydospora than to Ph. zymoides. A more exact determination of the phylogenetic relationships of Ph. pinifoliorum certainly will require further studies on additional strains.
It is relatively common in modern phylogenetic studies that anamorphic species of reduced morphological complexity are included in genera that primarily contain closely related species forming more elaborate structures (Burnett 2003). For example the anamorph of Pseudeurotium desertorum Mouchacca, which produces only solitary aleurioconidia on short stalks was placed into a new phylogenetically delimited genus, Teberdinia Sogonov, Summerbell & Schroers, that included various anamorphs of Pseudeurotium as well as a species with no known teleomorph (Sogonov et al 2005). Teberdinia anamorphs produce complex branching conidiophores with both discrete and intercalary conidiogenous cells that show a Sporothrix-like, sympodial, conidiogenous process (Sogonov et al 2005). Other anamorphs are simpler in construction, but Ps. desertorum is distinctive in being so highly simplified that it certainly could have been placed into a separate genus in conventional morphological taxonomy. Similarly, Onychocola, characterized by the distinctive, robust, erect arthroconidial chains of the type species Onychocola canadensis Sigler & Congly, was extended to include some Malbranchea-like anamorphs that fell into the same phylogenetic group along with teleomorphs of Arachnomyces (Sigler et al 1994). In the current taxonomy of fungal anamorphs it is increasingly difficult to conserve generic concepts for simplified or reduced morphological forms that phylogenetically group with more elaborate types with same basic ontogenetic structures.
Phaeomoniella chlamydospora, as an endophyte of woody stems, might have evolved under ecological pressures differing from those that brought Ph. zymoides and Ph. pinifoliorum into an evolutionarily convergent similarity as epiphytes. The two pine-inhabiting taxa of reduced form might represent a plesiomorphic morphology, as suggested by the closer relationship of Ph. pinifoliorum to Ph. chlamydospora than to Ph. zymoides, whereas Ph. chlamydospora might represent an apomorphic, newly elaborated, evolutionary derivative. Our sequences showed a clear relationship, at least in the ITS region, between our two Phaeomoniella clades and the undescribed Moristroma quercinum ined. that is known only from GenBank and thus notionally connected with the genus Moristroma. More distantly, our isolates had a degree of affinity with Capronia Sacc. and other typical members of the family Herpotrichiellaceae, including anamorphic taxa such as Cladophialophora Borelli and Exophiala J.W. Carmichael. In this context it is interesting that Ph. zymoides had, in part, a black, viscous colony appearance somewhat reminiscent of the "black yeast" members of the Herpotrichiellaceae (Kocková-Kratochvílová 1990, Guarro et al 1999, Groenewald et al 2001). Discomycetous fungi such as the members of Cadophora Lagerberg & Melin (related to the teleomorphs of Mollisia [Fr.] P. Karsten), which regularly emerged in NCBI BLASTN searches done with ITS1, ITS2 and 5.8S rDNA sequences of Ph. zymoides and Ph. pinifoliorum, grouped more distantly than with the members of the Herpotrichiellaceae.
At present we have no information about whether these fungi are found only in the phylloplane as epiphytic colonizers or they are able to act as endophytes or as pathogens in pine trees. To determine this the pathogenicity and host range of Ph. zymoides and Ph. pinifoliorum need to be studied soon. Moreover quantitative and qualitative compositions of the whole phylloplane mycota on pine trees also need to be investigated. Further ecological studies on epiphytes and endophytes of the Pinaceae are definitely needed, especially on the bioactivities of phylloplane fungi in respect to pine pathogens, and more fungi in this group need to be discovered and evaluated before comprehensive conclusions can be established.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: minervas{at}snu.ac.kr
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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