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Eupenicillium bovifimosum, a new species from dry cow manure in Wyoming

     Department of Botany, University of Wyoming, Laramie, Wyoming 82071

Jens C. Frisvad

     Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION LITERATURE CITED

 

A new species, Eupenicillium bovifimosum, was isolated from dry cow manure collected in Wyoming, USA. The outstanding morphological characteristics of this species are its robust, dense penicilli bearing long, broad columns of conidia, and its smooth-walled, unflanged ascospores which are produced within 3 wk of inoculation onto MEA. The new species produces penicillic acid, a fumagillin-like molecule, patulodin-like compounds CK2108A and CK2108B, and a compound resembling 2-[(2-hydroxypropionyl)amino]benzamide. Phylogenetic analysis using 1100 bases of the ITS and LSU DNA regions indicates that E. bovifimosum is most closely related to E. baarnense, and also to E. crustaceum, E. egyptiacum, and E. tularense.

Key words: dung fungi, ITS, LSU DNA, Penicillium, phylogeny, secondary metabolites, systematics, Trichocomaceae

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION LITERATURE CITED

 
During an investigation of soil microfungi, a middle school student isolated what appeared to be a new species of sclerotial Penicillium. The fungus was characterized by robust penicilli that produced prodigious numbers of blue-gray conidia in long, dense columns. The appearance of ascospores within three weeks was surprising, given that robustness and prolificacy are exceptional within Eupenicillium. The species is described as new, and compared on the basis of morphology, secondary metabolites and rDNA sequences to other Eupenicillium species (Gochenaur and Cochrane 1986 , Pitt 1979 , Stolk and Samson 1983 , Takada and Udagawa 1983 , Ueda 1995 ).

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION LITERATURE CITED

 
Dry cow manure and soil from beneath it were collected and placed into a plastic bag. Samples were refrigerated for two wk prior to use. The manure and soil, although stored together, were analyzed separately. All fungi were isolated from Martin's medium (Martin 1950 ) and transferred to PDA tubes. The new fungus was found twice, in the manure sample only.

Morphological characterization of the new Eupenicillium strain followed the protocol of Christensen et al (1999) . Morphological features are described from 6 to 10-d-old cultures grown on MEA at room temperature (20–24 C). Capitalized color names, followed by plate number, are those of Ridgway (1912 , and see Christensen et al 1994 ). The SEM photograph was obtained by squashing fresh cleistothecia onto double sticking tape mounted on an aluminum stub. The sample was coated with gold and examined with a Jeol JSM-5800LV scanning electron microscope.

For secondary metabolite analysis, isolates of E. bovifimosum, E. baarnense (NRRL 2086, CBS 339.61, CBS 105.68, CBS 315.59), P. matriti (NRRL 3452, CBS 347.61, IMI 96506, IBT 3320, IBT 15079, CBS 170.81) and P. concentricum (CBS 477.75, CBS 191.88, IMI 293197, IBT 20230, IBT 20229) were grown on CYA, MEA, YES and oat meal agars in the dark at 25 C (Samson et al 2000 ). The isolate of E. bovifimosum and NRRL 2086, CBS 105.68, CBS 315.59, NRRL 3452, CBS 191.88, IBT 20230, IBT 20239 and CBS 170.81 were extracted and analyzed according to the micro-scale agar plug HPLC method of Smedsgaard (1997) . NRRL 2086, NRRL 3452, CBS 170.81, CBS 191.88 and the remaining isolates were extracted and analyzed according to the HPLC method of Frisvad and Thrane (1987) . Briefly, in both methods the fungal material was extracted with methanol-dichlormethan-ethyl acetate (1:2:3) containing 1% formic acid. In the micro-scale method small agar plugs are extracted using ultrasonication and for the older method the whole content of the Petri dishes was extracted in a Stomacher-type blender. After filtration and evaporation of the organic solvent the dried extract was dissolved in methanol, filtered, and injected into the HPLC. The extracts were analyzed using reversed phase 100 mm x 4 mm columns at 40 C, using an acetonitril-water gradient. Both eluents contained triflouroacetic acid. All spectra were monitored at 225 nm, but UV spectra of all compounds detected were recorded from 200–600 nm. The data of metabolites detected were compared to both retention time, retention indices and UV spectra of standards, including penicillic acid (Sigma-Aldrich, USA), fumagillin (Leo Pharmaceuticals, Ballerup, Denmark), barnol (DC Aldridge, ICI, Macclesfield, Great Britain), patulin (Sigma-Aldrich, USA), CK2108A and CK2108B (from LI Kruse, Chektec Corp., USA) and 2-[(2-hydroxypropionyl)amino]benzamide (MC Dai, Université de Neuchâtel, Switzerland).

DNA was extracted following the protocol of Lee and Taylor (1990) , with two additional phenol-chloroform extractions and a final extraction using chloroform: isoamyl alcohol (24:1). Nuclear DNA was amplified using the primers ITS5 (White et al 1990 ) and LR3 (Vilgalys and Hester 1990 ). The 100 µL PCR reactions consisted of: 1 x buffer (Sigma; 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin), 2 nmol dNTPs, 50 pmol each primer, 2.5 units Taq polymerase and 50–100 ng template DNA. Thermal cycling parameters were: initial denaturing for 2 min at 96 C, followed by 25 cycles of 1 min at 96 C, 1 min at 53 C and 2 min at 72 C, and a final elongation at 72 C for 7 min. Amplification products were viewed and quantities were estimated on 1.5% agarose stained with ethidium bromide. Residual primers, salts, and dNTPs were removed using Centricon 100 filters (Millipore Corp., Bedford, Massachusetts). Sequencing was done by Davis Sequencing, Davis, California, using primers ITS5 and ITS4 (White et al 1990 ) for the ITS region and LS1 (Hausner et al 1993 ) and LR3 for the LSU region.

The sequence alignment included all Eupenicillium species available from GenBank, plus three species of Penicillium that have either similar morphology or a similar sequence in the ITS-LSU region (Table I , Peterson 2000 ). Several taxa were investigated for the outgroup, based on previous studies of Trichocomaceae (Berbee et al 1995 , Ogawa et al 1997 , Tamura et al 2000 ), but members of Dichlaenoideae failed to provide support for monophyly of the ingroup (bootstrap values much less than 50% for Eurotium rubrum, Neosartorya fischeri and Monascus purpureus, singly or in combination). The final selection included Talaromyces bacillisporus (Trichocomoideae) and Monascus purpureus. These two species together provided much better ingroup support than did T. bacillisporus alone.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION LITERATURE CITED

 
Eupenicillium bovifimosum Tuthill et Frisvad, sp. nov.

Figs. 1–4 .

View larger version (78K): [in this window] [in a new window]    FIGS. 1, 2. Penicilli and conidia of Eupenicillium bovifimosum grown on malt for one week. FIGURE 1. x640. FIGURE 2. x1600. FIGURE 3. Asci and ascospores from six week old culture, x1600

View larger version (146K): [in this window] [in a new window]    FIG. 4. SEM of ascospores, x7400.

Conidiophorae superficie agari vel mycelio aerio exorientes, ferentes columnum unicam latam conidiorum. Stipites 50–150 µm alti et 3.5 µm lati, glabrotunicati; metulae cuneiformes, 10–13 x 3.5–5.0 µm, dense contiguae in verticillis usque 6–7, interdum septatae et longiores; phialides densissime contiguae, uniformes, 7 x 2.3–2.5 µm; conidia globosa, glabrotunicata, portata in columnis 25–30 µm latis et usque 600 µm longis. Cleistothecia abundantia, prima pallida, brunnescentia, 100–160 mm in diametro, intra hebdomades tres maturescentia; ascopsorae 3.3–4.0 x 2.7–3.0 µm, laeves, carentes cristis aequatoriis.

Isolata e sterco sicco vaccae in Wyoming, USA.

Conidiophores (Fig. 1 ) arising directly from the agar, or from aerial mycelium at colony center, bearing a single broad column of conidia which extends for greater length than the conidiophore. Stipes 50–150 µm tall by 3.5 µm in width, smooth-walled; metulae (Fig. 2 ) short, broad and densely packed in whorls of 6–7 members, 10–13 x 3.5–5 µm, wedge-shaped, occasional metulae septate and longer or giving rise to additional metulae; phialides very densely packed, often arising from the side of the vesicle and in that case sometimes nearly perpendicular to the conidiophore axis, uniform in size, 7.0 x 2.3–2.5 µm; conidia (Fig. 2 ) globose, appearing smooth in liquid mounts but faintly wrinkled in air, 2.2–3.0 µm in diameter, forming compact columns 25–35 µm wide, commonly 350 µm and up to 600 µm in length.

Cleistothecia abundant, at first cream-colored but quickly becoming brown, 100–160 (200) µm in diameter, maturing within 3 wk, surface consisting of polygonal, pseudoparenchymatous cells; asci in helical chains, 6–7 µm in diameter; ascospores (Figs. 3 and 4 ) 3.3–4.0 x 2.7–3.0 µm, smooth-walled, equatorial flanges visible on immature ascospores but absent at maturity.

Cultural features – MEA. Colony 16–21 mm in diameter after 7 d at room temperature. Cleistothecia abundant, intermixed with conidiophores and overgrown with aerial mycelium at colony center; some sectors producing fewer cleistothecia and more conidiophores; conidia blue-gray, near Dark Glaucous Gray (XLVIII); reverse dirty yellow near Olive Ocher (XXX), yellow pigment diffusing into the surrounding medium.

CYA. Colony 25–29 mm after 7 d. Mycelium white; cleistothecia at first not apparent, but present beneath conidia and abundant pale yellow exudate, eventually becoming conspicuous and causing the colony to become dark brown; reverse pale at 7 d, becoming brownish orange near Tawny (XV) after two weeks.

CYA 37C. Colony 6 mm in diameter after 7 d; light yellow pigment diffusing into surrounding agar.

The outstanding characteristics of this species, which together make it unique among described species of Eupenicillium, are its production of robust conidiophores and dense penicilli consisting of 6 or 7 metulae and very densely packed phialides, and production within 21 d of ascospores which are smooth and lack flanges or furrows.

Specimens examined. HOLOTYPE. USA. WYOMING: Albany Co. RMF 9598, in the Laramie Range, approximately 11 miles east of Laramie, Wyoming, isolated from sample of dry cow manure by Mr. Tomy Jansen, September 1999, elevation 7800 ft (2400 m), 41°16.3'N, 105°18.1'W. ISOTYPE. RMF 9599. Living cultures of RMF 9598 have been deposited at Centraalbureau voor Schimmelcultures (CBS 102825).

Etymology. bovi = cow, fimosum = dungy, describing the habitat from which it was isolated.

The PCR amplification product was approximately 1100 bases long, and contained the complete ITS1, ITS2, and 5.8S rDNA regions and about 600 bases of the LSU DNA gene. The final alignment (TreeBASE number S629) included 1193 sites, of which 138 were variable but phylogenetically uninformative and 176 were informative. Tree length distribution, based on 100 000 randomly generated trees, was significantly skewed to the left (g1 = -0.6795, P < 0.01; Hillis and Huelsenbeck 1992 ), indicating strong phylogenetic signal in the data set. Phylogenetic analysis yielded three equally parsimonious trees of 717 steps (Fig. 5 ).

View larger version (32K): [in this window] [in a new window]    FIG. 5. Strict consensus of the three equally most parsimonious trees. Tree length = 718. For the original trees, length = 717, CI = 0.5941, RI = 0.6745, RC = 0.4007. Bootstrap values (1000 reps) of 60% or greater are shown. Bar = 10 nucleotide changes

Eupenicillium bovifimosum produced penicillic acid, a fumagillin-like compound, the patulodin-like compounds CK2108A and CK2108B, a compound with UV spectrum resembling 2-[(2-hydroxypropionyl)amino]benzamide, and some unknown indol alkaloids. Eupenicillium baarnese produced penicillic acid, the fumagillin-like compound, barnol and a compound with UV spectrum similar to the "Raistrick phenols" (Andersen 1991 ). Penicillium matriti also produced penicillic acid, but the fumagillin-like compound was produced by only a single strain (IBT 15709). In addition, all strains of P. matriti produced the unknown metabolite "A." The patulodin-like compounds CK2108A and CK2108B were consistently produced by P. concentricum, as was patulin.

The so-called fumagillin-like compound had a UV resemblance to fumagillin, but the retention index (RI) was much higher (RI 1547 as compared to RI 1193 of fumagillin) and the new compound had UV maxima at 296 (shoulder), 311 and 320 nm, different from those of fumagillin (UV maxima at 322, 336 and 351). Some isolates of P. matriti and E. baarnense produced a compound with a UV spectrum similar to that at RI 1547, but at RI 1338 (NRRL 2086, IMI 96506, and IBT 3320). The compound at RI 1547 was only produced by E. bovifimosum, the ex-type culture of E. baarnense (NRRL 2086) and a single strain of P. matriti (IBT 15709).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION LITERATURE CITED

 
According to the generic subdivisions of Stolk and Samson (1983) , E. bovifimosum should be placed into section Javanica, which includes the Eupenicillia that lack equatorial ridges on the ascospores. The species included in that section, E. inusitatum and four varieties of E. javanicum, have rough or spinose ascospores, with the exception of E. javanicum var. levitum. That taxon, more commonly called E. levitum (Pitt 1979 , Pitt and Samson 1993 ), has ascospores very similar to those of E. bovifimosum in size, shape and smoothness of the surface. Although their conidiophores may be biverticillate, as well as monoverticillate, E. inusitatum, E. javanicum, and E. levitum have more delicate and irregular penicilli than E. bovifimosum. The conidiophores of the latter are more similar to those found within Stolk and Samson's section Eupenicillium, particularly those of E. baarnense and E. shearii, both of which may have whorls of up to five metulae. However, these species are not as burly as E. bovifimosum, nor do they compare in details of conidial size and shape, metula shape, or size, shape and ornamentation of the ascospores.

Pitt's (1979) classification of the Eupenicillia, based on growth rates and morphological characters, can accommodate E. bovifimosum in series Crustacea. Inclusion in Crustacea suggests a relationship to E. crustaceum, E. egyptiacum, E. baarnense, E. molle Malloch & Cain and E. shearii.

The phylogenetic analysis indicates that E. bovifimosum is indeed most closely related to E. baarnense, E. crustaceum and E. egyptiacum. Eupenicillium shearii, however, is only distantly related to those species (Fig. 5 ; E. molle was not included in the analysis). Ornamentation of the ascospores does not appear to be a reliable indicator of relatedness among Eupenicillia. Of the five Eupenicillia within the clade, three have flanged ascospores with rough or spiny walls, and two have smooth walls lacking flanges. The presence of broad, shallow furrows on the smooth-walled ascospores of E. egyptiacum makes them distinct from those of E. bovifimosum. Eupenicillium levitum, on the other hand, has ascospores very similar to E. bovifimosum‘s, but is not closely related to the latter species.

Secondary metabolite analysis also indicates a close relationship between E. bovifimosum and E. baarnense: both species produce penicillic acid and a fumagillin-like compound. Penicillium matriti, perhaps related as well, produces penicillic acid, and one isolate (IBT 15709) also produces the fumagillin-like metabolite. All three species, however, produce metabolites not shared by the other two.

Despite similarities in secondary metabolite production and rDNA sequences, Eupenicillium bovifimosum, E. baarnense and P. matriti have very different morphologies. In comparison to E. bovifimosum, E. baarnense has longer, narrower stipes bearing no more than four metulae, and its conidia are larger, ellipsoidal and accumulate in disordered chains. In addition, it produces larger ascospores with conspicuous flanges and echinulate walls. Eupenicillium baarnense does not grow at 37 C as does E. bovifimosum. Penicillium matriti is also morphologically distinct from E. bovifimosum: it is characterized by rapidly growing colonies, pale gray-green conidia borne in short, narrow columns, and penicilli with 3–5 nearly parallel metulae.

Eupenicillium bovifimosum is not an ascosporic form of any known Penicillium species. A BLAST search using the ITS-LSU DNA sequence indicated that the Penicillium species to which E. bovifimosum may be most closely related is P. turbatum, with which it shares few or no morphological features. Penicillium turbatum has nonvesiculate, monoverticillate conidiophores, long phialides and large ellipsoidal conidia. Stolk and Samson (1983) suggested that P. turbatum is the anamorph of E. baarnense. Those two species share a number of traits, including colony appearance, growth rate and large ellipsoidal conidia, and have identical sequences in the region investigated for this study. However, the suite of secondary metabolites produced by P. turbatum is very different from that of E. baarnense (or E. bovifimosum; Michel et al 1974 ).

Eupenicillium bovifimosum is morphologically similar to P. paxilli, to which it keys with Pitt (1979) . Like E. bovifimosum, P. paxilli has dense whorls of 5–8 apically inflated metulae, closely packed phialides and small, globose, smooth conidia. However, P. paxilli differs in several significant ways: it has longer stipes, the number of phialides per metula is much less than in E. bovifimosum, the conidia form disordered chains and the suite of secondary metabolites produced by P. paxilli is very different (Frisvad and Filtenborg 1990 ). In addition, phylogenetic analysis indicated that P. paxilli is a member of the clade which contains E. anatolicum and E. shearii (Fig. 5 , Peterson 2000 ).

Eupenicillium bovifimosum also has the distinction of being the only Eupenicillium species thus far isolated from dung. The new species is similar to some coprophilic fasciculate Penicillia such as P. concentricum (Samson et al 1976 , Frisvad et al 2000 ) in producing both an antibacterial antibiotic and the antifungal, patulodin-like compounds CK2108A and CK2801B. Penicillic acid, produced by E. bovifimosum, and patulin, produced by P. concentricum, are broad spectrum antibacterial compounds (Korzybski et al 1967 ). CK2108A and B are fungicides known to inhibit Oomycetes, Trichoderma, Alternaria, Fusarium, Ascobolus and Phoma species (Broedel and Kruse 1999 ). These metabolites could thus confer advantages to fungi competing for the rich but temporary resources of dung. Indeed, coprophilic fungi have proven to be an especially rich source of antifungal agents (Gloer 1995 ).

	ACKNOWLEDGMENTS

 
The University of Wyoming Lab School provided the isolates of the new species and location information. The authors appreciate the help of Dr. Gregory K. Brown with SEM photography and Dr. Philip G. Holt with the Latin. Many thanks to Dr. Steve Peterson for making so many sequences available. The comments of two anonymous reviewers were very helpful for improving the manuscript. This research was supported in part by the National Science Foundation (grant # DEB-9632880), a NASA grant from the Wyoming Space Grant Consortium to the University of Wyoming Lab School (#NGT 5–40008), and the Danish Technical Research Council, Programme for Predictive Biotechnology.

	FOOTNOTES

 
¹ Corresponding author, Email: dtuthill{at}uwyo.edu

Accepted for publication July 13, 2001.

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tuthill, D. E. Articles by Frisvad, J. C. Search for Related Content PubMed Articles by Tuthill, D. E. Articles by Frisvad, J. C. Agricola Articles by Tuthill, D. E. Articles by Frisvad, J. C.