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Mycological Progress 3(1): 13–18, February 2004

A new species from tropical soils, Eupenicillium tropicum Dorothy E. TUTHILL1 and Jens C. FRISVAD2

Forty-three strains of Eupenicillium tropicum sp. nov. were isolated from soils collected in India, Costa Rica and Galapagos, Ecuador. The species is characterized by biverticillate penicilli, slightly rough, subglobose to ovate conidia, brownish cleistothecia that become brown-gray with age, and ascospores with two equatorial flanges and slightly roughened valves. All strains produced a large number of indole alkaloids, and many types of unknown secondary metabolites with characteristic chromophores were produced by a majority of strains. Eupenicillium tropicum is morphologically most similar to E. shearii, but based on ITS-LSU sequences, is most closely related to Penicillium citrinum, P. sartoryi and P. westlingii. Eupenicillium shearii strains consistently produce paxillin, paspalinine and shearinins, while the latter three penicillia all produce citrinin consistently. Taxonomical novelty: Eupenicillium tropicum Tuthill & Frisvad

O

ur interest in molecular variation at the population level prompted isolation of large numbers of Eupenicillium strains from several tropical soils. These strains were all originally identified as E. shearii, based on cultural characters and morphology of the anamorphic structures. However, ISSR fingerprinting indicated that we were actually dealing with two taxa, and subsequent phylogenetic analysis using ITS-LSU sequences showed that the two taxa were distinct species (TUTHILL, this issue). One of the species was clearly E. shearii, since the ex-type strain of that species was included in the cluster, but the other, larger group could not be assigned to any known species of Eupenicillium. An additional strain in our collection, from the Galapagos Islands, matched the new species in every respect. The new species is described here as Eupenicillium tropicum sp. nov.

Material and methods Isolation of strains has been described previously (TUTHILL, this issue). Morphological characterization of the new Eupenicillium species followed the protocol of CHRISTENSEN, FRISVAD & TUTHILL (1999). Morphological features are described from 6 to 10-d-old cultures grown on malt extract agar (MEA) at room temperature (20–24 ºC). Capitalized color names, followed by plate number, are those of Ridgway (1912, and see CHRISTENSEN, MILLER & TUTHILL 1994). The strains were also characterized using the standardized methods by

1 2

Department of Botany, PO Box 3165, University of Wyoming, Laramie, WY 82071. Email: [email protected]. BioCentrum, Building 221, Technical University of Denmark, DK2800 Kgs. Lyngby.

PITT (1979), including growth on Czapek yeast autolysate (CYA) agar at 37 °C and growth at 25 °C on MEA, yeast extract sucrose (YES) agar, oatmeal agar, and creatine sucrose agar measured after one week. (See SAMSON et al. 2002 for formulations of the media.) 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. Several strains of E. tropicum and E. shearii were analyzed for their profile of secondary metabolites (Table 1) using the HPLC-diode array detection (DAD) method of FRISVAD & THRANE (1987) as modified by SMEDSGAARD (1997). Three plugs were extracted with organic solvents as described by SMEDSGAARD (1997) on the media CYA and YES. All metabolites detected were compared to standards of known secondary metabolites and characterized by their UV spectra from 200–600 nm and their retention indices (RI). The RI value can be compared to those reported by NIELSEN & SMEDSGAARD (2003). Strains, including ex-types, of P. citrinum, P. sartoryi and P. westlingii were also analyzed by HPLC-DAD. The phylogenetic analysis included all of the Eupenicillium ITS-LSU sequences available from GenBank, plus Penicillium members of the E. shearii clade. Talaromyces bacillosporus was selected for the outgroup. All of the strains used have been previously listed (TUTHILL & FRISVAD 2000, TUTHILL, this issue). The alignment was done with ClustalX (THOMPSON et al. 1997) with all multiple alignment parameters used at default settings, then corrected manually. Phylogenetic analyses were performed with PAUP*4.0 (SWOFFORD 2000) set for maximum parsimony, using outgroup rooting. The heuristic search included the following options: starting trees obtained via stepwise addition, simple addition sequence, branch © DGfM 2004

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TUTHILL & FRISVAD: Eupenicillium tropicum sp. nov.

Tab. 1: Strains of Eupenicillium tropicum and E. shearii sequenced or examined for secondary metabolites. Many more strains were included in the ISSR analysis than are indicated here (TUTHILL, this issue)

E. tropicum:

E. shearii:

Origin

Isolate #

IBT #

Sequence

ISSR

HPLC

Costa Rica India India India India India India India India India India Ecuador

SS133-17 SC18-13 SC18-17 SC42-1 SC42-23 I 154 I2-B13 I2-C9 I2-F5 I2-G3e I2-L2 DMG 1004

24584 24604 24610 24580 42582

x

x x x x x

16462

x

x x x x (type) x x x x x x x x

Australia Australia Colombia Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Honduras India India India India India India Ivory Coast Papua New Guinea Venezuela Zaire Zaire

RMF 9573 RMF 9574 CBS 502.78 CR9-1 CR10-1 CR10-2 CR14-1 CR14-7 CR16-7 CR18-17 SS133-1 CBS 290.48 SC18-3 SC18-12 SC18-19 SC42-5 SC45-3 I2-J5 CBS 488.66 CBS 513.73 Ven97 M75-3 CBS 343.54 CBS 342.68A

18201 18202 24589 33433 22281 22286 22993 22432 23357 23404 24585 24588

x x

swapping by tree bisection-reconnection, and steepest descent modification off.

Results Eupenicillium tropicum Tuthill and Frisvad Figs. 1-5 Nova species a Eupenicillium shearii cleistotheciis plus brunneis, plus tarde crescens ad 25 ºC, non crescens ad 37 ºC et metabolitibus secondariis proprius distinguenda.

Conidiophores arising from mycelial mat, smooth, 150–250 µm in length; metulae in whorls of 3–4, rarely 5, even in length, 14–17 x 3–3.5 µm, cylindrical or inflated at the tip to 4 µm; phialides nearly parallel, 9–11 x 2–2.8 µm; conidia subglobose to ovate, finely roughened, 2.2–2.6 x 2–2.3 µm. Cleisto© DGfM 2004

24608 24607 24586

14694 14698 20983 14695 14786

x

x x

x x x x x x x x x x x x x x x

x x x x x x x x x x x x (ex-type) x x x x x x x x x x x

thecia sclerotioid, 200–300 µm diam, at first orange-tan but becoming brown-gray within 3 weeks, maturing in six weeks on MEA or oatmeal agar. Asci borne singly, 5–6 µm diam; ascospores ellipsoidal, 2.5–3 x 2–2.5µm, with two narrow, closely appressed equatorial flanges and valves that appear smooth under the light microscope, but appear to be slightly roughened with SEM. Cultural features MEA: Colonies slow-growing, reaching diameters of only 10–16 mm at 7 d. Cleistothecia abundant, white to light orangetan, conidia abundantly produced in some strains, less so in others, blue-gray near Greenish Glaucous-Blue (XLII), darkening to Slate Olive (XLVII). Reverse uncolored at 7 d, but becoming very light yellow by 10 d.

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Mycological Progress 3(1) / 2004

Figs. 1-4: Liquid slide mounts of Eupenicillium tropicum. 1: Typical 3-metulate and exceptional 5-metulate, biverticillate penicilli. Bar = 10 µm. 2: Conidia. Bar = 5 µm. 3: Wall of mature cleistothecium. Bar = 10 µm. 4: Ascospores. Bar = 3 µm

Oatmeal agar: Colonies 22–33 mm, with abundant pale brown cleistothecia, exudates in droplets, and light conidium production. CYA: Colonies 19–24 mm diameter, with pale orange-tan cleistothecia heaped at center, exudate in droplets, clear or very light yellow. Conidial color similar to that on MEA. Reverse uncolored. No growth on CYA at 37 ºC. YES agar: Colonies 29–40 mm, appearance similar to colonies on CYA, reverse creamish yellow. Creatine-sucrose agar: Poor growth, colony diameter after one week 14–20 mm with no acid production. Secondary metabolites Eupenicillium tropicum produces a large number of indole alkaloids, the most prominent having a RI of 1567, with other indoles at RIs 981, 1101, 1301, 1371, 1603, 1621, 1767, 1790 and 1987. All indoles have UV maxima at 225 nm (100 %), 275 nm (shoulder; 23 %), 282 nm (25 %), and 292 nm (21 %). In addition, the uncharacterized metabolites Emon-1088, HOLOX and Isom1 were produced by all strains examined, while Aflo1 and 2, Gx, RAIMO and STOT were produced by a majority of strains, and CIT and KO-1067 were produced by a few strains. Structures of the uncharacterized metabolites have not been elucidated, but the UV maxima and RIs are as follows: Emon-1088 (also produced by Emericella spp.): 225 nm (100 %), 260 nm (41 %), 370 nm (31 %), RI = 1088;

HOLOX: 253 nm (100 %), 284 nm (37 %), RI = 710; Isom1: 202 nm (100 %), 222 nm (shoulder; 25 %), 275 nm (11 %); Aflo1: 205 nm (100 %), 231 nm (61 %), 256 nm (52 %), 300 nm (shoulder; 32 %), 348 nm (58 %), RI = 826; Aflo2: 210 nm (100 %), 245 nm (42 %), 340 nm (88 %), RI = 789; Gx (produced by several Penicillium spp.): 206 nm (100 %), 256 nm (20 %), 330 nm (45 %), RI = 738; RAIMO (also produced by P. raistrickii): 206 nm (100 %), 256 nm (shoulder; 27 %), 280 nm (shoulder; 11 %), RI = 922; STOT: 200 nm (100 %), 222 nm (55 %), 260 nm (34 %), RI = 1372; CIT (also produced by P. citrinum): 247 nm (100 %), 255 nm (shoulder; 71 %), 324 nm (23 %), RI = 849; KO-1067: 244 nm (100 %), RI = 1067. All strains examined of E. shearii produced paxillin, paspalinine and shearinins in agreement with the data on one strain examined by BELOFSKY et al. (1995), and strains of P. citrinum consistently produced citrinin and tanzawaic acid in agreement with MALSTROM, CHRISTOPHERSEN & FRISVAD (2000). Specimens examined TYPE. SC42-1, isolated from soil collected in 1996 from between plants of Coffea arabica, at Merthi Subbangudigy Estate near Basarikatte, Karnataka, India. A live culture of SC42-1 has been deposited at Centraalbureau voor Schimmelcultures (CBS 112584) and at BioCentrum-DTU (IBT 24580). A number of strains were isolated from the same soil sample. Additional strains were isolated from soil collected © DGfM 2004

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Fig. 5: SEM of ascospores, showing roughened wall and appressed flanges. Bar = 2 µm

beneath coconut palm at Netrakonda coffee farm near Balehonnur, Karnataka, India, and from soil collected beneath trees in a residential area, Escazu, Costa Rica. A total of 43 strains were identified as members of this species through ISSR fingerprinting (TUTHILL, this issue). An additional strain, DMG 1004, was isolated in 1970 by Dr. Dan Mahoney from soil collected in the Galapagos Islands. DMG 1004 represented a number of isolates that were not saved; all were obtained after treatment of the soil with 60 % ethanol for 10 minutes, and with or without heat treatment of 35 minutes at 60 ºC (D. Mahoney, personal communication). DMG 1004 has been retained at BioCentrum-DTU as IBT 16462. Alignment of the 38 taxa (TreeBASE study accession number = S957, matrix accession number = M1588 http:// www.treebase.org/treebase/) included 1185 sites, of which 140 were variable but phylogenetically uninformative and 186 were informative. Phylogenetic analysis yielded 6 equally parsimonious trees of 785 steps. Eupenicillium tropicum is a member of the well-supported clade that also includes E. anatolicum, E. shearii and a number of Penicillium species, among them P. miczynskii, P. paxilli and P. westlingii (Fig. 6). It is most closely related to P. westlingii, P. citrinum and P. sartoryi.

Discussion PITT (1979) divided Eupenicillium into eight series based on subjectively estimated overall similarity, including anamorphic, teleomorphic and colony characters and growth rates under standard conditions. The new species may fit into either Crustacea or Tularensia series, both of which are characterized by singly-borne asci, flanged ascospores, biverticillate penicilli and lack of growth (usually) at 37 ºC. The growth rates of E. tropicum on MEA and CYA are intermediate between the two series. According to the taxonomic scheme of STOLK & SAMSON (1983), E. tropicum is a member of the section Eupenicillium, based on the presence of flanges on the © DGfM 2004

TUTHILL & FRISVAD: Eupenicillium tropicum sp. nov.

ascospores, weakly pigmented and nearly smooth-walled conidia and three or more metulae on the conidiophore. However, since neither of these schemes represent evolutionary relationships, it is most useful to consider E. tropicum as a member of the E. shearii clade (Fig. 6; PETERSON 2000). Eupenicillium anatolicum, also a member of the E. shearii clade, has growth rates on MEA and CYA similar to those of E. tropicum, as well as brown cleistothecia and slightly roughened, flanged ascospores. However, its predominantly monoverticillate penicilli, strongly pigmented colonies in reddish-brown or yellow shades and cleistothecia that become dark brown at maturity, make E. anatolicum distinct. Additionally, E. anatolicum produces a different array of secondary metabolites, including xanthocillins and some unique, unknown compounds. Eupenicillium tropicum is more similar to E. shearii in its production of orange-tan cleistothecia, greenish conidia borne on biverticillate penicilli, pallid reverses on both MEA and CYA and multiple indole-alkaloids. However, the sclerotia of E. shearii, while starting out brownish, become distinctively gray on CYA; the cleistothecia of E. tropicum also become gray, but of a notably warmer shade. In addition, E. shearii grows more quickly than E. tropicum on both MEA and CYA, and also grows at 37 ºC, unlike E. tropicum (PITT 1979, STOLK & SAMSON 1983). Furthermore, E. shearii produces an array of secondary metabolites quite different from that of E. tropicum. It produces shearinins A and B, paspalinine, paxillin (BELOFSKY et al. 1995, confirmed here for all isolates of E. shearii examined using HPLCDAD), the uncharacterized metabolites XX and XX’, and several indole alkaloids different from those of E. tropicum. The closest known relatives of E. tropicum are P. westlingii, P. citrinum and P. sartoryi. Like E. tropicum, they possess biverticillately branched penicilli. However, they differ from E. tropicum in the absence of a teleomorph, in various micromorphological features including globose conidia, and in the secondary metabolites that they produce. Penicillium citrinum and P. sartoryi, which have been placed in synonymy (PITT 1979), produce citrinin, tanzawaic acid (KURAMOTO et al. 1997, MALSTROM, CHRISTOPHERSEN & FRISVAD 2000) and the uncharacterized metabolites CIT, CITY, CIB, AQ949gl and AQ-1100gl. Penicillium westlingii produces citrinin, territrems, arisugacins (FRISVAD & FILTENBORG 1990) and several other compounds distinct from those of P. citrinum, P. sartoryi and E. tropicum. All three Penicillium species fail to produce any of the indole alkaloids characteristic of E. tropicum. In addition, the three Penicillium species have a 40 base deletion in the ITS1 region that is not present in E. tropicum, nor in any other sequenced Penicillium or Eupenicillium species. Eupenicillium tropicum appears to be widely-distributed, having been isolated from soils collected in Costa Rica, Galapagos Islands (Ecuador), and India. Multiple strains were obtained from all of the soil samples, indicating that the species is common in those soils. Evidence indicates that the species reproduces strictly clonally, despite its ability to produce prodigious numbers of ascospores (TUTHILL, this issue).

Mycological Progress 3(1) / 2004

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Fig. 6: Strict consensus of 6 trees of 785 steps. Bootstrap values (100 reps) > 70 % are shown on branches. Branches with bootstrap values < 50 % have been collapsed. For the original trees CI = 0.5694, RI = 0.7236, RC = 0.4121. Bar = 10 base changes

Acknowledgements We thank Dr. R. Naidu for allowing us to sample from the Coffee Research Station in Balehonnur and coffee estates in Karnataka, N. Swoboda-Colberg for help with the SEM photo and Dr. Philip Holt for the Latin translation. As ever,

thanks to Dr. Stephen Peterson for making so many DNA sequences available. This work was funded by a National Science Foundation grant (DEB 6932880), the Department of Botany, University of Wyoming, and the Technical Research Council of Denmark in the project Program for Predictive Biotechnology. © DGfM 2004

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TUTHILL & FRISVAD: Eupenicillium tropicum sp. nov.

PETERSON SW (2000) Phylogenetic analysis of Penicillium species based on ITS and LSU-rDNA nucleotide sequences. In: SAMSON RA, PITT JI, eds. Integration of modern taxonomic methods for Penicillium and Aspergillus classification, pp 163178. Hargrove Academic Publishers, Amsterdam. PITT JI (1979) The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London. RIDGWAY R (1912) Color standards and color nomenclature. Selfpublished, Washington. SAMSON RA, HOEKSTRA ES, FRISVAD JC, FILTENBORG O, eds. (2002) Introduction to food- and airborne fungi. Sixth edition. Centraalbureau voor Schimmelcultures, Utrecht. SMEDSGAARD J (1997) Micro-scale extraction procedure for standardized screening of fungal metabolite production in culture. – Journal of Chromatography A 760: 264-270. STOLK AC, SAMSON RA (1983) The ascomycete genus Eupenicillium and related Penicillium anamorphs. – Studies in Mycology 23: 1-149.

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KURAMOTO M, YAMADA K, SHIKANO M, YAZAWA K, ARIMOTO H, OKAMURA T, UEMURA D (1997) Tanzawaic acids A, B, C, and D: inhibitors of superoxide anion production from Penicillium citrinum. – Chemistry Letters 1997: 885-886.

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MALSTROM J, CHRISTOPHERSEN C, FRISVAD JC (2000) Secondary metabolites characteristic of Penicillium citrinum, Penicillium steckii and related species. – Phytochemistry 54: 301-309.

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© DGfM 2004

Accepted: 15.10.2003