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1 Center for Microbial Biotechnology, BioCentrum-DTU, Building 221, Søltofts Plads, DK-2800 Kgs, Lyngby, Denmark
2 Biodiversity (Mycology and Botany), National Programme on Environmental Science, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada
3 Department of Chemistry and Biochemistry and the Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742, USA
4 Institute of Biological Sciences, Edward Llwyd Building, University of Wales, Aberystwyth, Ceredigion SY23 3DA, UK
Correspondence
Jens C. Frisvad
jcf{at}biocentrum.dtu.dk
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the ITS and 
-tubulin gene sequences for the strains of the four Penicillium species examined in this study are DQ285608–DQ285627 and DQ267904–DQ257924, respectively.
A phylogenetic tree based on ITS gene sequences and a description of the methods used for X-ray analysis are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Recently, it was shown that a high degree of fungal diversity exists in tundra soils, even those which are covered by snow for most of the year (Schadt et al., 2003). However, despite these findings, few novel species are currently being described from cold regions. In the fungal genus Penicillium (with the ascomycetous state Eupenicillium), only one novel species has been described from very cold regions. This species, Penicillium antarcticum, is not particularly psychrotolerant (McRae et al., 1999). Many species of Penicillium grow rather well at 5 °C, especially most of the food-borne terverticillate penicillia, but most of these species have an optimum temperature for growth at around 25 °C (Pitt, 1979). Those Penicillium species that have been reported from cold regions have been found to be common ubiquitous fungi (McRae et al., 1999) and no penicillia have been reported to be geographically confined to the Arctic or Antarctic; even P. antarcticum occurs worldwide (J. C. Frisvad and others, unpublished observations). Despite this, a recent paper (Gunde-Cimerman et al., 2003) indicates that there may be several novel cold-tolerant species of Penicillium in Arctic areas. Currently there are no data concerning the chemical diversity of cold-tolerant fungi; however, the chemical diversity within the genus Penicillium is considered to be generally high (Frisvad & Filtenborg, 1989, 1990a, b). It is our hypothesis that not only tropical species, but also psychrotolerant species have high chemical diversity.
Many novel interesting metabolites have been described from unidentified species of common genera such as Aspergillus and Penicillium. One such example is the biosynthetic family of cyclic peptides, cycloaspeptide A, B and C, from an Aspergillus species (Kobayashi et al., 1987). The original Aspergillus strain was not available for study. No producers were revealed after we performed a screen of the whole genus Aspergillus for cycloaspeptide producers by HPLC with diode array detection. In contrast, by screening the genus Penicillium, we found four species producing cycloaspeptides: two known species and two novel species (Table 1). We isolated this compound from the novel species described below in order to confirm the structure of cycloaspeptide A. Interestingly, these four species were all isolated from cold regions (alpine, northern temperate and Arctic regions). Species nova characterizations were derived from extrolite, morphological and colony data and supported by phylogenetic analyses of internal transcribed spacer (ITS) and -tubulin gene sequences.
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). The filamentous fungi isolated from the soil samples from cold areas showed a high diversity of Penicillium species, but a low diversity of Aspergillus species. Only Aspergillus versicolor was detected in a sample from Nuuk airport, Greenland. The remaining fungi consisted of Geomyces pannorum, Cladosporium spp., Mucor spp. and Mortierella spp. Among the species most often recovered in the samples were Penicillium soppii and Penicillium lanosum and the two novel species, described here as Penicillium ribium sp. nov. and Penicillium jamesonlandense sp. nov. P. ribium was found only in samples taken at different locations in Wyoming, USA, while P. jamesonlandense was recovered in samples from Greenland (both east and west) at low elevations and from Wyoming in tundra soil at a high elevation (2500–2800 m). P. jamesonlandense could not be recovered from DG18 plates incubated at 25 °C (see Table 1
for a list of representative isolates of the four species).
According to one of the definitions of psychrophilic fungi, the most cold-tolerant species described here, P. jamesonlandense, is not a true psychrophilic species as it can grow at 25 °C, albeit very weakly. It is, however, close to being a true psychrophile (Weinstein et al., 1997), as its optimum temperature for growth is 18 °C. We propose to call such species quasipsychrophilic, as they are clearly different in their temperature profile from the psychrotolerant species P. ribium, P. lanosum and P. soppii and several psychrotolerant species from foods that actually grow and sporulate well at 25 °C (Pitt, 1979). P. jamesonlandense is the first species described in the genus Penicillium that grows slowly, or not at all, at 25 °C and it can be distinguished solely on that basis from any other Penicillium species. Some species of Eupenicillium, e.g. Eupenicillum fractum, also grow quite slowly at 25 °C (Pitt, 1979), albeit not as slowly as P. jamesonlandense, but these fungi are xerotolerant, not psychrotolerant, and they grow and sporulate well on media with a lowered water activity, such as the medium G25N, at 25 °C (Pitt, 1979). Moreover, P. jamesonlandense does not sporulate at 25 °C, another indication that this species is psychrophilic. Pringle & Taylor (2002) have suggested that the fitness of filamentous fungi can be measured by their ability to sporulate and, if their suggestion is accepted, P. jamesonlandense is not fit at 25 °C. The three other species, also found in cold alpine or Arctic areas, grow well and sporulate well at 25 °C, except P. soppii, which produced a large number of sclerotia and relatively few conidia on the media used. P. jamesonlandense is thus the first quasipsychrotolerant species found in the genera Eupenicillium and Penicillium.
New sequences for the ITS gene and partial sequences of the -tubulin gene were prepared for strains of P. ribium, P. jamesonlandense, P. lanosum and P. soppii using DNA isolated from conidia and mycelia produced on malt extract agar (MEA) using the FastPrep FP120 (BIO 101) or UltraClean microbial DNA isolation (Mo Bio Laboratories) kits. PCRs were performed in 25 µl volumes using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech) and 2 µl template, using a Techne Genius thermocycler (Techne). PCR cycling parameters included 30 cycles of denaturation at 95 °C for 1.5 min, annealing at 56 °C for 1 min and extension at 72 °C for 2 min, with an initial denaturation of 4 min and a final extension step of 10 min. Amplified products were purified using the UltraClean microbial PCR purification kit (Mo Bio Laboratories) and DNA concentrations were estimated from fragments stained by ethidium bromide and separated by agarose gel electrophoresis. Sequencing reactions were performed using the BigDye Terminator cycle sequencing system (Applied Biosystems) with the recommended cycling parameters. Reactions were purified by ethanol/sodium acetate precipitation. The sequences were determined using an ABI PRISM 3100 DNA automated sequencer (Applied Biosystems). The complete ITS and 5.8S rRNA genes were amplified using ITS1 and ITS4 primers, with the addition of ITS2 and ITS3 for cycle sequencing when necessary (White et al., 1990). Exons 3–6 of the -tubulin gene were amplified using T1, T10 and T224 or T222 primers (O'Donnell & Cigelnik, 1997) and sequenced using Bt2a and Bt2b primers (Glass & Donaldson, 1995). Consensus sequences were determined from overlapping sequence data for both DNA strands, except where noted, using SEQUENCHER software (Gene Codes).
Datasets were compiled of sequences of the novel species and selected ITS gene sequences of species of Penicillium, subgenus Furcatum, mostly from the study of Peterson (2000), with individual sequences from the studies of Haugland et al. (2004), Rakeman et al. (2005) and H. A. Sabev, P. S. Handley & G. D. Robson (unpublished; GenBank accession number GI 53125189). Additional ITS and -tubulin gene sequences from ongoing studies in the Seifert/Louis-Seize lab were included as relevant. The two datasets were not completely congruent due to the inclusion of ITS gene sequences from GenBank and the unavailability of a few strains for reciprocal sequencing. Both analyses were rooted with sequences for Penicillium chrysogenum, but using different strains. GenBank accession numbers for all sequences used are included in Fig. 1 and in Supplementary Fig. S1, available in IJSEM Online. Initial alignments were calculated using CLUSTAL W and adjusted using SE-AL (version 1.d1; http://evolve.zoo.ox.ac.uk/software/Se-Al/main.html) to maximize alignment. Both data matrices were subjected to parsimony analysis using the heuristic search option of PAUP version 4.0b10 (Swofford, 1999) with simple stepwise addition of taxa, tree bisection-reconnection branch swapping, gaps treated as missing data and uninformative characters removed. The maximum number of trees to be saved to memory was set to 5000 to prevent saturation of the computer's memory, most relevant for the ITS dataset. The robustness of the phylogenies was tested using bootstrap analysis (1000 replications, ‘fast’ stepwise searches).
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. Both of the novel species, P. jamesonlandense and P. ribium, had invariant and unique ITS gene sequences that distinguished them from their closest neighbours, although P. jamesonlandense differed from P. swiecickii by only a single base pair change.
The -tubulin gene sequence dataset was more variable (Fig. 1), with 68 parsimony-informative characters present in the 513 bp alignment (13 %). A poly (T) run of about 13 characters was omitted from the 5' end of the alignment because of uncertainties in reading the particular sequences. The heuristic analysis yielded 20 equally parsimonious trees of 113 steps. This confirmed the close phylogenetic relationship between P. ribium, P. swiecickii, P. jamesonlandense and P. lanosum suggested by the ITS gene sequence analysis. However, P. soppii and Penicillium raistrickii were not clearly allied with this clade in the -tubulin gene sequence analysis and P. scabrosum was sister to P. raistrickii, rather than to P. soppii. The three strains of P. soppii were invariant in their -tubulin gene sequences, whereas there was a single base pair substitution among the four sequenced strains of P. ribium. There was a 15 bp insertion in two of the strains of P. jamesonlandense (strains IBT 22005 and IBT 21984T), which accounted for the dichotomy within this species in Fig. 1. There was a fair amount of divergence among the strains of P. lanosum sequenced, including three base pair differences between two different versions of the type strain, IBT 4172T and NRRL 2009T (obtained from D. Malloch, University of Toronto, in 1995). These polymorphisms were confirmed by direct comparison of the sequence files. Evidently, there are two different strains in circulation as the type culture, an issue that requires investigation.
Culture extracts of at least two strains of each named species of Aspergillus and Penicillium from the IBT collection, as well as the novel strains isolated from soil samples, were screened for secondary metabolite production using HPLC with diode array detection according to Frisvad & Thrane (1987, 1993) as modified by Smedsgaard (1997). The screening of all Penicillium and Aspergillus species in our collection revealed only four Penicillium species that produced cycloaspeptide: P. jamesonlandense, P. ribium, P. lanosum and P. soppii. All four species produced a large number of both known and unknown secondary metabolites. P. jamesonlandense produced the glucose-derived kojic acid, the polyketides griseofulvin and penicillic acid, the amino acid-derived compounds cycloaspeptide A, tryptoquivalins and chrysogine and the phenylalanine- and hexaketide-derived pseurotin and some of the strains produced the terpene fumagillin. P. ribium produced kojic acid, the polyketides asperfuran, norlichexanthone, viridicatumtoxin, and an unknown anthraquinone in addition to cycloaspeptide A and D, psychrophilin A (Dalsgaard et al., 2004) and 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (Amade et al., 1994). P. lanosum produced kojic acid, the polyketide compactins, griseofulvins and pyripyropens, and the amino acid-derived cycloaspeptide A and sclerotigenin. P. soppii produced the polyketides asperentins, terrein and griseofulvin, in addition to the amino acid-derived cycloaspeptide A, benzomalvins, asperphenamate and pseurotins, and the terpene fumagillin.
The consistency in cycloaspeptide production by the strains examined here is high. This consistent production of cycloaspeptide in soil-borne psychrotolerant species could indicate an ecophysiological function of this metabolite. Cycloaspeptides have never been found in the psychrotolerant food-borne penicillia (Frisvad & Filtenborg, 1989; Frisvad et al., 2004; and this study). The other cyclic peptide, psychrophilin A, was only produced by P. ribium in this set of species and, like cycloaspeptide, psychrophilin A has not been found in any food-borne species of Penicillium.
For structural confirmation of the production of cycloaspeptide A, P. jamesonlandense strain IBT 21984T was cultured on 200 Czapek yeast autolysate (CYA) agar plates in the dark at 12 °C for 3 weeks. Cultures were then macerated and extracted with 2 l ethylacetate (16 h, 22 °C), evaporated and partitioned between dichloromethane and water (CH2Cl2/H2O; 55 : 45; v/v). The cycloaspeptide-enriched CH2Cl2 fraction was further separated on a Merck Lichroprep Si (310x25 mm i.d., 40–63 µm) column (30 : 69 : 1 to 0 : 99 : 1 heptane/ethylacetate/methanol gradient in 40 min at 14 ml min–1). The third Lichroprep fraction was subsequently fractioned on a Sephadex LH20 column (25x300 mm) using CH2Cl2/methanol (50 : 50) flowing at 1 ml min–1. Additionally, Sephadex fraction three was then further purified by HPLC on a Waters Prep Nova-Pak Si cartridge (100x25 mm i.d., 6 µm) using a gradient of CH2Cl2/methanol (99 : 1 to 95 : 5 gradient in 15 min at 14 ml min–1) to give 32 mg pure cycloaspeptide A. Since a crystal of cycloaspeptide A could easily be obtained from methanol, X-ray analyses were performed, confirming the original structure proposed by Kobayashi et al. (1987) (Fig. 2; for X-ray methodology, see supplementary material in IJSEM Online).
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-tubulin gene sequences both showed that the two novel species, P. ribium and P. jamesonlandense, formed a monophyletic clade with two other species producing cycloaspeptide, P. swiecickii and P. lanosum. The inclusion of the fifth cycloaspeptide producer P. soppii in this clade is equivocal in the -tubulin gene sequence analysis. Some of the species belonging to this clade, P. ribium, P. scabrosum, P. soppii and P. raistricki, do not produce kojic acid, but several species in the major clade produce griseofulvin (Table 2). Both analyses supported the species concepts for the cycloaspeptide-producing species, including the two novel species. As expected, the less variable ITS region revealed invariant gene sequences for the four species producing cycloaspeptide, whereas the -tubulin gene sequences had some infraspecific variation, most notably a 15 bp insert in two of the four strains of P. jamesonlandense. However, the two datasets provide support that the phenotypically delimited species also meet the criteria of the phylogenetic species concept (Taylor et al., 2000). In contrast with some other phylogenetic studies on complexes in Penicillium (Skouboe et al., 1999), but in agreement with others (Boysen et al., 1996), the ITS gene sequences provided species-level resolution for the species studied here. As in previous studies, partial -tubulin gene sequences were more variable and provided more robust support for species concepts, despite some infraspecific variation in the sequences (Samson et al., 2004b).
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). It is often claimed that most bioactive molecules are to be found in the tropics (Pointing & Hyde, 2001) but, among the secondary metabolites tested, griseofulvin, asperfuran, compactin, pyripyropens, benzomalvins, pseurotin and fumagillins have been suggested or used as drugs. Several compounds have antibiotic activity and some have been regarded as mycotoxins, including tryptoquivalins, viridicatumtoxin and penicillic acid (Cole & Cox, 1981). It is remarkable that bioactive secondary metabolites such as griseofulvin and viridicatumtoxin are found in tropical species such as Penicillium aethiopicum (Frisvad & Filtenborg, 1989) and also in the psychrotolerant species examined in this study. Apparently, climatic habitat is not necessarily a reliable indicator of production of particular bioactive secondary metabolites. On the other hand, metabolites such as cycloaspeptide A and D and psychrophilin A have only been found in psychrotolerant species. We conclude that polar regions are an untapped resource of biodiversity and chemical diversity.
For the purpose of species descriptions, isolated strains of P. ribium and P. jamesonlandense were cultured individually on multiple media [creatine-sucrose (CREA), CYA, MEA, oatmeal (OAT) and yeast extract-sucrose (YES) agar; for formulae see Samson et al., 2004a] by incubating in the dark at 15, 20, 25 and 37 °C. After 7 days growth, colony appearance, exudate production, pigmentation and reverse colouration were assessed and colony diameters were measured. A set of 25 micromorphological dimensions was obtained for each characteristic at 40x10 and 100x10 magnification using an Olympus microscope, DP20 digital camera and DP-Soft Image Analysis software.
The results obtained in this study show that strain IBT 21984T represents a novel species, Penicillium jamesonlandense sp. nov., and that strain IBT 16537T represents a second novel species, Penicillium ribium sp. nov. The two species were unique morphologically, physiologically and in their extrolite profiles. Furthermore, they were clearly different from other Penicillium species in ITS and partial -tubulin gene sequences. A list of the strains used in this study is provided in Table 1.
Latin diagnosis of Penicillium jamesonlandense Frisvad et Overy sp. nov.
Penicillio lanoso simile, sed crescentia lentissima (0.5–7 mm diametro, conidias absentibus post 7 dies 25 °C), ramis valde divergentibus distinctum. Acidum penicillicum formatur, neque sclerotigeninum et compactinum. Typus: DAOM 234087T (=IBT 21984T=IBT 24411T=CBS 102888T), isolatus ex solo, Jamesonlandii in Groenlandia. Herbarium specimen: C 60164T.
Description of Penicillium jamesonlandense Frisvad & Overy sp. nov. (Fig. 3)
Penicillium jamesonlandense (ja.me.son.lan.den'se. N.L. neut adj. jamesonlandense pertaining to Jamesonland, Greenland from where the type strain was isolated).
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). The type strain, DAOM 234087T (=IBT 21984T=IBT 24411T=CBS 102888T), was isolated from a soil sample from Greenland.
Latin diagnosis of Penicillium ribium Frisvad et Overy sp. nov.
Penicillio lanoso simile, sed stipitibus conidiophororum longissimis, rugosis, conidiis levibus distinctum. Psychrophilinum et asperfuranum formantur, neque sclerotigeninum, compactinum et pyripyropenum. Typus: DAOM 234091T (=IBT 16537T=IBT 24431T), isolatus ex solo alpino sub Ribes sp., Wyoming, USA. Herbarium specimen: C 60165T.
Description of Penicillium ribium Frisvad & Overy sp. nov. (Fig. 4)
Penicillium ribium (rib'i.um. N.L. adj. ribium of Ribes, isolated from around Ribes spp. growing in tundra soil).
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). The type strain, DAOM 234091T (=IBT 16537T=IBT 24431T), was isolated from around plants of Ribes spp. growing in tundra soil in Wyoming, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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