An Amendment of Aspergillus Section Candidi Based on Chemotaxonomical Evidence

Phytochemistry 53 (2000) 581±586 www.elsevier.com/locate/phytochem

An amendment of Aspergillus section Candidi based on chemotaxonomical evidence

Lisa Rahbñk a, Jens C. Frisvad b, Carsten Christophersen a,*

aDepartment of Chemistry, Marine Chemistry Section, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark bDepartment of Biotechnology, Building 221, Technical University of Denmark, DK-2800 Lyngby, Denmark Received 18 November 1998; received in revised form 11 November 1999

Abstract

A novel 2,2'-epoxy-terphenyllin, candidusin C, in addition to the well known secondary metabolites terphenyllin, 3- hydroxyterpenyllin and chlor¯avonin, has been isolated from the chemically unexplored fungus Aspergillus campestris. The latter three are known secondary metabolites from Aspergillus candidus and therefore a large number of Aspergilli were screened for production of these compounds to see whether they could be regarded as chemotaxonomical indicators of section membership in the monotypic Aspergillus section Candidi. The results indicated that A. campestris and A. taichungensis should be placed in Candidi and this was further con®rmed by morphological and physiological similarities. Three species outside the section Candidi produced candidusin related secondary metabolites: Aspergillus arenarius, A. ellipticus and Penicillium raistrickii. Chlor¯avonin, however, was only found in section Candidi. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Aspergillus candidus; A. campestris; A. taichungensis; Candidi; Chemotaxonomy; 2,2'-Epoxy-terphenyllin; Candidusin C

1. Introduction 1969), dechlorochlor¯avonin (Marchelli & Vining, 1973), xanthoascin (Takahashi et al., 1976b), kojic acid The genus Aspergillus has been subdivided into sev- (Kinosita & Shikata, 1969; Cole & Cox, 1981), 3-nitro- eral subgenera and sections based on conidial colour propionic acid (Kinosita, Ishiko, Sugiyama, Seto, Igar- and morphology. One of these sections, Candidi, con- asi & Goetz, 1968), citrinin (Kinosita & Shikata, 1969; tains only Aspergillus candidus (Gams, Christensen, Cole & Cox, 1981; Timonin & Rouatt, 1944), and 6- Onions, Pitt & Samson, 1985; Raper & Fennell, 1965) sulfoaminopenicillanic acid (Yamashita et al., 1983). which is a common cereal spoiling organism (Raper & Other Aspergillus sections are rich in species and we Fennell, 1965). A. candidus has been reported to pro- wanted to examine whether any newly described duce several secondary metabolites of the terphenyl- Aspergillus species with light coloured conidia might type (Marchelli & Vining, 1975; Kurobane, Vining, belong to section Candidi based on biochemical, mor- McInnes & Smith, 1979; Kobayashi, Takemoto, phological, and physiological evidence. Species such as Koshimizu & Kawazu, 1985; Takahashi, Yoshihira, A. campestris (Christensen, 1982) and A. taichungensis Natori & Umeda, 1976b) [including candidusins (Yaguchi, Someya & Udagawa, 1995) and other (Kobayashi, Takemura, Koshimizu, Nagano & species with light conidia have been placed in the sec- Kawazu, 1982) and terprenins (Kamigauchi et al., tions Cervini, Flavipedes, Terrei or Circumdati based 1998)], in addition to chlor¯avonin (Bird & Marshall, on light (white, fawn, avellaneus, ochre or yellow) conidium colours, but this placement is based on ease of identi®cation rather than on classi®catory or phyloge- * Corresponding author. Tel.: +45-35-32-01-57; fax: +45-35-32- 02-12. netic relatedness (Peterson, 1995). There are examples E-mail address: [email protected] (C. Christophersen). of misidenti®ed and misplaced isolates: an isolate

(NRRL 1955) identi®ed as A. candidus (subgenus Cir- of secondary metabolites in common with metabolites cumdati section Candidi ) was reported to produce the reported from Aspergillus candidus (terphenyllins and nephrotoxic mycotoxin citrinin (Timonin & Rouatt, chlor¯avonins) prompted us to examine other species 1944), but that particular isolate was later shown to be with white or light coloured conidia, as the results A. niveus (subgenus Nidulantes section Flavipedes ) indicated that A. campestris might belong in section (Raper & Fennell, 1965). Emphasising on species with Candidi rather than in section Circumdati as previously white, or yellow conidia, our goal was to screen a suggested by Christensen (1982). large number of species in Aspergillus for secondary Ten isolates of Aspergillus candidus, including the metabolites in order to see whether the results culture ex type, were examined. They all produced suggested another taxonomic placement of newly terphenyllin and 3-hydroxyterphenyllin and seven of described species. the ten isolates produced at least one of the terphenyllin derivatives candidusin A and/or B. In addition seven of ten isolates produced chlor¯avonin 2. Results and discussion and one produced xanthoascin in agreement with Takahashi, Sekita, Yoshihira and Natori (1976a). A. 2.1. Isolation and characterization of compounds from campestris produced terphenyllin and 3-hydroxyter- Aspergillus campestris phenyllin in common with A. candidus, but consist- ently produced candidusin C in contrast to Initial interest in A. campestris was based on the candidusin A and B produced by A. candidus (see unusually large number of secondary metabolites pro- Table 1). Although some cultivations were per- duced by this species as judged by HPLC diode array formed up to 10 years ago the isolates were culti- analysis of extracts as part of a large chemotaxonomical study on species of the genus Aspergillus. Separ- ation of the EtOAc extract of Petri dish cultures of A. Table 1 Strains examined for production of Aspergillus candidus related sec- campestris IBT 13382 resulted in the isolation of the ondary metabolites, kojic acid, and citrinin on CYA and YES agar new compound candidusin C (1), in addition to 3- (x means metabolite detected)a hydroxyterphenyllin and chlor¯avonin, previously reported from A. candidus (Kurobane et al., 1979; Bird Section Candidi 123456 7891011 & Marshall, 1969). The molecular formula of 1, A. candidus CBS 566.65b xx xx xx C21H18O6, was established by NMR analysis in combi- CBS 567.65 xx xx + nation with HREIMS (M , 366.109, C21H18O6, D 4 CBS 102.13 xx xx xx ppm). The characteristic UV-spectrum of 1 and 1H CBS 266.81 xxxx x IBT 21789 xx xx x NMR data indicated a structure similar to candidusin c A(2) and B (3), two other metabolites produced by A. UAMH 1325 xxxx x NRRL 5214 xxx x candidus (Kobayashi et al., 1982). Thus, the NMR IBT I 239y xx xx analysis, including APT, HMQC and HMBC exper- IBT 12659 xxx iments, provided evidence for a 2,2'-epoxy-terphenyl- IBT as5 xx xx x b,d structure substituted with three methoxy and two A. campestris CBS 348.81 xxxx hydroxy groups. The positions of the substituents were IBT 17867 xxx UAMH 1324e xxx determined by comparison of the NMR data of 1 with IBT I 174 xxx those of 2,3'-demethoxy-6'-desmethyl-5'-methoxycan- IBT 16773 xxx didusin B (4) (Belofsky, Gloer, Gloer, Wicklow & A. taichungensis IBT 19404b,f xxxg Dowd, 1998) and 6'-desmethyl-candidusin B (5) Other sections (Belofsky et al., 1998), in addition to analysis of long- A. ellipticus CBS 707.79 xx A. helicothrix CBS 677.79 xx 1 13 range H± C-correlations (from C1, C4 and C5 to H6, A. ¯avus ATCC 44054a x from C1' to H5', from C3' to H5' and 3'-OMe, from A. niveus NRRL 1955b x C4' to H5' and H20/H60, from C6' to H5'and 6'- A. carneus NRRL 527b x

OMe, and from C40 to 40-OMe) observed in the a HMBC spectrum of 1. Thus, the structure of 1 was 1 = Candidusin C, 2 = candidusin A, 3 = candidusin B, 4 = candidusin analogues, 5 = terphenyllin, 6 = 3-hydroxyterphenyllin, established as 2,2'-epoxy-3',40,6'-trimethoxy-1,1':4',10- 7 = chlor¯avonin, 8 = chlor¯avonin analogue, 9 = xanthoascin, 10 terphenyl-4,5-diol. = kojic acid, and 11 = citrinin. b Type culture. c 2.2. Examination of species and isolates with white and Received as A. niveus. d IBT 13382. yellow conidia e Received as A. sulphureus. f PF 1167. Having found two biosynthetically dierent groups g Received as A. candidus. L. Rahbñk et al. / Phytochemistry 53 (2000) 581±586 583 vated on the same media. However, variations in amended here, and chlor¯avonin is produced by iso- production of secondary metabolites may be due to lates in two species. However, kojic acid and citrinin variations in the composition of Difco yeast extract were not produced by any of the examined strains of in the course of 10 years. For example A. candidus A. candidus. IBT as5 (no longer available in culture) was cultivated in 1989 and produced xanthoascin as the 2.3. Examination of Aspergillus and Penicillium species only strain out of the 10 strains examined (see with other conidium colours than white and yellow Table 1). An isolate claimed to be A. candidus, ATCC During a screening of approximately 2000 isolates of 44054, indicated to be a mutant of A. ¯avus with Penicillium and approximately 500 isolates of Aspergil- white conidia. It produced kojic acid, a metabolite lus, terphenyllins and candidusins were found in the commonly produced by isolates of A. ¯avus (Cole isolates listed in Table 1 inclusive A. ellipticus CBS & Cox, 1981). Another isolate claimed to be A. 707.79 and a synonym of A. ellipticus: A. helicotrix candidus, NRRL 1955, proved to be A. niveus. This CBS 677.79, in addition to Penicillium raistrickii FRR isolate produced citrinin and gregatins as three 1576 and CBS 215.71. other isolates of A. niveus do (see Table 1). Other The terphenyl-type of secondary metabolites has species related to A. niveus in sections Flavipedes also been found in the sclerotia of other species such and Terrei, including A. carneus and A. terreus also as A. arenarius (Oh, Gloer, Wicklow & Dowd, 1998) produce citrinin (Cole & Cox, 1981, see Table 1) and Penicillium raistrickii (Belofsky et al., 1998). Our con®rming the close relatedness of these species as screening con®rmed that these compounds were pre- indicated by Raper and Fennell (1965). sent in those two species and were only found in one Previously isolates have been referred to Aspergil- further species outside section Candidi: A. ellipticus in lus sections based primarily on conidium colour, section Nigri of Aspergillus. These compounds were, presence of metulae, vesicle shape and arrangement however, not found in any of the other species of of conidia (Raper & Fennell, 1965). For example Aspergillus analyzed. For example, none of the several section Candidi is characterized by conidial heads tested strains of all known species in the sections Cir- with persistently white conidia or conidia becoming cumdati, Flavi, Wentii, Ornati, Usti, Flavipedes, Terrei, yellowish cream in age (Raper & Fennell, 1965) and Aspergillus, Clavati, Fumigati and Cremei were able to section Circumdati (formerly the A. ochraceus group) produce either terphenyllin and related compounds nor by yellow or ochraceous conidia (Christensen, 1982) chlor¯avonin under the culture conditions used. Sev- and both sections by globose heads as opposed to eral strains of species in Nidulantes and Versicolores the columnar heads in sections Terrei and Flavi- were also examined and no producers of terphenyllins pedes. A. campestris was placed in section Circum- or chlor¯avonins were found. However, it cannot be dati because of its yellowish white conidia and it ruled out that other species not yet examined in the was not considered closely related to A. candidus by latter two sections or the section Sparsi produce these Christensen (1982). A. taichungensis was equivocally compounds. A. arenarius was placed in section Versico- placed in either section Versicolores, Terrei or Flavi- lores, since the dark yellow-green conidia and metulae pedes (Yaguchi et al., 1995). However, the chemo- covering only the upper part of the vesicle found in taxonomical evidence presented here in Table 1 this species are too dierent from the characteristics of indicates that both species belong to section Can- the three species in Candidi to be considered a member didi. This is strongly supported by all the morpho- of this section. DNA sequence data could be useful in logical characteristics that are characteristic of the order to ®nd out the correct phylogenetic placement of section Candidi: slow growing colonies with globose A. arenarius. In species of Candidi the production of conidial heads having white to yellowish conidia terphenyllin like compounds is not dependent on scler- (amended description suggested here), conidiophores otium production: sclerotia were not observed in any smooth, small conidiophores common, metulae pre- of the cultures used for extraction. One family of sec- sent and covering the entire vesicle, some large ondary metabolites alone does not necessarily indicate Aspergillus heads with large metulae, conidia smooth close taxonomic or phylogenetic relationship (Geiser, or nearly so with a subglobose to ovoid shape Frisvad & Taylor, 1998). However, when chemotaxo- (albeit slightly ellisoidal in A. campestris ), and scler- nomical and morphological characters are combined, otia present (dark brown) in A. taichungensis. Scler- the species in the section Candidi seem to be a natural, otia have not been observed in A. campestris, but albeit polythetic (Sneath & Sokal, 1973), group of have been observed in A. candidus (light cream species that are closely related. Many secondary coloured turning purple to black in age). metabolites are known that occur in species in dierent Thus, terphenyllin, 3-hydroxyterphenyllin and candi- genera (Frisvad, Thrane & Filtenborg, 1998), but it dusins are common in all species in section Candidi as has also been shown that closely related species in 584 L. Rahbñk et al. / Phytochemistry 53 (2000) 581±586 series or sections in Penicillium and Aspergillus pro- species in sections Circumdati, Terrei and Flavipedes. duce several secondary metabolites in a polythetic The isolates examined in section Candidi are listed in fashion (Frisvad et al., 1998; Lund & Frisvad, 1994). Table 1. The following further isolates with white coni- Furthermore, secondary metabolite production is dia were examined: CBS 115.27, CBS 114.33, IMI rather consistent in dierent isolates of the same 165060, IBT 16747, IBT 16357, and IBT 18518 (all A. species examined here, as have been shown for other niveus ). Furthermore the Aspergillus isolates examined fungal species previously (Lund & Frisvad, 1994). included cultures ex type listed in Pitt & Samson (1993). The strains examined were all from the IBT collection of the Dept. of Biotechnology, Technical Univer- sity of Denmark, Lyngby, Denmark and from other major fungal culture collections such as CBS (Cen- traalbureau voor Schimmelcultures, Baarn, the Nether- lands; ATCC (American Type Culture Collection, Rockville, Maryland, USA; NRRL (National Center for Agricultural Utilization Research, Peoria, Illinois, USA); IMI (CABI Bioscience, Egham, UK) and UAMH (University of Alberta Microfungus Collection and Herbarium, Edmonton, Alberta, Canada). All strains were grown on Czapek yeast autolysate agar (CYA) (Samson, Hoekstra, Frisvad & Filtenborg, 1995), Blakeslee malt extract agar (MEA) (Raper & Fennell, 1965; Samson et al., 1995), yeast extract sucrose agar (YES) (Samson et al., 1995) at 258Cand CYA at 378C for morphological examinations. CYA and YES agar were used in combination for extraction of secondary metabolites as detailed by Frisvad and Thrane (1987, 1993) and analyzed by HPLC-DAD. One culture of A. candidus (IBT 21789) from a salt pan in Slovenia was grown on CYA and YES agar and analyzed separately for each medium as detailed by Smedsgaard (1997). Five strains of A. campestris were examined (see Table 1). The strain used for isolating pure candidusin 3. Experimental C(1), terphenyllin 3-hydroxyterphenyllin and chlor¯a- vonin was ex type culture, CBS 348.81 = IBT 13382, NMR spectra were recorded in DMSO-d6 solution which was originally isolated from prairie soil, North on a Varian 400 FT-NMR spectrometer. EIMS spectra Dakota (Christensen, 1982). The fungus was cultivated and high-resolution data were obtained on a JEOL in large scale on 440 Petri dishes with yeast extract AX505W instrument. sucrose (YES) agar (total 1200 ml) with an incubation time of 12 days at 258C. 3.1. Collection strains and growth conditions 3.2. Extraction and separation of secondary metabolites 2000 isolates of Penicillium (covering all accepted species in the genus) and 500 isolates of Aspergillus The culture (A. campestris ex type culture, CBS (covering all species accepted and publically available, 348.81 = IBT 13382) and growth media were except tropical species in the sections Nidulantes, Versi- extracted two times with EtOAc containing 0.5% for- colores and Sparsi ) were screened for terphenyllin, mic acid giving 13.61 g of crude extract. The extract candidusins, xanthoascin and chlor¯avonin screened (13.56 g) was partitioned between 10% aq. MeOH and using HPLC with diode array detection according to hexanes and then between 50% aq. MeOH and the method of Frisvad and Thrane (1987, 1993). A CH2Cl2. A fraction of the CH2Cl2 soluble part (1.38 g) number of strains (the culture ex type and when avail- was separated using vacuum liquid chromatography able from culture collections more isolates of each with a 5 Â 4 cm column packed with Silica gel 60G taxon) in each of most species in the genus Aspergillus from Merck and eluted with mixtures of EtOAc/hep- were screened for production of chlor¯avonins and ter- tane of rising polarity. All separations were guided by phenyllin like compounds with an emphasis on the the UV spectra of the candidusin analog using analyti- L. Rahbñk et al. / Phytochemistry 53 (2000) 581±586 585

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