Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi

Mycol. Res. 107 (10): 1201–1209 (October 2003). f The British Mycological Society

1201

DOI: 10.1017/S0953756203008487 Printed in the United Kingdom.

Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi

Antonio GONZALEZ DEL VAL1*, Gonzalo PLATAS1, Francisco ARENAL1, Juan Carlos ORIHUELA3, Marı´ a GARCIA1, Pilar HERNANDEZ1, Inmaculada ROYO1, Nuria DE PEDRO1, Lynn L. SILVER2, Katherine YOUNG2, Marı´ a Francisca VICENTE1 and Fernando PELAEZ1 1

Centro de Investigacio´n Ba´sica, Merck Sharp & Dohme Research Laboratories, Josefa Valca´rcel 38, E-28027 Madrid, Spain. Department of Infectious Disease Research – Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA. 3 Instituto de Productos Naturales y Agrobiologı´a, Consejo de Investigaciones Superiores Cientı´ficas-CSIC Apartado de Correos 195, E-38206 La Laguna Tenerife, Spain. E-mail : ant_ [email protected] 2

Received 30 August 2002; accepted 2 July 2003.

The illudins are a family of fungal sesquiterpenes that have been studied as anti-tumor agents, and they also have antibacterial activity. Over a four-year period, 25 304 fungal isolates (approximately 97 % ascomycetes and 3 % basidiomycetes), were screened for antibacterial activity against methicillin-resistant Staphylococcus aureus. Illudin-like compounds with antibacterial and cytotoxic activity against tumor cell lines were observed in 10 basidiomycete strains. The isolates were recovered from different types of substrata using indirect methods and only formed sterile mycelia in pure culture. The isolates were genetically related but not identical, based on PCR-based fingerprinting techniques. DNA sequencing of the ITS1-5.8 S-ITS2 region of the strains revealed that nine had identical sequences, indicating that they were conspecific. The sequence of the remaining isolate was 96.34 % similar, suggesting that it was a closely related species. The D1–D2 region of the 25 S rRNA gene of the two strain types was also sequenced. Both sequences were 99.39 % similar, and Coprinopsis gonophylla (syn. Coprinus gonophyllus) was the closest match for both. Strains were grown in pure culture on a rice-based medium that allowed the development of basidiomata from one culture of the main strain type, which was identified as C. episcopalis, a close relative of C. gonophyllus. Both species (or strain types) produced different types of illudin-like compounds. Three novel illudins (I, I2 and J2) were found to be produced by the cultures identified as C. episcopalis, while only illudinic acid was produced by the other Coprinopsis sp. The taxonomical relationships of the Coprinopsis species identified in this study with other illudin producers previously reported in the literature are discussed.

INTRODUCTION The illudins are a group of sesquiterpene antibiotics that have been widely reported as antibacterial and antitumor agents (Anchel, Hervey & Robbins 1950, McMorris & Anchel 1965). These low molecular weight natural products are cytotoxic to human tumor cells in vitro. Their analogs show differences in selectivity against a variety of multidrug-resistant tumor cell lines (Kelner et al. 1996). Although they do not induce remission of primary tumors (McMorris et al. 1996), studies to improve their therapeutic index have been carried out in order to develop them as potential antitumor agents for humans (Kelner et al. 1997). * Corresponding author.

A derivative of illudin S, hydroxymethylacylfulvene (also designated MGI 114), has been tested in Phase II human clinical trials (Murgo et al. 1999). These compounds are synthesized from farnesyl pyrophosphate via humulene to obtain the protoilludane skeleton (Turner & Aldridge 1983, Morisaki et al. 1985). Several basidiomycetes have been reported to produce different classes of illudins and related compounds (Nakanishi et al. 1963, McMorris & Anchel 1965, Turner & Aldridge 1983, McMorris et al. 1989, Arnone et al. 1991, Cardillo, Nasini & Vajna De Pava 1992, Lee et al. 1996, Dufresne et al. 1997, Burgess, Zhang & Barrow 1999, Kirchmair, Po¨der & Huber 1999). In the course of our natural products screening programme for the detection of new antibacterial

Illudin-like compounds produced by basidiomycetes compounds against methicillin-resistant Staphylococcus aureus, three new members of the illudin family were found, the illudins I, I2 and J2 (details on the isolation and structural elucidation of these compounds will be published elsewhere), produced by nine basidiomycetous strains. These were taxonomically identical, and were identified as Coprinopsis episcopalis (syn. Coprinus episcopalis). In addition, they were very similar to another basidiomycetous strain that first produced illudinic acid, another member of the illudin family previously discovered in our laboratory (Dufresne et al. 1997). We have now identified that strain as a different Coprinopsis species. This report assesses the relationships among these illudins producing fungi, by means of molecular fingerprinting and rDNA sequences. The biological activity of the compounds is also described. MATERIALS AND METHODS Fungal isolation The illudins-producing strains tested were recovered from leaf litter, twigs, bark and decayed fungal fruit bodies, collected in various ecological niches in Spain and Norway. Thus, MF6183 was isolated from leaf litter under Quercus pyrenaica collected in El Escorial (Madrid); F-043615 and F-050152 from twigs of Q. ilex in El Pardo (Madrid); F-043616 from the bark of Pinus sp. in Fuenfrı´ a valley (Madrid); and F-043898 from twigs of an undetermined bush in Puerto de Canencia (Madrid). The remaining isolates were recovered from fruit bodies of different basidiomycetes ; F-036104 and F-065192 from Coltricia perennis collected in San Rafael (Segovia) and Sierra de Aracena (Huelva), respectively ; F-064720 from Omphalina pyxidata in Rı´ o Pelagallinas (Guadalajara) ; and F-062769 and F-061559 in Akershus (Norway) from Hyphoderma setigerum and Tylospora asterospora respectively. The isolations were made following standard procedures (Pela´ez et al. 1998, Worrall 1991). All the strains were maintained as PDA slants (Difco) until used for fermentation studies, and are preserved as frozen agar plugs in glycerol 10 % at x80 xC in the CIBE culture collection. Fungal fermentations To prepare the inocula for fermentation studies, the upper third of the agar slants from the cultures was removed and transferred aseptically to unbaffled 250 ml Erlenmeyer flasks, containing 50 ml of the seed medium formulated as in Bills et al. (1996). The seed flasks were incubated at 25 x for 3–4 d with agitation (220 r.p.m.). 2 ml aliquots of the resulting cultures were used to inoculate the same type of flasks containing a production medium which contained sucrose, 80 g lx1 ; yellow corn meal, 50 g lx1 and yeast extract (Difco), 1 g lx1. The production flasks were incubated for 21 d at 25 x with agitation (220 r.p.m.).

1202 For the production of basidiomata from the cultures producing illudins, a medium containing brown rice was used (Jayasuriya et al. 1995). These flasks were inoculated from seed flasks obtained as described above, and incubated statically up to 4 months at 25 x. Screening for antimicrobial activity In vitro antimicrobial susceptibility tests were performed using a methicillin-resistant Staphylococcus aureus (MRSA) strain (Davis & Stone 1986). The assay plates used for the antimicrobial susceptibility tests were prepared following the procedure described by Pela´ez et al. (1998). Methanol extracts (25 ml), prepared as described (Pela´ez et al. 1998), were applied onto the surface of the assay plates seeded with the S. aureus strain, which was incubated overnight at 28 x. Inhibition zones around the application points were measured after 24 h. Antibiotics with positive and negative responses were used as internal controls for the plates. Evaluation of biological activity Fungal extracts exhibiting anti-MRSA activity were further evaluated in a variation of the SOS chromotest assay (Auffray & Boutibbones 1986, Quillardet et al. 1982). SOS positive extracts were selected and the presence of a UV-mimetic activity, assumed to be due to production of illudin-like compounds (Kelner et al. 1994), was determined by zone size differential on an isogenic set of Escherichia coli uvrA+ and uvrAx strains (Green & Muriel 1975, Ishi & Kondo 1975, Quillardet et al. 1982, Fram, Sullivan & Marinus 1986). Mitomycin C and an extract from an illudinic-acid producing strain (Dufresne et al. 1997) were used as controls. Determination of minimum inhibitory concentrations MICs of illudins I, I2 and J2 against Staphylococcus aureus, MRSA, Escherichia coli, Bacillus subtilis and Proteus vulgaris were determined by the microdilution technique using cation-adjusted Mueller–Hinton broth in accordance with the guidelines of the National Committee for Clinical Laboratory Standards (1993). The inoculum was prepared so as to obtain a final concentration of bacteria of 5r105 colony forming units in each well of the microdilution trays. The microtitre plates were incubated at 37 x for 18–20 h. Chloramphenicol, fosfomycin and oxytetracycline were used as controls. Cytotoxicity assay For evaluation of cytotoxicity, the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test was applied to three human cell lines, A-549 (ATCC CCL-185), HT-29 (ATCC HTB-38), and

A. Gonzalez Del Val and others Hep G2 (ATCC HB-8065), following the procedures described by Corine et al. (1998). The samples to evaluate (10 ml) were incubated with 190 ml of a cell suspension for 24 h in an atmosphere of 5% CO2. Every compound was tested in a range of concentrations from 250 to 1.95 mg mlx1, per duplicate. Absortion at 490 nm was measured in a Titertek Multiskan. Actinomycin and MMS (methylmethasulfonate) were used as controls.

1203 Table 1. Orders and families of basidiomycetous isolates screened. The species were grouped by suprageneric taxa according to the criteria established in the 8th edition of the Ainsworth & Bisby’s Dictionary of the Fungi (Hawksworth et al. 1995).

Order

Isolates tested

Family

Isolates tested

Poriales

242

Coriolaceae Polyporaceae Lentinaceae

183 43 16

Agaricales

170

Tricholomataceae Coprinaceae Strophariaceae Agaricaceae Bolbitiaceae Entolomataceae Amanitaceae Pluteaceae

89 27 22 14 7 5 4 2

Molecular phylogenetic studies DNA extraction and microsatellite-primed PCR was performed following Bills et al. (1999). The rDNA region containing the two ITSs and the 5.8 S rRNA gene was amplified using primers ITS1F and ITS4 (White et al. 1990, Gardes & Bruns 1993) following standard procedures (40 cycles of 30 s at 93 x, 30 s at 53 x and 2 min at 72 x). Partial sequences were obtained using primers ITS1F, ITS4, ITS2 and ITS3. A fragment from the 25 S rRNA gene containing about 1000 nucleotides adjacent to the ITS-2, including the two variable regions D1 and D2, was amplified using primers LR0R, LR16 (Hopple & Vilgalys 1994). Partial sequences of this fragment were obtained using primers LR0R, LR3R, LR6 and LR16. About 0.1 mg mlx1 of the double stranded amplification products were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster city, CA) following the procedures recommended by the manufacturer. The separation of the reaction products by electrophoresis and the reading of the results was performed in an ABI 373 Automatic Sequencer (Applied Biosystems). The sequences obtained were aligned visually and the phylogenetic analysis was performed by parsimony analysis using PAUP 3.1.1. (Swofford 1993). To assess branch support, the data were resampled with 1000 bootstrap replicates (Felsenstein 1985) by using a heuristic search option of PAUP. The sequences obtained in this work were deposited in GenBank with the accession numbers AY145853 (partial sequence of the 25 S rRNA gene of isolate F-062769, a representative of the strains producing illudins I, I2 and J2); AY145854 (partial sequence of the 25 S rRNA gene of isolate MF6183, the illudinic acid producer) ; AY145855 (ITS region of isolate F-062769) and AY145856 (ITS region of isolate MF6183).

Stereales

61

Stereaceae Meruliaceae Peniophoraceae Hyphodermataceae Corticiaceae Steccherinaceae Botryobasidiaceae Atheliaceae Aleurodiscaceae Sistotremataceae Podoscyphaceae

21 10 10 8 4 3 1 1 1 1 1

Hymenochaetales

57

Hymenochaetaceae

57

Ganodermetales

28

Ganodermataceae

28

Cortinariales

16

Cortinariaceae Crepidotaceae

9 7

Boletales

15

Boletaceae Paxillaceae Coniophoraceae Hygrophoropsidaceae Rhizopogonaceae

3 3 3 3 3

Lycoperdales

13

Lycoperdaceae Mycenastraceae

10 3

Cantharellales

11

Sparassidaceae Cantharellaceae Typhulaceae Clavulinaceae

6 2 2 1

Hericiales

11

Hericiaceae Auriscalpiaceae Lentinellaceae Gloeocystidiellaceae

5 2 2 2

Schizophyllales

10

Schizophyllaceae

10

Thelephorales

8

Thelephoraceae Bankeraceae

5 3

Nidulariales

6

Nidulariaceae

6

Dacrymycetales

5

Dacrymycetaceae

5

Tremellales

4

Tremellaceae Exidiaceae

2 2

RESULTS AND DISCUSSION

Sclerodermatales

3

Discovery and biological activity of illudins I, I2 and J2

Astraeaceae Sphaerobolaceae

2 1

Gomphales

2

Ramariaceae

2

Russulales

1

Russulaceae

1

Auriculariales

1

Auriculariaceae

1

Hymenogastrales

1

Gastrosporiaceae

1

During the search for new antibacterial compounds against methicillin-resistant Staphylococcus aureus (MRSA), 25 304 fungal isolates distributed among 24 586 ascomycetes and 718 basidiomycetes, with a broad taxonomical, ecological and geographical diversity, were screened. Table 1 shows the distribution of

Undetermined Total

53 718

Illudin-like compounds produced by basidiomycetes

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Table 2. Biological activity of illudins I, I2 and J2. The data shown are the minimum inhibitory concentrations against several bacteria and the IC50 (concentration providing 50% of the maximum response) in the MTT cytotoxicity assay. Activity (mg mlx1)

MIC

Cytotoxicity IC50 a b

Organism Staphylococcus aureus MRSA Escherichia coli Bacillus subtilis Proteus vulgaris Cell lines A-549 MDBK Hep G2

Illudin I

Illudin I2

Illudin J2

Controlsb

NTa 150 80 80 625

300 150 150 150 625

300 300 150 150 62.5

CLM 3 6.25 1.5 2.5 3.12

FOS 1 15.5 9.25 30.5 9.25

MMS 70 80 90

ACT 110 90 100

32 55 13

174 >250 104

>250 >250 84

OXT 2 125 30.5 2.5 15.6

NT, not tested. CLM, chloramphenicol; FOS, fosfomycin; OXT, oxytetracycline; MMS, methylmethasulfonate; ACT, actinomycin D.

the basidiomycetes screened among orders and families. Representatives of 54 families corresponding to 20 different orders were assayed. The best represented orders were Poriales, Agaricales, Stereales and Hymenochaetales (74 % of the isolates tested). About 25 % of the strains tested showed anti-MRSA activity, but only 10 undetermined basidiomycetes strains inhibited the growth of S. aureus and were positive in the SOS counterscreen (see Materials and Methods). The compounds responsible for this activity were isolated and identified as illudinic acid (Dufresne et al. 1997) for the strain MF6183 and the novel illudins I, I2 and J2 for the rest of the strains (Fig. 1). All the producers of these illudanes were basidiomycetes, none of the ascomycetes tested produced this class of compounds. Illudins I2 and J2 were only moderately active against S. aureus, Escherichia coli and Bacillus subtilis. Illudin I was slightly more active against E. coli and B. subtillis (Table 2), but still weaker than the control antibiotics. In contrast, the MIC of illudinic acid (1 mg mlx1 against S. aureus), was in the same range as fosfomycin, chloramphenicol and oxytetracycline (Dufresne et al. 1997). Illudins I, I2 and J2 were also tested for cytotoxicity against A-549, MDBK and Hep G2 cells (Table 2). Illudins I2 and J2 showed similar cytotoxicity against Hep G2 cells as the positive controls actinomycin and MMS, which inhibited the growth of the three mammalian cell lines used in the test with IC50s in the range of 70–110 mg mlx1. The activity of illudin I2 was lower against A-549 cells, and illudin J2 was inactive in this cell line at the maximum concentration tested. Also, these illudins exhibited no significant activity against MDBK cells. In contrast, illudin I presented remarkable cytotoxicity against the three cells lines, with greater potency than the control compounds. The stereochemical differences among the structures of these novel illudins (Fig. 1) could account for their different biological activity.

O COOH OH Illudinic acid

O CH2OH OH Illudin I2

O CH2OH OH Illudin I

O

CH2OH OH Illudin J2

Fig. 1. Structures of illudinic acid and the novel illudins I, I2 and J2.

Identification of the fungi As a normal consequence of the high numbers of samples tested during the screening effort, none of the producing fungi were identified to species before screening. However, all of them had been initially recognized as basidiomycetes, using PCR with primer

A. Gonzalez Del Val and others 1

2

3

4

5

6

7

8

1205 9 10 11 12 13

85

MF6183 F-062769

93

65 Coprinopsis gonophylla AF041502

100 53

Coprinopsis friesii AF045103 Coprinopsis kimurae AF041500

Coprinopsis utrifer AF041501 52

Coprinopsis macrocephala AF041489 Coprinopsis atramentaria AF041484 Coprinopsis cinerea AF041494 Coprinopsis radiata AF041493 Coprinopsis semitalis AF041508

Coprinopsis trispora AF041504 Psathyrella candolleana AF041531

Fig. 2. DNA fingerprint of the producing organisms. Lane 1, 250 bp ladder ; lane 2, F-065192; lane 3, F-043898; lane 4, F-062769; lane 5, F-061559 ; lane 6, F-043615; lane 7, 250 bp ladder; lane 8, F-064720 ; lane 9, F-050152 ; lane 10, F-036104; lane 11, F-043616; lane 12, MF6183 ; lane 13, 250 bp ladder.

ITS 4B as a DNA probe specific for this fungal phylum (Gardes & Bruns 1993) (Fig. 2). Microsatellite-primed PCR revealed that none of these isolates were genetically identical, although a general relatedness among them was apparent, as evidenced by the presence of several shared amplification bands. These data, and that these strains were isolated over a period of more than 36 months, suggest that these isolates were not vegetative clones derived from a single strain, but rather independently derived isolates from different mycelia. In order to clarify the phylogenetic relationships among the producing isolates, the complete ITS region of the rDNA, including the 5.8 S rRNA gene (619 nt), was sequenced. Nine out of the ten isolates had identical sequences, and they all produced the three novel illudins I, I2 and J2. The remaining isolate, which was the producer of illudinic acid (Dufresne et al. 1997), was 96.3 % similar. These results suggest that at least the nine isolates with identical sequences belong to the same fungal species, whereas the remaining strain, formerly reported as an undetermined basidiomycete (Dufresne et al. 1997), could be a closely related species. No good matches were found when these sequences were searched at GenBank. In a further attempt to classify these strains, the sequences from the D1–D2 regions of the 25 S rDNA of two cultures representing the two different ITS sequences were obtained. As expected, the two sequences showed a high degree of homology, 99.4 % in 820 bp.

Fig. 3. Phylogenetic tree showing the relationship between the illudinic acid producer (MF6183), one of the novel illudins I, I2 and J2 producers (F-062769) and several Coprinus species. Accession numbers are indicated for the sequences retrieved from GenBank. Bootstrap indexes (higher than 50 %) are indicated above each branch node.

The comparison of these sequences with those stored in GenBank showed a high homology with sequences from members of the genus Coprinopsis, a lineage within Coprinus s. lat. now segregated and referred to the Psathyrellaceae (Redhead et al. 2001). The best match was C. gonophylla, with 99.9 and 99.3 % of similarity for a representative strain of the nine identical cultures (F-062769) and the illudinic acid producer, respectively. A phylogenetic tree based on parsimony analysis of the D1–D2 nucleotide sequences of these fungi and other Coprinopsis spp. was constructed (Fig. 3). The two producers fell in the same branch with C. gonophylla (bootstrap index 85 %). All the species of the clade supported with 53 % bootstrap (Fig. 3), are members of Coprinopsis (syn. Coprinus sect. Picacei ; Citerin 1992, Redhead et al. 2001). The basal position of C. utrifer in this clade is consistent with its former placement in a different subsection (Nivei) of Coprinus sect. Picacei from the rest of the species in that group (subsect. Alachuani), according to Ulje´ & Bas (1988, 1991). In an attempt to induce the formation of fruit bodies from the producing isolates, which would permit a more accurate identification, the ten isolates were grown on a rice-based medium. However, only one of the nine identical isolates (F-050152) developed basidiomata after 30 d incubation. The other strains never fruited, even after prolonged incubation periods, in different culture media and changing incubation parameters (light, temperature) nor after pairing mycelial mats. Morphological examination of the basidiomata identified the fungus as Coprinopsis episcopalis (syn. Coprinus episcopalis, Orton 1957, Orton & Watling 1979, Citerin 1992) based on the following combination of characteristics (Fig. 4).

Illudin-like compounds produced by basidiomycetes

1206

Fig. 4. Micromorphology of Coprinopsis episcopalis (F-050152) fruited in pure culture. (A–C) Basidiospores. (D) Veil hyphae. (E–F) Marginal cystidia. (G) Facial cystidia. Bar=10 mm.

Pileus cylindrical to conical when young, pale to grey with paler or greyish cream centre, first covered by a white veil, then grey, breaking up into apressed patches on disk yellowish brown. Lamellae white when young, finally black. Stipe white with bulbous base. Marginal cystidia vesiculose to clavate, 30–40(–60)r16–30 mm. Facial cystidia fusiform-cylindric often narrowed at the apex, 90–180r20–60 mm. Veil of hyaline branched clamped hyphae up to 9 mm broad. Cap cuticle of cylindric cells up to 30 mm broad, often with thick brownish walls. Clamp connections present. Basidia 4-spored. Basidiospores ellipsoid-amygdaliform to mitriform, 7–10r7–8r5–6.5 mm, with a central truncate germ pore. Coprinopsis episcopalis and C. gonophylla are closely related species. Both belong to the same section (Picacei) within subgen. Coprinus, and both have been described as similar to a small C. picacea (Orton 1957, Citerin 1992). However, they differ in some microscopical features, such as the mitriform shape of the spores of C. episcopalis, while in C. gonophylla spores are ovoid; and in their habitat, C. gonophylla is a carbonicolous species, whereas C. episcopalis has been reported from forest soils. The set of isolates described in this work provides evidence that C. episcopalis has a wider ecological

distribution than previously reported, being detected as an endophyte of Quercus ilex and other plants, and in leaf litter of Q. pyrenaica. The observation that some of the strains of C. episcopalis were isolated from several fruit bodies of basidiomycetes, collected in different locations and isolated at different dates, raises the interesting question of whether this species may also exhibit mycoparasitic behaviour. The isolates were from apparently clean and undamaged internal tissues of the fruit bodies. However, it is impossible to say if these isolates could have been derived from spores deposited in the external surface of the sample, during its manipulation in the laboratory, or from internally parasitic mycelium. More systematic studies would be needed to address the reason for the association of C. episcopalis with basidiomycetes of other species. Distribution of illudanes among filamentous fungi The producers of illudin-like compounds reported in the literature are distributed among a very few species of Basidiomycota. In spite of intensive screening of Poriales, Stereales and other groups (Table 1) which are known to produce other types of protoilludane derivatives (Turner & Aldridge 1983), these compounds were not detected in this screening. All the known producers of illudin-like compounds belong to

A. Gonzalez Del Val and others

1207

Table 3. Distribution of illudin-like compounds among basidiomycetes. Species

Family

Order

Illudin type

Reference

Omphalotus illudens or O. oleariusa

Paxillaceae

Boletales

M, S

O. olivascens

Paxillaceae

Boletales

O. nidiformis

Paxillaceae

Boletales

Clitocybe phosphorea Coprinopsis atramentaria C. episcopalis Coprinopsis sp.

Tricholomataceae Psathyrellaceae Psathyrellaceae Psathyrellaceae

Agaricales Agaricales Agaricales Agaricales

Anchel et al. (1950) Hara et al. (1987) McMorris & Anchel (1965) McMorris et al. (2000) Morisaki et al. (1985) Nakanishi et al. (1963) Turner & Aldridge (1983) Arnone et al. (1991) Kirchmair et al. (1999) McMorris et al. (1989) Burgess et al. (1999) Kirchmair et al. (1999) Cardillo et al. (1992) Lee et al. (1996) This work This work

A, B M, S S F, G, H M, S M C, C2, C3 I, I2, J2 Illudinic acid

a O. illudens and O. olearius have been frequently taken as the same species, and there are several synonyms in the literature, e.g. Clitocybe olearia (Turner & Aldridge 1983, Morisaki et al. 1985, Arnone et al. 1991); Clitocybe illudens (Anchel et al. 1950, McMorris & Anchel 1965); Lampteromyces japonicus (Nakanishi et al. 1963) and Pleurotus japonicus (Hara et al. 1987). However, Hughes & Petersen (1998) have reported that O. illudens and O. olearius should be considered as different species based on analysis of restriction sites in the ribosomal ITS1-5.8 S-ITS2 region.

(a)

(b) O

O

O

OH

HO

HO COOH OH

Illudin A

Illudinic acid O

OH

OH

O

O

Illudin G

OH

O HO

HO

OH

CH2OH OH

OH

OH

Illudin C

Illudin I

Illudin B

O

O

OH

OH Illudin F

O

O HO

CH2OH

CH2OH

OH

OH

Illudin C2

Illudin I2

O

O

OH

OH

Illudin D

Illudin M O

O

OH HO

HO CH2OH

CH2OH

OH

OH

Illudin C3

Illudin J2

OH Illudin H

CH2OH OH

OH Illudin S

Fig. 5. Structures of illudin-like compounds isolated from : (a) Coprinopsis spp. ; and (b) Omphalotus spp.

Illudin-like compounds produced by basidiomycetes two relatively distant genera, Omphalotus (in Marasmiaceae) and Coprinopsis (in Psathyrellaceae ; Table 3). Furthermore, both groups produce different classes of illudins. Omphalotus spp. have been reported to produce illudins A, B, M, F, G, H and S, whereas Coprinopsis spp. are able to produce illudin C, I, I2, J2 and illudinic acid. That these compounds are not reported from other segregates of Coprinus s. lat. appears to support the taxonomy proposed by Redhead et al. (2001). Although Clitocybe phosphorea has been also reported to produce illudin M (Cardillo, Nasini & Vajna De Pava 1992), it is possible that this species could also be an Omphalotus, because other luminescent species of Clitocybe have been synonymized with Omphalotus (Table 3 footnote ; Singer 1986). All the illudins produced by Omphalotus species (except for illudin D) are hydroxylated in positions 2 and 6 (Fig. 5), whereas those isolated from Coprinopsis species are not, suggesting genus-specific biosynthetic pathways for these compounds.

ACKNOWLEDGEMENTS We are grateful for the technical assistance from the CIBE-MSD technicians who participated in this study, especially for their help with the assays, the preparation of the samples, and the fermentation of the producing organisms. We would also like to thank Karen Overbye and Gail Hammond for providing bacterial strains and to Gerald F. Bills for his critical review of the manuscript.

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Corresponding Editor: S. A. Redhead