This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Andersen, B. Articles by Jarvis, B. B. Search for Related Content PubMed Articles by Andersen, B. Articles by Jarvis, B. B. Agricola Articles by Andersen, B. Articles by Jarvis, B. B.

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

     Mycology Group, BioCentrum-DTU, Søltofts Plads, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

Tim Szaro
John W. Taylor

     Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102

Bruce B. Jarvis

     Department of Chemistry and Biochemistry and the Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20742

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.

Key words: atranones, beta-tubulin, chemotypes, chitin synthase, metabolite profiles, morphological species, phylogenetic species, roridins, satratoxins, sick-building syndrome, trichodiene synthase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Most Stachybotrys isolates found in water-damaged buildings in recent years have been recorded in the literature as S. chartarum (Ehrenb.) Hughes, or as its synonym S. atra Corda (Jarvis et al 1986; Johanning et al 1993; Nikulin et al 1997; Jarvis et al 1998; Vesper et al 1999, 2000b). However, different S. chartarum isolates from water-damaged buildings are reported in the literature to have different toxic, inflammatory and/or immunological effects (Jarvis et al 1998; Fung et al 1998; Routsalainen et al 1998; Vesper et al 1999, 2000a), which have resulted in taxonomic and medical confusion.

Jong and Davis (1976) reviewed Stachybotrys and treated 15 species primarily based on examination of living cultures. Their work has been the starting point for modern Stachybotrys systematics. Since then at least 25 additional Stachybotrys species have been described. Although there are now more than 40 described Stachybotrys species, only a few are reported frequently in literature.

Results of a study by Andersen et al (2002) of Stachybotrys isolates from water-damaged buildings in Northern Europe and the United States showed that isolates segregated into two distinct groups based on morphology, physiology and chemistry. Cruse et al (2002) independently showed a similar segregation into two distinct groups of another set of Stachybotrys isolates from the U.S.A., based on DNA sequence analyses. A collaboration was initiated to determine the relationship between these groups of isolates and their taxonomic placement. This paper reports the results of a combined phenotypic and phylogenetic study and the description of a new species of Stachybotrys.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Fungal description – Stachybotrys isolates were inoculated in three points (as described by Singh et al 1991) on cornmeal agar (CMA, DIFCO 1969) and Czapek yeast autolysate agar (CYA, Samson et al 2002) media. The unsealed, vented plates were put in perforated plastic bags and incubated for 7 d at 25 C in the dark. Micromorphological observations were made from CMA cultures, whereas cultural descriptions were based on CYA cultures. The isolates first were examined directly on the CMA plate and then mounted in a drop of lactophenol using tape preparations (Butler and Mann 1959) and examined at 400x and 1000x magnification. All isolates are held in the IBT culture collection at BioCentrum-DTU, Denmark, and maintained as dried soil cultures.

Physiological characterization – The isolates were three-point inoculated on alkaloid-forming agar (ALK) (Reshetilova et al 1992), CYA, potato-sucrose agar (PSA) (Samson et al 2002), Sigma yeast-extract sucrose agar (SYES) (Filtenborg et al 1990), and V8 juice agar (V8) (Simmons 1992) media. The inoculated, unsealed plates were put in perforated plastic bags and incubated in the dark at 25 C. After 7 d the colony diameter and pigment production were recorded as described by Andersen at al (2002). PSA cultures were re-incubated for an additional 7 d before they were used for chemical characterization.

Chemical characterization – Cultures on PSA were extracted after 14 d of growth at 25 C in the dark. Five to six agar plugs, approx. 1.5 cm² of colony and agar, were extracted with methanol as described in Andersen et al (2002) but without the polyethylene imine (PEI) clean-up step.

Methanol extracts (5 µL) were analyzed by liquid chromatography—photo diode array detection—positive electro spray high-resolution mass spectrometry (Nielsen and Smedsgaard 2003). Samples were separated by reversed-phase chromatography on a C₁₈ column by a water-acetonitrile gradient system on an Agilent HP 1100 Liquid Chromatograph (Waldbronn, Germany) interfaced with a Micromass LCT (Manchester, United Kingdom) Time of Flight mass spectrometer (MS). The MS was tuned to a resolution of 6000 and collected as centroid data from m/z 100 to 900, with a scan time of 1 s. Potential difference between the two skimmers was set to 6 V, to minimize fragmentation of the labile trichothecenes.

The two atranone precursors, 3,4-epoxy-6-hydroxy-dolabella-7,12-diene-one and 6-hydroxydolaballa-3,7,12-trien-14-one, and the atranones A, B and F were detected as their protonated molecular ions, [M+H]⁺ by plotting m/z 319.23, 303.23, 417.23, 447.24, 433.22, respectively. The simple trichothecenes, trichodermol and trichodermin, were detected as [M+H]⁺ by plotting m/z 251.16 and 293.18, respectively. The macrocyclic trichothecenes, roridin E and epi-roridin E, were detected as their [M+NH₄]⁺ ion m/z 532.29, and satratoxins G, H, and iso-F, as well as roridin L-2 and hydroxyroridin E, as their [M+H]⁺ ions by plotting m/z 545.20, 529.24, 543.22, 531.26, 531.26, respectively. Specificity was achieved by using a window of m/z ± 0.04 of each of the mentioned ions.

Cluster analysis – A data matrix of 25 objects (Stachybotrys isolates) and 10 variables (colony diameters on the five media and five metabolite families) was constructed. The matrix was standardized (the mean of each variable was subtracted and then divided by the standard deviation of each variable) and analyzed using the Manhattan coefficient and unweighted pair-group method, arithmetic average (UPGMA) in NTSYS 2.02j (Applied Biostatistics Inc., New York).

Molecular characterization – Methods of growing mycelium for DNA extraction, DNA extraction, PCR amplification, DNA sequencing, sequence alignment and phylogenetic analysis are as described by Cruse et al (2002).

DNA sequencing – Mycelium was grown in yeast broth for 3–4 d, lyophilized and stored. Lyophilized mycelium was broken in a bead beater and DNA extracted using a CTAB protocol followed by the use of solvents and a Qiagen Dneasy kit. The trichodiene synthase 5 fragment (tri5), the beta-tubulin 1 fragment (tub1) and the chitin synthase 1 fragment (chs1) were PCR amplified using primers and conditions described in Cruse et al (2002). PCR products were prepared for sequencing with Qiagen's QIAquick PCR purification kit or with an isopropanol precipitation. Purified PCR product was sequenced with an ABI model 3100 Sequencer and ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California). Sequences were analyzed and aligned with Sequencing Analysis 3.0 and Sequence Navigator 1.01 (Applied Biosystems, Foster City, California).

Phylogenetic analysis – Sequences obtained from tri5, tub1 and chs1 were aligned and checked visually with Sequence Navigator 1.01. There were no gaps in the alignments. The aligned sequences were exported to a NEXUS file and analyzed with PAUP 4.0b8 (Swofford 2001). All sequences were placed in a single NEXUS file and partitioned by each locus to create the complete dataset. These data then were analyzed by maximum parsimony using settings described in Cruse et al (2002). Analysis was done individually on each locus and on all loci combined. Heuristic searches were carried out with tree-bisection reconnection and 1000 random sequence additions. Support for internal branches was assessed with a heuristic parsimony search of 1000 bootstrapped datasets. The trees shown are rooted at the midpoint.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Stachybotrys chlorohalonata Andersen et Thrane, sp. nov. Figs. 1–6

View larger version (86K): [in this window] [in a new window]   FIGS. 1–6. Stachybotrys chlorohalonata. 1. Sporulation pattern directly on CMA (no scale bar). 2–3. Simple and branched conidiophores and phialides on CMA (scale bar = 50 µm). 4–6. Conidiophore, phialides and conidia on CMA (scale bar = 20 µm)

In agaris CMA et CYA descripta. Coloniae in CMA 35 mm diam a 7d, 25 EC, luce excluso; mycelium superficiale, hyalinum, arachnoideum, capitulis atris, mucosis manifestis; pigmenta extracellulosa nulla. Coloniae in CYA a 7 d, 25 EC, luce excluso, 14 mm diam, glaucae, pubescentibus, margine angusto, albido; pigmento extracelluloso atroviridente, in agaro trans marginem coloniae 4–5 mm diffuso. Conidiophora in CMA erecta, stricta, vulgo ramosa, (0–)1–2(–3) septata, cellulis basalibus tumidis exortis, basi plerumque laevibus, hyalinis, saepe apice versus fuscatis et irregulariter verruculosis; vulgo 44–69 µm, basi ad 5 µm, apice deminutis. Phialides apicales, 3–5 fasciculatae, clavatae vel obovoideae, basi hyalinae, apice fuscatae, laevibus, eseptatae, monophialidicae, vulgo 8–11 x 4–6 µm. Conidia juvenilia ellipsoidea, citrino-viridia; matura late ellipsoidea vel obovoidea, basi saepe inconspicue papillata, eseptata, atroviridia, laevia, plerumque 8–10.5 x 4–5.5 µm. Teleomorphosis ignota.

Typus: pars ex cultura IBT 9467 ex tabula gypsea, lecta Kristian F. Nielsen, X-1997, Selandia, Dania, desiccata et in C 60160 (Holotypus) conservanda. Culturae ex typo IBT 9467, CBS 109285.

Colonies on CMA attain 35 mm diam after 7 d of growth at 25 C in the dark. Mycelium is superficial, hyaline and cobweb-like with visible black slime heads (Fig. 1) thinning toward the edge. No extracellular pigment is produced on CMA. Colonies on CYA are 14 mm diam after 7 d at 25 C in the dark. They are grayish green to dull green (color plates 28 E 4–5 in Methuen Handbook of Color, Kornerup and Wanscher 1978) with narrow white edges and a downy texture. A dark green to blackish green (color plates 27–28 F 6, Kornerup and Wanscher 1978) extracellular pigment is produced on CYA, which extends 4–5 mm beyond the edge of the colonies. On CMA, conidiophores are erect, straight or slightly flexuous and mostly branched once or twice with (0–)1–2(–3) septa (Figs. 2–3). Solitary conidiophores usually arise from swollen basal cells. Conidiophores are mostly smooth and hyaline at the base, often darker toward the apex, and the upper portion is sometimes irregularly verrucose (Fig. 5).

The whole conidiophore apparatus may be up to 100 µm long. Most conidiophores are 44–69 µm long from first point of branching to apex, up to 5 µm at the base and tapering toward the apex. Phialides, which are produced in groups of 3–5, are clavate to obovoid. They are smooth, aseptate and monophialidic. The phialides are hyaline at the base and darker toward the apex. Most phialides are 8–11 x 4–6 µm (Figs. 5–6). Immature conidia initially are ellipsoid and yellowish green, becoming broadly ellipsoidal to obovoid, often inconspicuously papillate at the base. Mature conidia are aseptate, blackish green, opaque and smooth. Most conidia are 8–10.5 x 4–5.5 µm (Figs. 4–6). Teleomorph unknown.

Habitat: Wet cellulose-containing material such as fabric, hay, seaweed, grain, paper and soil. Known distribution: Belgium, Denmark, Finland, Iraq, New Guinea, Spain and U.S.A.

Etymology: chlorohalonata, refers to green halo of extracellular pigment around the colonies that can be seen on CYA medium.

Type specimen. DENMARK: Sjælland, cardboard on gypsum board, OCT 1997, Kristian F. Nielsen. (HOLOTYPE: C 60160; Living cultures EX-TYPE: IBT 9467 (ALK pl. 2.), CBS 109285).

Additional cultures examined. Stachybotrys chlorohalonata (IBT 9225, IBT 9226, IBT 9293, IBT 9294, IBT 9299 [= CBS 109284], IBT 9714 [= HT-016], IBT 9755, IBT 9756, IBT 9757, IBT 9824, IBT 9826, IBT 9827, IBT 10219, IBT 40285, IBT 40287, IBT 40290, IBT 40292, IBT 40294, IBT 40295, IHEM 2248 [= ba 173], IHEM 9905 [= IBT 9754], ATCC 201860 [= JS51–08 = IBT 9823], ATCC 201863 [= JS58-06 = IBT 9825] and NRRL 29940 [= QM 94d = IBT 9767]).

Other species examined. Stachybotrys albipes (CBS 100343); S. bisbyi (CBS 142.97); S. chartarum (CBS 414.95); S. cylindrospora (IHEM 17451); S. dichroa (CBS 526.50); S. microspora (CBS 186.79); S. nephrospora (CBS 796.95); S. nilagirica (IHEM 17453); S. parvispora (CBS 173.97) and S. theobromae (IHEM 17456).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Morphology – In this study, a subset of 12 Stachybotrys isolates from Cruse et al (2002) and a subset of 13 isolates from Andersen et al (2002) were examined (Table I) and compared with isolates of 10 other Stachybotrys species from CBS and IHEM culture collections (see Taxonomy section). These cultures that arrived as S. albipes, S. bisbyi, S. chartarum, S. cylindrospora, S. dichroa, S. microspora, S. nephrospora, S. parvispora and S. theobromae all fit the descriptions of those species in Jong and Davis (1976), and S. nilagirica fit the description by Subramanian (1957). The culture from CBS culture collection, CBS 414.95 (= IBT 9460) and 14 other Stachybotrys isolates from Denmark and the U.S.A. (see Table I), were similar morphologically on CMA and fit the description of S. chartarum in Jong and Davis (1976).

The morphological appearance of these 10 questionably identified isolates, however, was consistent with a description of a "Stachybotrys sp." isolated from water in Iraq by Muhsin and Al-Helfi (1981). Unfortunately, it was not possible to obtain this isolate. A new Stachybotrys species, S. chlorohalonata Andersen & Thrane, therefore, has been described to accommodate these isolates. The S. chlorohalonata isolates in Table I and cultures cited in earlier works ("Stachybotrys sp. Group A" isolates in Andersen et al [2002] and "small clade" isolates in Cruse et al [2002]) are morphologically identical to S. chlorohalonata and have the same distinctive colony appearance on CYA. We have encountered more than of 25 Stachybotrys isolates that belong to S. chlorohalonata (See Additional cultures examined).

Morphologically on CMA, S. chlorohalonata most closely resembles S. chartarum sensu lato. The conidiophore apparatus of S. chlorohalonata are similar in appearance to that of S. chartarum, but individual conidiophores are shorter (up to 70 µm and 90 µm, respectively). The phialides of S. chlorohalonata also resemble those of S. chartarum but are shorter (see Table II). The conidia of S. chlorohalonata are ellipsoidal to broadly ellipsoidal with a smooth surface and papillate at the base, in contrast to those of S. chartarum, which are slightly longer, ellipsoidal and have a rough surface. Several Stachybotrys species are reported to produce smooth conidia, but S. chlorohalonata can be distinguished from these species either by its conidial shape and size and/or the shape and size of its phialides (see Table II).

View larger version (53K): [in this window] [in a new window]   FIG. 7. Dendrogram produced by Manhattan coefficient and UPGMA cluster analysis based on diameters on five media and five metabolite families. The top cluster contains S. chartarum chemotype A isolates (cha-A), the hatched cluster contains S. chlorohalonata isolates (chloro) and the bottom cluster S. chartarum chemotype S isolates (cha-S)

The metabolite production, recorded as metabolite families, of all 25 Stachybotrys isolates is shown in Table III. Spirocyclic drimanes (stachybotrys-lactams, lactones and dialdehydes and Mer5003 terpeneoids), produced by all 25 Stachybotrys isolates, were the dominant compounds in all metabolite profiles. All S. chlorohalonata and all S. chartarum (cha-A) isolates, except IBT 9754, produced atranones (A, B, F and E) and their precursors (3,4-epoxy-6-hydroxy-dolabella-7,12-diene-one and 6-hydroxy-dolaballa-3,7,12-trien-14-one), but the quantities of these varied by nearly three orders of magnitude. The LC-MS method used in this study showed that the three main trichothecenes produced by S. chartarum (cha-S) were satratoxin H, roridin E and L-2, followed by lower quantities of satratoxin G, iso-F and occasionally isosatratoxin H, verrucarins J and B, epiroridin E. A new metabolite, hydroxyroridin E, was found in all the extracts of S. chartarum (cha-S). Hydroxyroridin E tentatively was identified by accurate mass by LC-MS (m/z 531.2594 [M+H]⁺ and m/z 548.2860 [M+NH₄]⁺), retention time (4 min before roridin E, Nielsen and Smedsgaard 2003) and its UV-spectrum (almost identical to roridin E).

Differences in colony diameters distinguished S. chlorohalonata (chloro) from isolates of S. chartarum (cha-A), as seen in the dendrogram (Fig. 7). Growth of S. chlorohalonata isolates was more restricted on all five media than S. chartarum (cha-A and cha-S) isolates. The mean diameter (± twice the standard deviation) of S. chlorohalonata on CYA was 15 mm (± 4 mm) compared to the mean diameters of the cha-A isolates and the cha-S isolates of 23 mm (± 3 mm) and 20 mm (± 3 mm), respectively. It was not possible to differentiate between S. chartarum (cha-A) and S. chartarum (cha-S) on the basis of colony diameter alone, although the cha-A isolates appeared to grow slightly faster on all media than the cha-S isolates.

Phylogeny – The results of maximum-parsimony analysis based on the trichodiene synthase 5 gene (tri5), beta-tubulin 1 gene (tub1) and chitin synthase 1 gene (chs1) are shown in Figs. 8–10, respectively. The differentiation between S. chlorohalonata and S. chartarum was strongly supported by all three gene fragments. There were 28 fixed nucleotide substitutions between isolates in the two species for tri5, 27 for tub1 and 9 for chs1, for a total of 64 in the combined analysis (Fig. 11). In all single-gene genealogies, the branch separating the two species was supported in 100% of bootstrap resampled datasets, and the same result was found in parsimony trees based on the data from all three gene fragments. In S. chartarum, the cha-A and cha-S isolates were separated by a single nucleotide substitution in the tri5 gene fragment. With chs1, all cha-A isolates except one (IBT 9466) could be distinguished from the cha-S isolates by a single nucleotide substitution. With tub1, there were no consistent differences between cha-A and cha-S isolates. In the combined analysis, both the tri5 and chs1 single nucleotide substitutions supported a clade of cha-A isolates emerging from a nonmonophyletic assemblage of cha-S isolates, although the latter nucleotide substitution showed a reversal in isolate IBT 9466.

View larger version (20K): [in this window] [in a new window]   FIG. 8. The single most-parsimonious tree for tub1. The combined clade of cha-A plus cha-S and the chloro clade are monophyletic, however, neither cha-A nor cha-S, alone, are monophyletic

View larger version (20K): [in this window] [in a new window]   FIG. 9. The single most-parsimonious tree for tri5. The combined clade of cha-A plus cha-S, and the cha-A clade and the chloro clade are monophyletic. The clade cha-S is paraphyletic

View larger version (20K): [in this window] [in a new window]   FIG. 10. One of two most-parsimonious trees for chs1, both of which support the monophyly of the combined clade cha-S plus cha-A and the monophyly of the clade chloro. The difference between the two topologies involves the position of cha-S-07711, which groups with cha-A-09460 in the other most-parsimonious tree. Note that neither cha-A nor cha-S are monophyletic, due to the placement of cha-A-09466 among cha-S isolates

View larger version (23K): [in this window] [in a new window]   FIG. 11. One of 18 most-parsimonious trees for the combined data, all of which support the monophyly of the cha-A plus cha-S, cha-A (alone) and chloro clades. The variation in topology concerns the position of isolates cha-A-09466 and cha-A-09633 (three topologies), cha-S-07711 (two topologies), and chloro-09299 and chloro-09467 (three topologies). Note that cha-S is paraphyletic

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

Both phenotypic and phylogenetic methods have proven useful for recognizing fungal species because they have demonstrated cryptic species within a single morphological species or a species complex (Taylor et al 2000, Larsen et al 2001). These cryptic species often have distinctive and important phenotypes; for example, phylogenetic species found within the human pathogenic fungus Histoplasma capsulatum correlate in their ability to cause systemic or superficial disease and in their ability to cause disease in immuno-competent or immuno-compromised hosts (Kasuga et al 1999). Similarly, phylogenetic species within the toxigenic fungus Aspergillus flavus correlate in the size of their reproductive propagules (sclerotia) and in the production of mycotoxins (aflatoxins B and G) (Geiser et al 1998, 2000).

There are several types of phylogenetic species recognition (Mayden 1997), and the type applied in these studies and in previous work on S. chartarum (Cruse et al 2002) is by congruence of multiple gene genealogies. In recombining organisms, genealogies for different genes will conflict within species because different genes are inherited from different parents. However, as emerging species become genetically isolated, drift will reduce the ancestral variation in most genes to one ancestral allele, so that all newly developed alleles will coalesce to one ancestral allele. The result is that branches between species will be congruent for most gene genealogies and the shift from conflict to congruence in gene genealogies can be used to identify the limits of phylogenetic species (Avise and Ball 1990, Taylor et al 2000). The phenotypic approach used for Stachybotrys in this and previous studies (Andersen et al 2002) is based on the presence or absence of different metabolite families rather than quantities of individual metabolites and the use of growth characteristics under standardized growth conditions. These measures have been taken to minimize the influence of environmental factors, which always has been a major argument against the phenotypic species concept (Lumbsch 1998). Combinations of different and independent phenotypic characters in multivariate analyses can reveal cryptic species, often determined to be new species based on subsequent morphological and phylogenetic re-examination.

The separation of S. chlorohalonata from S. chartarum sensu lato, suggested by Andersen et al (2002) based on secondary metabolite production, colony diameter and morphology, is shown here to agree exactly with the separation suggested by Cruse et al (2002) based on three gene genealogies. In this case, morphological species recognition and phylogenetic species recognition arrived at the same conclusion and any of 64 fixed nucleotide substitutions in the three gene fragments could be used to distinguish the two species. We also have found that the two species can be recognized by the single ITS nucleotide polymorphism reported among isolates of S. chartarum by Haugland and Heckman (1998); for those individuals sampled, S. chartarum is represented by GenBank ITS sequence AF081469 and S. chlorohalonata by GenBank sequence AF081468.

Within S. chartarum, differences in metabolite production identified the S. chartarum chemotype S capable of producing satratoxins and phylogenetic analysis of the tri5 gene fragment identified the S. chartarum chemotype S as a monophyletic clade distinguished from S. chartarum chemotype A by a single nucleotide substitution in the tri5 gene fragment. With tub1, no distinction was seen; with chs1 all S. chartarum chemotype A isolates, except IBT 9466, had a common nucleotide substitution not seen in the S. chartarum chemotype S. The tri5 and chs1 nucleotide substitutions are important from a toxicological point of view because the two chemotypes of S. chartarum elicit very different toxicological responses (Nielsen et al 2001). It is interesting to note that it has been only S. chartarum chemotype A, the nonsatratoxin producer, that has been found in both air and on material samples from case homes in which infants were diagnosed with pulmonary hemosiderosis, both in Belgium (Nielsen, 2002) and the U.S.A. (Vesper et al 2000b, Johanning, Gareis and Nielsen unpubl).

In this study we showed the presence of a tri5 gene in all 25 Stachybotrys isolates examined, although the sequence in S. chartarum and S. chlorohalonata differed with 28 fixed nucleotide substitutions. In a study by Peltola et al (2002), the tri5 gene was amplified in S. chartarum but not in the group of Stachybotrys sp. isolates identical with Stachybotrys Group A (= S. chlorohalonata). The failure of the tri5 gene to amplify in isolates from Group A probably is due to the placement of the SCTOX5–1 primer (Peltola et al 2002) in a region now known to contain five nucleotide substitutions in the S. chlorohalonata sequence, as compared to the S. chartarum sequence (nucleotides 510–530 in GenBank sequences AF468155 and AF468154, respectively). None of the 10 isolates of S. chlorohalonata investigated in this study or the 17 isolates analyzed by Andersen et al (2002) produced satratoxins or any other macrocyclic trichothecenes, although it was possible to amplify their tri5 gene. The eight Stachybotrys isolates analyzed by Peltola et al (2002) did not produce satratoxins, either. However, 30% of all S. chlorohalonata isolates produced detectable quantities of trichodermol and trichodermin (simple trichothecenes), a result that correlates with the presence of the tri5 gene in S. chlorohalonata. The ability to produce trichodermol and trichodermin (the precursor for roridins and satratoxins), however, is not always consistent from inoculation to inoculation, a situation that was also observed with GC-MS/MS detection (Andersen et al 2002).

The improved LC-MS analysis method in this study revealed that satratoxin G and roridin L-2 co-elute in S. chartarum chemotype S extracts but that they can be distinguished by their different molecular masses and a difference of retention time of 0.09 min. Therefore, the peak that previously was interpreted as satratoxin G by LC-UV (Nielsen et al 2001, Andersen et al 2002) is mainly roridin L-2. Also, there has been very little focus on the spirocyclic drimanes, such as stachybotrys-lactams, lactones and di-aldehydes, Mer5003 terpenoids (Andersen et al 2002, Nielsen 2002) and the bisabosquals (Minagawa et al 2001). They are produced by S. chlorohalonata and both chemotypes of S. chartarum when growing on building materials, agar substrates and especially, on their natural habitat, hay and straw (Nielsen 2002).

This study has focused on the separation of S. chlorohalonata from S. chartarum. Further research is needed on the toxicity and phylogeny of the two chemotypes of S. chartarum. Because they have such different metabolite profiles, there likely will be more genes like the tri5 gene that can distinguish between these two important chemotypes of S. chartarum. We recommend these isolates as best representatives for the three taxa: S. chlorohalonata: IBT 9467, IBT 9825 and 103 (= IBT 40292); S. chartarum chemotype A: IBT 9290, IBT 14915 and 007 (= IBT 40288); and S. chartarum chemotype S: IBT 7711, 201 (= IBT 40293) and 206 (= IBT 40291).

	ACKNOWLEDGMENTS

 
This study is a part of the Danish "Mold in Buildings" research program partly supported by the Danish government and private companies through the Danish Research Agency. The analytical work was supported by the Danish Technical Research Council under Program for Predictive Biotechnology: "Functional biodiversity in Penicillium and Aspergillus" (Grant No. 9901295). The phylogenetic work was supported by grants from the NIH and NSF to JWT.

	FOOTNOTES

 
¹ Corresponding author. E-mail: ba{at}biocentrum.dtu.dk

Accepted for publication March 24, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Andersen B, Nielsen KF, Jarvis BB., 2002 Characterization of Stachybotrys from water-damaged buildings based on morphology, growth and metabolite production. Mycologia 94:392-403[Abstract/Free Full Text]

Avise JC, Ball RM.Jr, 1990 Principles of genealogical concordance in species concepts and biological taxonomy. In: Futuyma D, Antonovic J, eds. Oxford surveys in evolutionary biology Vol. 7. Oxford: Oxford University Press. p 45–67

Barron GL., 1961 Studies on species of Oidiodendron, Helicodendron and Stachybotrys from soil. Can J Microbiol 39:1563-1571

Batista AC, Bezerra JL, Peres GEP., 1960 Singera n. gen. e outros fungos Moniliales. Publicacoes do Instituto de Micologia da Universidade do Recife 298:1-33

———, Vital AF., 1957 Novas diagnoses de fungos Dematiaceæ. Anais da Sociedade de Biologia de Pernambuco 15:373-397

Bisby GR., 1943 Stachybotrys. Trans Brit Mycol Soc 26:133-143

Butler EE, Mann MP., 1959 Use of cellophane tape for mounting and photographing phytopathogenic fungi. Phytopathology 49:231-232

Cruse M, Teletant R, Gallagher T, Lee T, Taylor JW., 2002 Cryptic species in Stachybotrys chartarum. Mycologia 94:814-822. Erratum: Mycologia 95(3):559 (2003) [Abstract/Free Full Text]

DIFCO Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures. 1969 B386, p 246. DIFCO Laboratories Incorporated Detroit

Domsch KH, Gams W, Anderson T-H., 1980 Compendium of soil fungi. London: Academic Press. p 742–747

Dorai M, Vittal BPR., 1986 A new Stachybotrys from eucalyptus litter. Trans Brit Mycol Soc 87:642-644

Ellis MB., 1971 Dematiaceous hyphomycetes. Surrey: CAB Commonwealth Mycological Institute. p 540–545

———. 1976 More Dematiaceous Hyphomycetes. Surrey: CAB Commonwealth Mycological Institute. p 463–464

Filtenborg O, Frisvad JC, Thrane U., 1990 The significance of yeast extract composition on metabolite production in Penicillium. In: Samson RA, Pitt JI, eds. Modern concepts in Penicillium and Aspergillus classification. New York: Plenum Press. p 433–441

Fung F, Clark R, Williams S., 1998 Stachybotrys, a mycotoxin-producing fungus of increasing toxicological importance. Clinical Toxicology 36:79-86

Geiser DM, Dorner JW, Horn BW, Taylor JW., 2000 The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae. Fungal Genet Biol 31:169-179[Medline]

———, Pitt JI, Taylor JW., 1998 Cryptic speciation and recombination in the aflatoxin producing fungus Aspergillus flavus. Proc Natl Acad Sci (USA) 95:388-393[Abstract/Free Full Text]

Haugland RA, Heckman JL., 1998 Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum. Mol Cell Probes 12:387-396[Medline]

Jarvis BB, Lee Y-W, Comezoglu SN, Yatawara CS., 1986 Trichothecenes produced by Stachybotrys atra from Eastern Europe. Appl Env Microbiol 51:915-918[Abstract/Free Full Text]

———, Sorenson WG, Hintikka E-L, Nikulin M, Zhou Y, Jiang J, Wang S, Hinkley SF, Etzel RA, Dearborn DG., 1998 Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants. Appl Env Microbiol 64:3620-3625[Abstract/Free Full Text]

Johanning E, Morey PR, Jarvis BB., 1993 Clinical-epidemiological investigation of health effects caused by Stachybotrys atra building contamination. Proceedings in Indoor Air '93 1:225-230

Jong SC, Davis EE., 1976 Contribution to the knowledge of Stachybotrys and Memnoniella in culture. Mycotaxon 3:409-485

Kasuga T, Taylor JW, White TJ., 1999 Phylogenetic relationships of varieties and geographical groups of the human pathogenic fungus, Histoplasma capsulatum Darling. J Clin Microbiol 37:653-663[Abstract/Free Full Text]

Kong H-Z., 1997 Stachybotrys yunnanensis sp. nov. and Neosartorya delicata sp. nov. isolated from Yunnan, China. Mycotaxon 62:427-433

Kornerup A, Wanscher JH., 1978 Methuen handbook of color. 3rd ed. London: Methuen

Krzemieniewska H, Badura L., 1954 Przyczynek do znajomosci mikroorganizmów sciólki i gleby lasu bukowego (A contribution to the knowledge of the microorganisms from the litter and soil of beechwood). Acta Societatis Botanicorum Poloniae 23:727-781

Larsen TO, Svendsen A, Smedsgaard J., 2001 Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium. Appl Env Microbiol 67:3630-3635[Abstract/Free Full Text]

Lumbsch HT., 1998 The use of metabolic data in lichenology at the species and subspecies level. Lichenologist 30:357-367

Matsushima T., 1985 Matsushima Mycological Memoirs No. 4. Kobe. 68 p

———. 1989 Matsushima Mycological Memoirs No. 6. Kobe 100 p

Matsushima K, Matsushima T., 1995 Fragmenta Mycologica—I. Matsushima Mycological Memoirs 8:45-54

Mayden RL., 1997 A hierarchy of species concepts: the denouement in the saga of the species problem. In: Claridge MF, Dawah HA, Wilson MR, eds. Species the units of biodiversity. London: Chapman & Hall. p 381–424

McKenzie EHC., 1991 Dematiaceous hyphomycetes on Freycinetia (Pandanaceae). 1. Stachybotrys. Mycotaxon 41:179-188

Mercado-Sierra A, Mena-Portales J., 1988 Nuevos o raros hifomicetes de Cuba. V. Especies de Stachybotrys. Acta Botanica Cubana 55:1-8

Minagawa K, Kouzuki S, Nomura K, Kawamura Y, Tani H, Terui Y, Nakai H, Kamigauchi T., 2001 Bisabosquals, novel squalene synthase inhibitors—II. Physico-chemical properties and structure elucidation. J Antibiot 54:896-903[Medline]

Misra PC., 1975 A new species of Stachybotrys. Mycotaxon 2:107-108

———. 1976 Stachybotrys renispora sp. nov. Mycotaxon 4:161-162

———, Srivastava SK., 1982 Two undescribed Stachybotrys species from India. Trans Brit Mycol Soc 78:556-559

Morgan-Jones G, Karr GWJr., 1976 Notes on hyphomycetes. XVI. A new species of Stachybotrys. Mycotaxon 4:510-512

———, Sinclair RC., 1980 Notes on hyphomycetes. XXXIII. Stachybotrys sphaerospora sp. nov. from South Africa. Mycotaxon 10:372-374

Muhsin TM, Al-Helfi MA., 1981 Hyphomycetes of Iraq—the genus Stachybotrys. Sydowia 34:130-134

Munjal RL, Kapoor JN., 1969 Some hyphomycetes from the Himalayas. Mycopath Mycol Appl 39:121-128

Nielsen KF., 2002 Mould growth on building materials. Secondary metabolites, mycotoxins and biomarkers [Doctoral Thesis]. ISBN 87-88584-65-8. BioCentrum-DTU, Technical University of Denmark

———, Huttunen K, Hyvärinen A, Andersen B, Jarvis BB, Hirvonen M-R., 2001 Metabolite profiles of Stachybotrys spp. isolates from water damaged buildings, and their capability to induce cytotoxicity and production of inflammatory mediators in RAW 264.7 macrophages. Mycopathol 154:201-205

Nielsen KF, Smedsgaard J., 2003 Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for de-replication by standardised liquid chromatography—UV—mass spectrometry methodology. Chromatogr A 1002:111-136

Nikulin M, Reijula K, Jarvis BB, Veijalainen P, Hintikka E-L., 1997 Effects of intranasal exposure to spores of Stachybotrys atra in mice. Fund Appl Tox 35:182-188

Peltola J, Niessen L, Nielsen KF, Jarvis BB, Andersen B, Salkinoja-Salonen M, Möller EM., 2002 Toxigenic diversity of two different RAPD groups of Stachybotrys chartarum isolates analyzed by potential trichothecene production and for boar sperm cell motility inhibition. Can J Microbiol 48:1017-1029[Medline]

Reshetilova TA, Soloveva TF, Baskunov BP, Kozlovskii AG., 1992 Investigation of alkaloid formation by certain species of fungi of the Penicillium genus. Mikrobiologiya 61:873-879

Rifai MA., 1964 Stachybotrys bambusicola sp. nov. Trans Brit Mycol Soc 47:269-272

Ruotsalainen M, Hirvonen M-R, Nevalainen A, Meklin T, Savolainen K., 1998 Cytotoxicity, production of reactive oxygen species and cytokines induced by different strains of Stachybotrys sp. from mouldy buildings in RAW2647 macrophages. Env Tox Pharm 6:193-199

Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O, eds 2002 Introduction to food- and air borne fungi. 6th ed. Utrecht: Centraalbureau voor Schimmelcultures. 379 p

Simmons EG., 1992 Alternaria taxonomy: current status, viewpoint, challenge. In: Chelkowski J, Visconti A, eds. Alternaria: biology, plant diseases and metabolites. Amsterdam: Elsevier. p 1–35

Singh K, Frisvad JC, Thrane U, Mathur SB., 1991 An illustrated manual on identification of some seed-borne Aspergilli, Fusaria, Penicillia and their mycotoxins. Copenhagen: Danish Government Institute of Seed Pathology for Developing Countries. p 10–11

Subramanian CV., 1957 Hyphomycetes—IV. Proceedings of the Indian Academy of Sciences 46, no. 5, Sec. B:324–335

Swofford DL., 2001 PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4.0b8. Sunderland, Massachusetts: Sinauer Associates

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC., 2000 Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31:21-32[Medline]

Udaiyan K., 1991 Some interesting hyphomycetes from the industrial water cooling towers of Madras. J Econom Taxonom Bot 15:627-647

Vesper SJ, Dearborn DG, Elidermir O, Haugland RA., 2000a Quantification of siderophore and hemolysin from Stachybotrys chartarum strains, including a strain isolated from the lung of a child with pulmonary hemorrhage and hemosiderosis. Appl Env Microbiol 66:2678-2681[Abstract/Free Full Text]

———, ———, Yike I, Allen T, Sobolewski J, Hinkley SF, Jarvis BB, Haugland RA., 2000b Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemmorrhage: quantitative assessment before, during, and after remediation. J Urb Health 77:68-85[Medline]

———, ———, ———, Sorenson WG, Haugland RA., 1999 Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains. Appl Env Microbiol 65:3175-3181[Abstract/Free Full Text]

Whitton SR, McKenzie EHC, Hyde KD., 2001 Microfungi on the Pandanaceae: Stachybotrys, with three new species. New Zealand Journal of Botany 39:489-499

This article has been cited by other articles:

				D.-W. Li Stachybotrys eucylindrospora, sp. nov. resulting from a re-examination of Stachybotrys cylindrospora Mycologia, March 1, 2007; 99(2): 332 - 339. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Andersen, B. Articles by Jarvis, B. B. Search for Related Content PubMed Articles by Andersen, B. Articles by Jarvis, B. B. Agricola Articles by Andersen, B. Articles by Jarvis, B. B.