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The Connecticut Agricultural Experiment Station, Valley Laboratory, 153 Cook Hill Road, Windsor, Connecticut 06095
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ABSTRACT |
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The holotype of Stachybotrys cylindrospora was examined and the morphological characters were found to fit the description of Stachybotrys chartarum. Thus Stachybotrys cylindrospora is a synonym of S. chartarum. However a number of isolates and specimens subsequently described and studied by several mycologists have typical cylindrical conidia with longitudinal striations. The conidia are much longer than those of S. chartarum. These conidial characters showed that those isolates and specimens are notably different from S. chartarum and of the holotype of S. cylindrospora. Therefore a new name, Stachybotrys eucylindrospora sp. nov., is proposed to accommodate these isolates and specimens.
Key words: Stachybotrys chartarum, synonym, type
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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collected an isolate of Stachybotrys from arable soil in North Cohocton, New York, in Aug 1911 and described it as a new species, S. cylindrospora, in 1912. Bisby (1943) suggested that S. cylindrospora might be a synonym of S. chartarum. Rayss and Borut (1958) demoted S. cylindrospora to Stachybotrys atra var. cylindrospora (C.N. Jensen) Rayss & Borut. Barron (1961) isolated several strains in Canada, which were thought to fit the description of S. cylindrospora. However the unusually wide range of S cylindrospora conidia size, which spanned the range of conidial size of S. chartarum, raised the question about validity of the species. The holotype specimens of S. cylindrospora of C.N. Jensen, the specimen of Stachybotrys atra var. cylindrospora of Rayss and Borut and additional specimens of S. cylindrospora and S. chartarum were borrowed from several major herbaria. The objective of the present study was to re-examine S. cylindrospora and to determine its taxonomic status. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). The specimen IMI 76515 of Stachybotrys atra var. cylindrospora, which Borut deposited in IMI was examined (TABLE I). Specimens of S. cylindrospora and S. chartarum were borrowed from IMI, DAOM, CUP and BPI for comparative examination (TABLE I).
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, Haugland et al 1999).
An isolate, ATCC 18851 ( = IMI 85334 and OAC 8603), under the name of S. cylindrospora (TABLE I
) was cultured on malt-extract agar (MEA: 15 g malt-extract broth (Difco), 15 g agar (Oxoid), 0.075 g chloramphenicol (Fisher), 750 mL distilled water, 0.75 mL trace metal solution (1 g ZnSO4 · 7H2O, 0.5 g of CuSO4 · 5H2O, 100 mL distilled water, 1 mL 1N NaOH) and cornmeal agar (CMA: 12.75 g cornmeal agar (Difco), 0.075 g chloramphenicol (Fisher), 750 mL distilled water) at 25 C for 15 d to observe its characteristics. It is the same isolate used by Haugland et al (1999, 2001) in their studies of QPCR and phylogenetic relationships of Stachybotrys spp.
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RESULTS |
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is reproduced (FIG. 1), while his original description of the species Jensen (1912) is quoted here: "Colonies round, thin, diffuse, becoming black with age; mycelium branched, septate, hyaline, 0.5–3 µ ; conidiophores hyaline at base, fuliginous toward apex, branched, sepate, attenuate toward tip, 40–65 µ high, bearing on the summit from 3.9 sterigmata; sterigmata subclavate, with or without short papilla, 8–11 by 4–5 µ ; conidia borne singly, smooth, subcylindrical to sometimes ovate, 6–16 by 3.8–5 µ , hyaline when young, becoming fuliginous and nearly opaque with age."
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). Most conidia oval, ellipsoid, (7.4–)7.5–11.1(–12.8) (mean = 9.8 ± 1.3) x (2.4–) 3.5–5.6(7.4) (mean = 4.5 ± 1.1) µ m, length/width ratio 2.3; while a few were subcylindrical, or globose. Mature conidia were roughened with ridged ornamentation on the surface, olivaceous (FIGS. 2, 3), while immature conidia were lighter in color and smooth or minutely roughened. The conidia were not truly cylindrical. It is unambiguous that these characteristics match that of S. chartarum because the conidia of S. chartarum are colorless when young, becoming dark olivaceous when mature, ellipsoid, smooth or verrucose with ridged ornamentation on the surface, 7–12 x 4–6 µ m (Jong and Davis 1976).
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Rayss and Borut (1958) described Stachybotrys atra var. cylindrospora as conidiophores 40–70 x 2.5–3.5 µ m; phialides 8–11 x 3.5–4.5 µ m; conidia cylindrical, 9–11 x 2.5–5 µ m. There is no indication that Rayss and Borut (1958) observed the type materials of S. cylindrospora. According to their description and the photograph in their paper, the isolate appears to be S. chartarum. An examination of IMI 76515, deposited by Borut, confirmed that S. atra var. cylindrospora is in fact S. chartarum. The conidia of IMI 76515 had a ridged surface, most were not cylindrical and a few were subcylindrical (FIG. 4). The significant variation among the isolates of S. chartarum and the conidial characters of IMI 76515 do not warrant its separation as a variety of S. chartarum.
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The comparative examination of specimens under the name of S. cylindrospora from BPI, CUP, DAOM and IMI showed that six out of eight specimens matched the isolate ATCC 18851 but not the holotype of S. cylindrospora (CUP-005925). DAOM208271 is S. chartarum. IMI 152718 and BPI 525146 do not fit either ATCC 18851 or CUP-005925. Conidia of IMI 152718 are smooth, oval, not cylindrical or subcylindrical. BPI 525146 is a mounted slide, which appears to match the isolate ATCC 18851 in size and shape but has no striation on the surface of conidia. The true identity of these two specimens remains to be clarified. Specimens DAOM 70309 and IMI 79062 originally identified as S. atra were found to match the isolate ATCC 18851.
According to the morphological characters, and QPCR analysis of the type of S. cylindrospora, the author concluded that the name S. cylindrospora proposed by Jensen is a synonym of S. chartarum. The specimen, description and photograph of S. atra var. cylindrospora all are reminiscent of S. chartarum. In the author’s opinion the conidial dimensions of IMI 76515 are within the variation of S. chartarum and do not warrant it as a new variety. However the isolate ATCC 18851 collected by Barron represents a distinct species of Stachybotrys. DNA sequence data of the isolate ATCC 18851 and analysis of phylogenetic relationships among 12 Stachybotrys and two species of Memnoniella supported the finding of the isolate ATCC 18851 as a new species (Haugland and Heckman 1998, Haugland et al 2001). Therefore the author proposes a new species.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Fungi mitosporici, Hyphomycetes.
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Conidiophora erecta, simplicia, septata, macronemata, solitaria vel interdum fasciculata, determinata, recta vel exigue curvata, deinde ramosa, prope apicem verrucosa et fusca, prope basi hyalina et latvia, usque ad 200 µ m longa et 3–5 µ m crassa.
Cellulae conidiogenae phialidicae, determinatae, discretae, laeviae, subclavatae, prope apicem fuscae olivaceae, (11.1–)12.4–14.6(–15) (Med. 13.5) x (2.5–)2.9– 4.5(–5.2) (Med. 3.6) µ m, collulo conspicuo praeditae, 4–7 in verticillo dispositae.
Conidia cylindrica vel cylindroellipsoidea, primo hyalina et laeviae, deinde atro-olivaceobrunnea, basi ± truncate, longitudinaliter striata, (10.3–)12.8–16(–18.5) (Med. = 14.4 ± 1.6, n = 51) x (2.5–)3.4– 5.5(–6.6) (Med. 4.4 ± 1) µ m, biguttata, in massam mucosam nigram lecta.
Teleomorphosis ignota.
Holotypus DAOM 87664 isolatus per GL Barron de humus, palus, ad Guelph, Ontario, Canada, de Nov 1960. Isotypus IMI 85334, CBS 203.61, et OAC 8603. Vivita cultura sustentare apud ATCC (ATCC 18851).
Colonies attaining 24–26 mm diam in 15 d at 25 C on malt-extract agar, becoming light yellow where no sporulation or grayish where sporulation of conidia occurred, granular, reverse light brown at the center (FIG. 5); attaining 45–46 mm diam in 15 d at 25 C on cornmeal agar, pale and velutinus, sporulation commencing within 3 d of incubation (FIG. 6).
Conidiophores determinate, macronematous, solitary or in groups, erect, straight or slightly curved, simple or irregularly branched, 3–5-septate, verrucose and slightly olivaceous at the upper part, hyaline and smooth at the lower part, variable in length, up to 200 µ m, 3–5 µ m wide, occasionally swollen to 7 µ m toward the base (FIGS. 7, 8).
Phialides determinate, discrete, subclavate, smooth, dark olivaceous at the tip, (11.1–)12.4–14.6(–15) (mean = 13.5 ± 1.1, n = 30) x (2.5–)2.9–4.5(–5.2) (mean = 3.6 ± 0.7) µ m, with or without conspicuous collarettes, unicellular, 4–7 in groups (FIGS. 7, 8).
Conidia cylindrical or cylindrical ellipsoid, rounded at the apex, rounded or truncate at the base, hyaline and smooth at first and becoming gray to dark olive gray when mature, with delicate longitudinal striations on the conidial surface (the striations visible only under oil lens), unicellular, (10.3–)12.8–16 (–18.5) (mean = 14.4 ± 1.6, n = 51) x (2.5–)3.4–5.5(–6.6) (mean = 4.4 ± 1) µ m, ratio of length/width 2.6–4.2 (mean = 3.4), often biguttulate, especially when young, aggregated in slimy masses (FIG. 9).
Teleomorph unknown.
Holotype: – DAOM 87664 collected by GL Barron from peat soil, cedar bog, near Guelph, Ontario, Canada, in Nov 1960. Isotypes are IMI 85334, CBS 203.61, and OAC 8603. Living cultures maintained at ATCC (ATCC 18851).
Etymology: – The specific epithet is chosen to indicate the unambiguous cylindrical shape of conidia of this species. The prefix "eu" is used for the purpose of distinguishing this proposed name from S. cylindrospora, one of the synonyms of S. chartarum.
Distribution: –
Canada, Japan, UK, Germany, Solomon Islands (Jong and Davis 1976, Matsushima 1971, Matsushima 1975).
Habitat: on Carya ovata husks, Dahlia stems, Heracleum sp., stems of Ligustrum officinale, peat soil, cedar bog, Populus log.
Specimens examined:.
CANADA. ONTARIO: St Lawrence Islands National Park, MacDonald Island. Carya ovata husks, 23 Jul 1979, SJ Hughes, DAOM 172376g. CANADA. ALBERTA: Waterton National Park, Aspen Parkland Picnic Area. On Populus log, 4 Aug 1980, GP White-696, DAOM 176800a (2 packets). CANADA, BRITISH COLUMBIA: South Burnaby. Dahlia stems, 9 Aug 1957, SJ Hughes, DAOM 56386d (2 packets). CANADA. ONTARIO: Ottawa. On stems of Ligustrum officinale, 26 May 1983, WI Illman, DAOM 186941. CANADA. ONTARIO: Nr. Guelph. From peat soil, cedar bog, Nov 1960, GL Barron, DAOM 87664. CANADA. QUEBEC: MacDonald College. From soil, 1 Jun 1955, OA Olsen, DAOM 70309 (labeled as S. atra). United Kingdom: ENGLAND, YORKSHIRE: Gundale. From Angelica sp., 18 Oct 1959, WG Bramley, IMI 79062 (labeled as S. atra). United Kingdom: ENGLAND, YORKSHIRE: Pickering. From Heracleum sp., 3 Jul 1964, WG Bramley, IMI 107222 (TABLE I). Other specimens and cultures examined are listed (TABLE I).
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DISCUSSION |
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described his S. cylindrospora with such a large range in conidial size (6–16 µ m) because the re-examination of his holotype showed that the size in fact is 7.5–11.1 x 3.5–5.6 µ m. Jensen (1912) described these conidia as smooth and subcylindrical, not cylindrical as indicated by the epithet of S. cylindrospora. The conidia from his holotype, on close observation, were found to be roughened with ridged ornamentation and were mostly oval or ellipsoid. These discrepancies might have originated from use of a culture that was not mature and might not have allowed him to observe all the conidial characters, such as the roughness. Because a live culture of CUP-005925 does not exist, it prevented the author from making more extensive examinations and conducting other analyses. These discrepancies remain to be clarified.
Barron (1961) obtained several strains (Nos. 8602, 8603, 8604) from organic soils at several localities in Ontario, Canada, and considered these isolates morphologically to match S. cylindrospora. He suggested that S. cylindrospora is a valid species. However he noticed that his isolates differed distinctively from Jensen’s description in two characters. First, conidia were primarily cylindrical, 12–16 x 4–5.5 µ m and the ratio of length/width of conidia was 2.5–4. Jensen’s inaccurate description and sketchy illustration (FIG. 1) had led Barron (1961) to explain the discrepancy as strain variation. Second, mature conidia of Barron’s isolates show fine striations running obliquely lengthwise along the conidia under the oil lens (Barron 1961). The striation on the conidial surface was the important character observed by Barron and others (Barron 1961, Jong and Davis 1976, Matsushima 1975). Barron did not examine the type material of S. cylindrospora.
Verona and Mazzucchetti (1968) published a monograph of Stachybotrys and Memnoniella. Their description of S. cylindrospora was based on that of Jensen (1912). However their illustration was redrawn from Barron’s illustration. There was no indication which specimens were examined.
Jong and Davis (1976) described S. cylindrospora according to their observation of the cultures ATCC 18851 and ATCC 16276 isolated by W. Gams from wheat field soil in Kittzeberg, Germany, and determined by G.L. Hennebert as S. cylindrospora. Understandably their description was in agreement with Barron’s. Ellis (1971) and Matsushima (1975) also agreed with Barron (1961) that S. cylindrospora is valid species. The S. cylindrospora studied in these publications is S. eucylindrospora.
Two phylogenic studies with DNA sequence data of 12 species of Stachybotrys, two species of Memnoniella, nine species of Stachybotrys and 3 species of Memnoniella respectively elucidated that the isolate ATCC 18851 under the name S. cylindrospora was well delineated from other Stachybotrys spp. including S. chartarum (Haugland and Heckman 1998, Haugland et al 2001). When the species specific primers and probe were developed for detecting S. chartarum sensu lato, the isolate ATCC 18851 was included as a species different from S. chartarum along with other species of Stachybotrys (Haugland and Heckman 1998). This confirms that the isolates observed by Barron (1961), Jong and Davis (1976) and Matsushima (1978) are different from the type material of S. cylindrospora and composed of a valid species, S. eucylindrospora. The quantitative result of 34 000 spore equivalents from QPCR analysis on the holotype of S. cylindrospora (CUP-005925) using S. chartarum sensu lato primers and probe verified the conclusion derived from morphological observation that S. cylindrospora is a synonym of S. chartarum.
Bisby’s (1945) opinion that S. cylindrospora is possibly a synonym of S. chartarum was correct. As Bisby (1943) indicated S. chartarum has extreme variability under standard culture conditions. Such variability has led to some confusion in species delineation among Stachybotrys. Anderson et al (2003) studied S. chartarum sensu lato with morphological, chemical and phylogenetic methods and subsequently described a new species, S. chlorohalonata Andersen et Thrane sp. nov. S. chlorohalonata differs morphologically from S. chartarum by developing smooth conidia that are ellipsoidal to broadly ellipsoidal and papillate at the basal end, in comparison with those of S. chartarum, which have a rough surface and are slightly longer, ellipsoidal (Anderson et al 2003). The holotype of S. cylindrospora with rough conidia matches S. chartarum sensu stricto and not its sister species, S. chlorohalonata. The study of Andersen et al (2003) refined the concept of S. chartarum sensu stricto. The species might not be as variable as it was considered before.
Isolates or specimens of S. cylindrospora also were reported from the USA (Gilman 1957, Matsushima 1975). Because specimens or cultures are not available for examination their true identities cannot be confirmed as either S. eucylindrospora or S. chartarum.
Other species of Stachybotrys have cylindrical and subcylindrical conidia. Conidia of S. yunnanensis are cylindrical or subcylindrical, but they are smaller (7.0–11 x 3.5–5.5 µ m) than those of S. eucylindrospora, which are smooth, or occasionally rough (Kong 1997). The cylindrical conidia of S. longispora are 8.8–12 x 2–2.4 µ m without ornaments or striations and L/W ratio >4 (Matsushima 1971).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: dewei.li{at}po.state.ct.us
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Barron GL. 1961. Studies on species of Helicodendron, Oidiodendron and Stachybotrys from soil. Can J Bot 39: 1563–1571.
Bisby GR. 1943. Stachybotrys. Trans Brit Mycol Soc 26:133–143.
———. 1945. Stachybotrys and Memnoniella. Trans Bri. Mycol Soc 28:11–12.
Ellis MB. 1971. Dematiaceous Hyphomycetes. Wallingford, UK: CABI.
Gilman JC. 1957. A manual of the soil fungi. 2nd ed. Ames, Iowa: Iowa State College Press.
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.[CrossRef][Medline]
———, Vesper SJ, Wymer LJ. 1999. Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqManTM fluorogenic probe system. Mol Cell Probes 13:329–340.[CrossRef][Medline]
———, ———, Harmon S. 2001. Phylogenetic relationships of Memnoniella and Stachybotrys species inferred from ribosomal DNA sequences and evaluation of morphological features for Memnoniella species identification. Mycologia 93:54–65.[CrossRef]
Jensen CN. 1912. Fungous flora of the soil. Bull Cornell U Ag Exp Stat 315:415–501.
Jong SC, Davis EE. 1976. Contribution to the knowledge of Stachybotrys and Memnoniella in culture. Mycotaxon 3: 409–485.
Kong H-Z. 1997. Stachybotrys yunanensis sp. nov. and Neosartorya delicata sp. nov. isolated from Yunnan, China. Mycotaxon 62:427–433.
Matsushima T. 1971. Microfungi of the Solomon Islands and Papua-New Guinea. Osaka, Japan: Nippon Printing & Publishing Co. 78 p + 169 figs. and 48 pls.
———. 1975. Icones Microfungorum: a Matsushima lectorum.. Kobe, Japan: Published by the author. 415 p.
Rayss T, Borut S. 1958. Contribution to the knowledge of soil Fungi in Israel. Mycopathol Mycolo Applicata (Mycopathologia) 10:142–174.
Verona O, Mazzucchetti G. 1968. I Generi Stachybotrys E Memnoniella. Roma: Publicazioni Dell’Ente Nazionale, Per La Cellulosa E Per La Carta, Laboratorio Di Cartotecnica Speciale. p 1–110.
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