[ 49 ] Trans. Br. mycol. Soc. 90 (1), 49--54
Printed in Great Britain
VARIABILITY IN CLADOSPORIUM HERBARUM BY K. PRASIL* AND G. S. DE HOOG Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG Baarn, The Netherlands Variability of Cladosporium herbarum in culture is described, with emphasis on conidiophore morphology. Lateral and terminal conidiophores are distinguished, the microscopical appearance of strains differing due to the preponderance of either type. Lectotypification of C. herbarum from Persoon's collections is discussed. Twenty-three previously described infraspecific taxa are reviewed and evaluated on the basis of authentic material or original descriptions. Fourteen have been found to be of doubtful identity, while nine are indistinguishable from C. herbarum. In the literature about 540 taxa have been described in the anamorph genus Cladosporium Link: Fr. Many of these are plant parasites, others are ubiquitous saprophytes. De Vries (1952) and Ellis (1971, 1976) treated a rather large number of Cladosporium species, but as yet no taxonomic revision of the genus is available. In particular, the common saprophytes (among which is the generic type species, C. herbarum (Pers.: Fr.) Link) are variable in morphology. Many older described taxa may be mere morphological aberrants of the common species. The aim of the present paper is to describe the plasticity of C. herbarum in culture, to review described infraspecific taxa and to evaluate these on the basis of authentic material or original descriptions. A comparative study of variability of specimens on the natural substrate and fresh isolates in culture is intended in future, elaborating Janczewski's (1894) early studies. MATERIALS AND METHODS All 19 available CBS strains of C. herbarum and 5 of Mycosphaerella tassiana (de Not.) Johanson enumerated in the CBS List of Cultures were inoculated centrally on 4 % malt extract agar (MEA), potato dextrose agar (PDA) and cherry decoction agar (ChA) in 8 em Petri dishes and incubated at 20-22°C in daylight. Gross cultural and morphological characteristics were recorded. Variability of the strains CBS 121.47 (ex deepfrozen Phaseolus vulgaris pods, The Netherlands, J. C. Mooi), CBS 177.71 (ex Thuja tincture, The Netherlands; de Hoog, 1982) and CBS 399.80 (ex skin of human patient, The Netherlands, G. A. de Vries) was studied in more detail. Slides were made in lactic acid at regular intervals from cultures * Present address: Department of Botany, Charles University, Benatska 2, 12801 Prague, Czechoslovakia.
grown on the media mentioned above during a period of 30-35 d. For simple slide cultures, consisting of inoculated drops of agar covered with a glass slip, all media mentioned above were used. Slides from herbarium material were mounted in lactic acid or polyvinyl alcohol. Conidiophore lengths were measured in two parts: a stipe (from the base to the first conidiogenous node) and a fertile part (from the first node to the apex). CLADOSPORIUM HERBARUM (Pers.: Fr.) Link, Mag. Ges. Naturforsch. Freunde, Berl. 7: 37 (1815). Dematium herbarum Pers., Usteri's Annis Bot. 7: 32 (1794)· Cladosporium herbarum (Pers.: Fr.) Link, Syst, my col. 3: 370 (1829). Lectotype. Dematium herbarum Pers. in herb. L, no. 9 10.225-733. For further synonyms, see Hughes (1958). The following description is based on CBS 177.71 grown on MEA at 22°. Daily growth rate of colony about 4 mm, ultimate diameter about 90 mm reached after 30-35 d. Fourteen-day-old colonies effuse, dark olivaceous green, with regular and sharp margin, powdery, lanose at the centre. Submerged expanding hyphae subhyaline to pale olivaceous, 2'5-4'0 pm wide, smooth- and thin-walled, regularly septate every 15-30 pm; older hyphae turning olivaceous green to olivaceous brown, at first often verruculose, later smooth-walled, septate every 10-25 pm, frequently branched; locally some very thick (about 4'5-7'5 pm), densely septate hyphae present, from which many lateral, narrower branches arise. Aerial hyphae usually 2'0-4'5 pm wide, less frequently branched. After 14-20 d dark, smooth, fasciculate hyphae are formed in the colony
Variability in Cladosporium herbarum
Fig. 1. Diagrammatic representation of conidiophore types. (a) Erect, from creeping hyphae; (b) erect, from hyphal fascicles ; (e) ascending, terminal on hypha.
centre. Conidiophores erect , nodose, smooth- and thick-walled, olivaceou s bro wn to brown , scarce ly septate ; stipe straight or slightly flexuou s, 3'0-5'5 ( 6) jfm thick; fertile part with 0-5 (- 7) proliferations, straight or geniculate at the nodes. Nodes 4'5-9'0 11m diam, with 2--6 thick, slightly prominent, dark-brown conidial scar s. Conidiophores of two type s : (1) lateral, ari sing at wide angles from intercalary cells of creeping hyphae, 3-5 pm wide and 55-250 j fm long , simple or rarely terminally branched, with 1-3 (- 5) proliferations ; if on fasciculate hyphae usually short (25- So pm) and with 0-2 proliferations; (2) terminal, gradually merging into pale , ascending hyphae, 3-6 lim wide , ( 100~) 1so -4So pm long , with 4- 6 (-7) proliferations, occasionally di- or trichotomou sly branched. Conidia catenate ; chains usuall y br an ched onl y in the lower part, comprising (2-) 3-5 (-8) conidia, the lower on es larger and sometimes septate. Conidia verrucose , rather thick-walled, pale olivaceous brown to golden brown, 0-1 (--2) septate. One-celled conidia ellip soidal to bro adl y fusiform, 5~12 ( - 15)X 3's - 6'o p m ; septate conidia cylindrical, usually not con stri cted at the septa, 9- 20 (- 23) x 3'5 - 7'0 (- 7-5) pm, with slightly protuberant, dark-brown scars. MORPHO L OGY AND TYPIFICATION O F
The species was first described by Persoon (1794) from rotten wood as D ematium herbarum, recla ssified by Link (1815) in Cl adosporium , and sanctioned by Fries (1829). D e Vri es (1952) incorrectly (Art . 7.S ICBN) indicated one of L ink 's seconda ry collections at herb . B as lectotype. An authentic spe cimen preserved at L , no. 910 .225~733 , is designated here as lectotype. It was the on ly specimen of Persoon's collections in which C. herbarum, th ough scanty, could be recogn ized . The live strain CBS 177'71 agrees with thi s spec imen, and ha s been selected here as representative strain for C. herbarum.
Re-examination of Persoon's collections labelled as Dematium epiphyllum Pers. (L, no. 910.225 -646) and Dematium gr aminum Pers. (no. 910.225 -723 ) confirmed Hughes's (1958) conclusion that th ese are identical to C. herbarum. In Persoon's specimen s of D emat ium [u scum Pers. (no. 910 .225 -720) and D . v ulgare Pers. o. typha rum Pers. (no. 910.225 -735) no C ladosporium could be found, but Hughes (1958) listed them as further synonyms of C. herbarum . Conidiogenesis in Cladosporium is uniform and has been adequately described in C . herbarum by Hashmi , Morgan-Jones & Kendrick (1973). The variability of conidia has been treated by de Vrie s (1952) and Riedl (1968). Mo st variation of the studied live strains has been found in colony and conidiophore character s. During the first 7 d of development a den se, low mat of conidiophores ma inly of type (1) is formed, giving the colony a powdery appearance. After about 10 d abundant aeri al hyphae are formed at the centre, overgrowing the first layer and producing conidiophores of the two type s (1) and (2). In a final stage of development, thi s secondary layer has a diameter of 3 5 ern, and dark hyphaI fascicl es with short conid iophores of type (1) appear. Cladosporium tenuissimum Cooke and C. chlorocephalum (Fres.) Mason & M. B. Ellis also have differ ent conidiophore layer s. These species are pleomorphic : in C. chlorocepha lum th e two coni dioph ore type s are markedly differ ent, while in C. tenu issimum the y are principally the sam e but of different length, intermediat e lengths being ab sent or very rare. In C. herbarum the macros cop ically visible layer s mainly differ by abundance of
: Ull~jlmja~~llil~JdA~\\!ll!!J I/l/iJWWI-b .
7 ' ~d~ c a Fig . 2 . Diagrammatic representation of colony growth after 7, 20 and 30 d. (a) Creeping hyphae ; (b) erect conidiophores; (e) ascending hyphae with (d ) terminal conidi oph ore s ; (e) fasciculate hyph ae. A, Primary conidioph ore layer ; B, seconda ry conidiophore layer.
K. Prasil and G. S. de Hoog
Fig. 3. Conidiophores of 30-day-old culture, CBS 177.71 on MEA. (a) Erect conidiophores on creeping hypha; (b) erect conidiophores on fasciculate hyphae; (e) terminal conidiophores on ascending hyphae.
Variability in Cladosporium herbarum
conidiophore types (1) and (2), and by arrangement of superficial hyphae and aerial mycelium, from which these conidiophores arise. Hence different isolate s ma y have markedly different appearance, as either conidiophore type (1) or (2) is preponderant. The teleomorph of C. herbarum is generally supposed to be M yco sphaerella rassiana (for reference s, see Barr, 1958), but th e evidence is not conclusive. Von Arx (1949) supposed that either one of the states of propagation may be lost, a process that would more or less coincide with climatic conditions. A cold climate would favour the tcleomorph, while the anamorph is more abundantly formed under warmer conditions. Von Arx (1949) noted variation in abundance of conidia of single ascospore isolates , even between strains from the same substrate. The five strains of M. tassiana in the CBS culture collection, among which several of von Arx's original anamorph and teleomorph strains, are variable in their cultural and microscopic characteristics . They all differ significantly from C. herbarum in the above sen se.
described small, subglobose conidia (2--3 x 1-2 Itm ) might indicate identity with C. sphaerospermum Penz ig. cerealiu m, - C. herbar um var. cerealium Sacc. , in Ferraris, A nni s my col. 7: 285 (1909).
Authentic material (PAD) contains C. herbarum with distinctly nodo se conidiophores and onecelled (y o- 9'5 x 4-5 pm) and z - g-celled ( 1422 x 5'5- 7'5 p m ) conidia. The taxon was described as a sub strate form on cereal s, without any real morphological difference, cit ricola . - C. herbarum var. citricola Fawcett & Burger, Phytopathology I : 165 (1911).
In the literature 23 infraspecific taxa of C. herbarum have been found (16 varieties and 7 formae ). Fifteen of these have been described as saproph ytes, in four cases a behaviour as opportunistic plant parasites can be supposed from the protologue and available literature, and four others were reported as parasitic on plants. None of the se taxa was available in culture.
Various C ladosporium taxa have been described on Citrus. Of C. v enrurioides Sacco var. citricola Sacco and C. [arnetiarum Sacco (= C. citri Briosi & Farneti) no material was available at PAD or PAV; the mentioned taxa are of doubtful identity . Cladosporium citri Massee is supposed to be the cause of citrus scab (F awcett , 1912, 1916), while C. herbarum var. citri Fawcett & Burger can cause scaly bark or nail head rust on Citrus aurantium (F awcett, 1911). Of the former taxon only a secondary specimen is pre ser ved at K , on which no Cladosporium conidiophores could be found . Of Fawcett' s taxon , a large collection of authentic material preserved at FLAS was anal ysed . The taxon is sufficient ly different from C. herbarum to be considered as a separate species . There are no pathogenic Cladosporium species on citrus, and none of the abov e name s are current in the literature (c. W. McCoy, per s. comm .).
aphidicola. - C. herbarum var. aph idi cola Mas sa!., in Sacc. , Madonna V erona 1918 : 21 (1918).
fascicular e. - C. herbarum var. fas ciculare Corda, le on . Fung. 3 : 9, tab. 1, fig. 24 (1839)·
This variet y was supposed to para sitize aphids. In the authentic specimen (PAD ) no aphids were present, and no Cladosporium conidia were found . Judging from the description (conidia polymorphic, z- j -septate, 6-20 x 3-6 pm) identity with C. herbarum seems unlikely.
No type material was available in PRM. Judging from the figure s, description and substrate the taxon seem s to be indistinguishable from C.
INFRASPECIFI C TAXA
aphidis. - C. herbarum var. aphidis Fuckel, Jb. nassau. Ver. Natuurh, 23: 356 (1869). No material has been preserved in G. The name was published without a diagnosis for a variety on aphids. cellulosae. -' C. herbarum var. cellulosae A. Sartory, R. Sartory, Meyer & Baumli, Papier, Paris 38 :
43 (1935)· According to Stafleu & Cowan (1983) the location of Sartory's herbarium and type s is unknown ; no mat erial was deposited in PC or STR. The
herbarum. fungorum. - Dematium herbarum var . fungorum Pers., Syn. meth . Fung.: 699 (1801).
Authentic material (L , no. 910.225--732) contains C. herbarum . The taxon seems to have been meant as a substrate form, growing on an old carpophore . lablab . - C. herbarum var . lablab Sacc., Philipp . J. S ci. 18 : 604 (1921).
The authentic specimen (PAD) contains a mixture of C. herbarum , C. cladosporioides, Alrernaria alt erna ta (F r . : Fr.) Keiss!. and Penicillium-like conidia. The description is too poor to dec ide which Cladosporium species was meant.
K. Prasil and G. S. de Haag C . herbarum var. ma crosporum Lagiere, Annis Ec, Natn , Agric , Grignon, ser . 3, S: 159 (1945)· The original material seems to be lost . The taxon was described as a parasite on Phleum pratense and had conidia of27-39 x 9-12 jim. Probably a H eterosporium species was concerned.
nigricans. - C. herbarum var. nigricans (Roth : Fr.) Fr., Syst. mycol. 3: 371 (1829), syn. Byssus nigricans Roth, Catal. bot . : 216 (1797).
No material is preserved in B or PRM. Arens (1945) recorded under this name a fungus colonizing oil-paintings, very similar to C. herbarum , phlei. - C. herbarum var. ph lei Lagiere, Annis Ec, nam . Agric. Grignon, ser. 3, S : 159 (1945)·
The original material is probably lost. Judging from the description, it was close to C. herbarum var. macrosporum, the only difference being the size of the 1-3-septate conidia, viz . (15-) 18- 24 (- 29) x (7'5-) 9-11 (- 12) usn. This indicates a possible identity with C. macrocarpum Preuss. rubi . - C. herbarum var. rubi Fragoso, Mems R. A cad . Cienc . Artes Barcelona, ser . 3, IS : 45 8 (1920).
In the type specimen (M A 3511 ) the irregular, mostly purplish spots on old Rubus leaves bear a Cladosporium species with fasciculate conidiophores arising from globose sclerotial bodies; the conidia are elongate, mostly smooth-walled. A second collection made by Fragoso (M A 3696 ) on Rubus ulmifolius bears numerous circular (u p to 3 mm diam) spots on abaxial sides of green leaves. The spots have a deep purple margin, and a brown, later blackish centre. The collection seems to represent a good taxon, which needs further study on the basis of fresh material. solutum . - C. solutum Link, Linne Spec. PI. I: 39 (1824), syn. C. herbarum var. solutum (Link) Sacc., Syll. Fung. 4: 578 (1886).
No authentic specimen is maintained in B. From the very brief original description without conidial dimensions, the identity of this fungus cannot be established. torulosum . - C. herbarum var. torulosum Berk . & Br ., Fungi Ceylon, no. 886 (1870).
The species was redescribed by Petch (1927). His data are in good agreement with the original specimen (K ), and his conclusions that the taxon is identical to C. herbarum are confirmed. On the type specimen dark, fasciculate hyphae were found,
resembling those found at the centre of C. herbarum cultures. v incetoxici. - C. herbarum var. v incetoxici Allesch., in Syd., Hedwigia 36, Beibl.: 163 (1897).
The type specimen (M ) and an original exsiccati specimen (Sydow, Mycotheca Marchica no. 4577; B) showed fasciculate conidiophores developing superficially on rotten Vincetoxicum purpurascens leaves; the conidia are short, verrucose. The taxon is indistinguishable from C. herbarum. vitricola . - C. herbarum var. vitricola Sacc. , in Ferraris, Annis mycol. 7: 286 (1909).
A study of the authentic specimen (PAD ) revealed that the small colonies (1-3 mm diam) on paper had very short, nodose and proliferating conidiophores . They produced golden brown conidia resembling those of C. herbarum. agave-echeveriae . - C. herbarum forma agaue-echeveriae Savelli, Annali Accad. Agric. Torino S: 114 (1914) (dated 1913).
No authentic material has been preserved in FI, PDA, PAV, RO , ROPV and TO . The taxon was described as parasitizing living leaves of Agave and Echeueria species. Judging from the size of conidia in the original description (12- 20 x 10-12/lm), it seems unlikely that this was a Cladosporium at all. carpophilum. - C. herbarum forma carpophilum Baccarini, Annali Bot. 4: 277 (1906).
No material is maintained in PAD. Judging from the original description (conidiophores 270 x 3-7 pm, one-celled conidia 9-10ltm long, two-celled conidia 20 pm long ), it might be identical to C. herbarum. An exsiccati specimen in herb. PAD (D . Saccardo, Mycoth. Ital. No. 590) contains a mixture of C. herbarum and C. cladosporioides on Cheiranthus. fimicola. - C. herbarum forma fimicola March., Bull. Soc . r. Bot. Belg. 24: 67 (1885) .
No authentic material is maintained in BR. Probably this was only a substrate form; the data in the original description correspond with C. herbarum. hormodendroides. - C. herbarum forma hormodendroides Ferraris, Fl. Ital. crypt. I, fasc, 8 : 332 (1912).
According to de Vries (1952) this taxon is identical with C. cladosporioides (F res.) de Vries . Synonymy was based on secondary material, the type specimen probably being lost.
Variability in Cladosporium herbarum
parasitica. - C. herbarum forma parasitica Sacc., Annls mycol. 13: 133 (1915). The authentic material (PAD) contains both C. herbarum and C. cladosporioides on Taphrina tosquinetii (Westend.) Tul. on leaves of Alnus sp. The morphological diversity described for this taxon was probably due to the fact that a mixture of Cladosporium species was concerned. The suspected parasitism remains questionable; the hyphomycetes seem to be only secondary invaders on broken, diseased leaves. saxicola. - C. herbarum forma saxicola Sacc., Michelia 2.: 578 (1882). The original specimen (PAD) comprises a stone without any fungus. The data in the description correspond with C. herbarum. stellariae. - C. herbarum forma stellariae Unamuno, Boln Soc. Esp. Hist. nat. 34: 146 (1934)· The original specimen (MA) contains fasciculate, nodose conidiophores and o-z-septate, verrucose conidia, fitting C. herbarum.
The authors are indebted to J. Holtman for technical assistance, and to F. Snippe-Claus for inking the drawings. We acknowledge the curators of the herbaria B, FLAS, K, L, M, MA and PAD for lending material for study. REFERENCES ARENS, K. (1945). Urn fungo destruidor de pinturas a oleo: Cladosporium herbarum (Pers.) var. nigricans (Roth). Summa Brasiliensis Biologiae I, 1-13. ARx, J. A. VON (1949). Uber die Ascusform von Cladosporium herbarum (Pers.) Link. Sydowia 4, 320-324. BARR, M. E. (1958). Life history studies of Mycosphaerella cassiana and M. typhae. Mycologia S0, 501-513. ELLIS, M. B. (1971). Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew.
ELLIS, M. B. (1976). More Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew. FAWCETT, H. S. (1911).Scaly bark or nail-head rust of citrus. Bulletin, Agricultural Experiment Station (Gainesville) 106, 1-41. FAWCETT, H. S. (1912). Citrus scab. Bulletin, Agricultural Experiment Station (Gainesville) 109, 51-60. FAWCETT, H. S. (1916). Citrus scab. Phytopathology 6, 44 2-445. FRIES, E. M. (1829). Systema mycologicum 3 (1). Lund. HASHMI, M. H., MORGAN-JONES, G. & KENDRICK, B. (1973)· Conidium ontogeny in hyphomycetes. The blastoconidia of Cladosporium herbarum and Torula herbarum. Canadian Journal of Botany 51, 10891091. HOOG, G. S. DE (1982). On the potentialIy pathogenic dematiaceous hyphomycetes. In The Fungi Pathogenic for Humans and Animals, part A (ed. D. H. Howard), pp. 149-216. New York, U.S.A.: Marcel Dekker. HUGHES, S. J. (1958). Revisiones hyphomycetum aliquot cum appendice de nominibus rejiciendis. Canadian Journal of Botany 36, 727-836. JANCZEWSKI, E. (1894). Recherches sur Ie Cladosporium herbarum et ses compagnons habituels sur le cereales. Bulletin de l' Academic des Sciences de Cracovie 27, 143-1 87. LINK, H. F. (1815). Observations in ordines plantarurn naturales. Dissertatio II, sistens nuperas de Mucedinum et Gasteromycetum ordinibus observationes, Magazin Gesellschaft Naturforschender Freunde zu Berlin 7, 25-45. PERSOON, C. H. (1794). Nahere Bestimrnung und Beschreibungen einiger sich nahe verwandter Pflanzen. Usteri's Annales Botanici 7, 1-32. PETCH, T. (1927). Revisions of Ceylon fungi: VIII. Annals of the Royal Botanic Gardens, Peradeniya 10, 161-180. RIEDL, H. (1968). Cladosporium herbarum und Cl. murorum. Sydowia 20, 331-338. STAFLEU, F. A. & COWAN, R. S. (1983). Taxonomic Literature, vol. IV. Utrecht, The Netherlands: Bohn et al. VRIES, G. A. DE (1952). Contribution to the Knowledge of the Genus Cladosporium Link ex Fr. Baarn, The Netherlands: HolIandia.
(Received for publication 15 April 1987)