Proc. Indian Acad. Sci. (Plant Sci.), Vol. 94, Nos 2 & 3, April & May 1985, pp. 273-280. 9 Printed in India.

Pigment Iocalization in Dematiaceous Hyphomycetes and the segregation of Pseudogliomastix gen. nov. from A cremonium

W GAMS and T BOEKHOUT Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG Baarn, Nctherlands

Abstraer. Conidia of polychromum and of the anamorph of Wallrothiella subiculosa were studied with TEM. The conidia of A. polychrornum have a very thick, fibrous, electron-dense outer layer, which fmaUy br coarsety warted. In W. subiculosa this layer remains thin and more compact and becomes minutely rou8hened. Bec.ause of these differences and the regular pigmentation of the vegetative hyphae and phialides, a new genus, Pseudogliomastix, is described for this anamorph. The differences between pigmented and hyaline conidia among species of Acremonium sect. do not seem to be so fundamental as to preclude their inclusion in one genus, Acremonium.

Keywords. Dematiaceous Hyphornycetes; ultrastructure; pigmentation; ; Acremonium; Gliomastix; Pseudoglioraastix; Wallrothiella.

1. Introduction

The delimitation of the genera Acremonium Link: Fr., Gliomastix Gu› and Saorahamala Subram. based on conidial pigmentation and chain formation has been a matter of dispute. The esteemed Prof. Dr C V Subramanian, whose contributions to mycology are honoured with this issue, has beena driving force in this discussion (Subramanian 1973; Subramanian and Pushkaran 1976). Features used to distinguish between false and true chains were dealt with in a previous paper (Gams 1978) and here we shall confine ourselves to pigrnentation. We hypothesize that the distribution of pigments within the conidial wall may have more taxonomic value than hitherto recognized. Ir is now generaUy accepted that within the Hyphomycetes the distinction between the form-families Dematiaceae with dark structures and Mucedinaceae with only hyaline structures is no longer tenable. Consequently EUis (1971, 1976) did not use these terms as rtames of families but only in adjectival formas convenient descriptive terms. To reach a workable classification0 pigment distribution must be more accurately defined, firstly, along different parts of the thallus (particularly con- idiophores and parts thereof and conidia) and, secondly, in different wall layers of the pigmented elements. In analogy with Stachybotrys Corda (see Jong and Davis 1976), Gams (1971) included species with both hyaline and pigmented conidia in one genus, Acremonium. Pairs of very similar species were repeatedly observed, which differed only in the presence or absence of conidial pigmentation. The species formerly classified in Gliomastix were classified in a separate section, Gliomastix; this section, however, was not delimited because of conidial pigmentation, but mainly because of the presence of thickened, chondroid hyphae, which render the colony very tough. Objections against this procedure were summarized by Hammill (1981). Gams (1971) was not entirely consistent in applying the criterion of chondroid hyphae and induded the A. striatisporum series with pigmented conidia which lacked thickened hyphae; this 273 p,,S. 24 274 W Gatos and T Boekhout series was subsequently (Gams 1978) transferred to a separate genus, Sagenomella, because of "connected" conidial chains (better "chains with connectives", according to Minter et al 1983). The species A. inflatum and A. atrogriseurn also lack chondroid hyphae and should be classified elsewhere. On the other hand, the anamorph of Watlrothietla subiculosa Hthnel, known as Gliomastix protea (Sacc.) Verona and Castella, was deliberately excluded from Acremonium because its vegetative hyphae are often pigmented and the pigmentation of the smooth yeUow-brown conidia differs strongly from the olivaceous-brown encrusted conidia of other Gliomastix species. Wallrothiella should therefore not be considered asa teleomorph ofGliomastix or even Acremonium, as was done in the symposium "The whole " (edited by Kendrick 1979) by Subramanian (p. 138) and Samuels and Rossman (p. t72), and also by Carmichael et al (1980). A separate genus is required for this fungus in order to preclude any misunderstanding. Whether the pigmentation develops in a similar way amongst species of Gliomaatix and whether this is fundamentaUy different from the development of pigmentation in conidia of Wallrothiella subiculosa, is the topic of this study.

2. Material and methods

The foUowing species and isolates were examined (in two-week-old cultures on 2 ~o M~): Acremonium polychromum (van Beyma) W. Gams, cas 511"63, conidia almost smooth, darldy pigmented; phialides hyaline, sometimes pigmented at the tip. Wallrothiella subiculosa H6hnel, cas 389.73, conidia smooth, graduaUy becoming yellow-brown, as dark as the phialides. Bleaching of pioment: Fragments of the agar cuttures were transferred into a concentrated solution of commercial sodium hypochlorite (containing t 0 % active C1) or in 5 % KOH. Transmission electron microscopy: The material was prefixed with2 % glutaraldehyde and 5 % dimethylsulphoxide in 0-05 M phosphate buffer (pH 7.2), for about 20 min. A subsequent treatment with snailgut juice (0-5 % v/v in a buffer containing 30 mM EOTA, 30 mM MgSO, and 50 mM mercaptoethanol) for about 30 min at 30~ was followed by fixation in 3 % glutaraldehyde in 0.05 M phosphate buffer (pH 7-2) for I hr. The material was then washed several times in buffer and postfixed in 1% OsO, in 0.05 M phosphate buffer (pH 7.2) for 1 lar. After fixation the material was rinsed in 50 % (v/v) acetone for 30 min and blockstained in 1-2 % uranyl acetate in 70 ~ (v/v) acetone for 30 min. This was foltowed by dehydration in a graded acetone sc¡ with a final addition of some drops of 2,2-dimethoxypropane (Linnemans et al 1977) and embedding in araldite. The sections were stained in 4 ~o aqueous uranyl acetate for 5 min, fotlowed by lead citrate for 1 ruin.

3. Results

3.1 Acremonium polychromum The hyphal wall is layered and consists of approximately 4 layers, which differ slightly in their e|ectron opacity (figure 3) and is covered by a thin electron-dense sheath. Like Segregation of Pseudogliomastix from Acremonium 275

Figures 1-5. Acremonium polychromum. 1, 2. Apex of phialide with conidium ( • 36000 and x 8400). 3. Layered hyphal walt. 4. Conidial scar of young conidium. 5. Wall ofyoung conidium (3-5 x 36000).

PoS. 25 276 W Gatos and T Boekhout other hyphae, the phia!ides contain an inner layer of low electron density; this layer gradually thickens towards the apex and forros a periclinal annuiar thickening characteristic of phiatidic conidiogenesis (figures 1,2). The conidia are extruded from the phialide opening. The walls of the young conidia are inconspicuously layered and of medium electron density. The outermost, electron-dense fibrillar sheath seems to be continuous with that covering the phialide (figures I, 2 arrow). A transverse septum is

".;..

Figures 6-9 (• 36000)6. Acremonium polychromum, wali of mature conidium. 7- 9. Anamorph of Waltrothiella subiculosa. 7. Hyphal septum. 8. Wall of young conidium. 9. Wall of mature conidium. Segregation of Pseudogliomastix from Acremonium 277

formed inside the phialide, just below its apex (figure 1 arrow), finally separating the conidium and the phialide. Young conidia appear nearly smooth. The walls of the young conidia are of medium electron density; an outer layer containing small electron-dense spots is covered by a thin electron-dense sheath (figures 4, 5). Mature conidia have essentially the same layers, but the electron-dense sheath has a homogeneous of fibrillar texture and has increased considerably in thickness. Finally the surface becomes warted due to the deposition of lumps of pigment. Just beneath this layer, a zone with small, dispersed, electron-dense spots oceurs in the less electron-dense layer of the conidial wall (figure 6), The pigmented sheath of the conidium is less developed near the conidial scar (figure 4). Bleaching experiments resulted in sudden disappearance of pigment in NaOC1 but no reaction to KOH. Probably the very thick warty pigmented sheath is a result of secondary excretion of pigment through the inner layers of the cell wall. This is schematically presented in figure 10A.

A

H

i Figure 10. Diagrammaticinterpretation of the development of A. the conidial wall in A. polychromumand B. the anamorph of W. subiculosa. Arrow heads indicate the supposed depositŸ of pigment.

P.$. 2a 278 W Gatos and T Boekhout

10 gro

]Figure 11. Pseudogliomastixprotea, the anamorph of Watlrothiella subiculosa, herb. IMI 31229a and 56093a.

3.2 Waltrothiella subiculosa

The hyphae are covered by a thick capsule (figure 7). Electron-dense lumps occur in the outer fibrillar layer adjacent to the septa, thus forming ah annular zone. The proper cell wall is layered and of medium electron density. Two thin outer zones occur which are slightly more electron-dense. The wall of presumably young conidia (figure 8) has a relative[y thin, electron-dense floccose outer layer anda rather thick inner layer of Iow electron density. In mature conidia the outermost electron-dense sheath remains thin, if compared with A. polychromum, and only small warts appear on the surface of the compact outer layer (figure 9 arrow). Just beneath this layer a zone of small electron-dense spots occurs in the less electron-dense inner layer, probably also indicating ah excretion of pigment through the inner iayer towards the outer layers of the wall. This is schematically presented in figure IOB. The conidia react more tardity to bleaching with NaOCI, which takes more than 10 sec, KOH has no noteworthy effect. This again indicates a deeper localization of the pigment.

4. Discussion

Reisinger and Gu› (1968), Reisinger (1969), Reisinger and Bonaly (1972), and Cole (1973) showed that in pigmented hyphae and conidia amorphous material or Segregation of Pseudoglioraastix from Acremonium 279 granules are oflen deposited at a later stage in and on the outermost wall layer (which they called layer A), followed by more or less pigment accumulation within the underlying layer B. This pigment localization becomes clearly visible in ultrathin sections under TEM after trypsin or HC1 treatment of the walls (Reisinger and Bonaly 1972). This situation is particularly pronounced even at the light-microscopic level in the chlamydospores of Humicola (Grifl]ths 1974). In Stachybotrys as well as in Memnoniella, Campbell (1972, 1975) showed by reM that conidia become pigmented in this way. This kind of secondary pigment deposition may explain the unusual feature that closely related fungŸ can either have hyaline of darkly pigmented conidia. The mechanisms of formation and deposition of pigment have not been elucidated in this study. It seems possible, however, that the pigment of the conidia of A. polychromum o¡ initially partly from the electron-dense sheath surrounding the hyphae and phialides, but the majority seems to be a seconda~" deposition from the cytoplasm onto the conidial waU surface. Probably a similar mechanism also operates in the conidia of W. subiculosa, although most of the pigment becomes more homogeneously tocalized within the wall layers. It can be concluded that the differences between Ve'. subiculosa and A. polychromum at the submicroscopic levet reflect those observed at the light-microscopic level. The conidia of the latter have a very thick electron-dense ou:~r layer, which finally becomes roughened by lumps of pigmented material, whereas in the conidia of W. subiculosa this layer remains thinner and more compact and the warts are smaller. As A. polychromum has less roughened conidia than most of the other species of the section with pigmented conidia, a generalization of our obser~'ations on these species seems to be justified. However, a more detailed study involving several species is required. These ¡ confirm the previous conclusion (Gatos 1971) that the anamorph of Wallrothiella subiculosa differs fundamentatly from other pigmented species of Acremonium sect. Gliomastix. Therefore we describe a new genus for it. Pseudogliomastix W. Gatos, gen. nov. (figure 11) Genus Hyphomycetum, Acremonii simile, sed hyphis vegetativis et conidiophoris pigmentatis differens. Conidia dilute brunnea in tota pa¡ colorata. Species typica Pseudogliornastix protea (Sacc.) W. Gams, comb. nov. (Basionym: Torulaprotea Sacc., Michetia 2 292. 1881, and Fungi ltal. delin, figure 95t 1881), syn. Gliomastixprotea (Sacc.) Verona and Castella, Annati Fac. Agr. Unir. Pisa, N.S. 5 383 1942. Teleomorph Wallrothiella subiculosa H6hnel, Sber. Akad. Wiss. Wien 121 381. 1912 (Fragm. Mykol. 763). Type material of anamorph on Arundo donax, Padova, Italy, Mycotheca veneta 1591, in PAD. Futl descriptions in Hughes and Dickinson (1968), Dickinson (1968) and Gatos (197t).

Acknowledgement

The authors are indebted to Mr K A Seifert for corrections of the English text. 280 W Gatos and T Boekhout

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