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Trans. Br , mycol, Soc. 69 (1) 125-131 (1977) Printed in Great Britain

CONIDIAL ONTOGENY IN ECHINOCATENA ARTHRINIOIDES

GEN. ET SP.NOV. (DEUTEROMYCOTINA: HYPHOMYCETES)

By R. CAMPBELL

Department of Botany, University of Bristol, BS8 1 UG

AND B. C. SUTTON

Commonwealth Mycological Institute, Kew

Echinocatena arthrinioides gen. et sp.nov. is described with micronematous, mononematousconidiophores, branched, acropetal chains of polyblastic conidiogenous cells, and solitary,spherical, brown, aseptate, echinulate conidia. It is compared and contrasted with TrichobotrysPenz. & Sacc., Sadasivania Subram. and Parapericonia M . B. Ellis. Conidiogenesis wasfollowed by light microscopy, scanning and transmission electron microscopy.

Amongst several collections of microfungi receivedfor identification at the C.M.I. from Dr KaranSingh Panwar, India, was included a distinctivehyphomycete on dead leaves. Initial observationsof habit and colony appearance suggested Arthri-nium Kze ex Fr. as a suitable genus, but thediscovery of echinulate conidia and a truly holo-blastic type of conidial ontogeny, both featureshitherto unknown in Arthrinium, necessitated arevision of these preliminary impressions of thefungus. Subsequent study by optical, scanning andtransmission electron microscopy has clearly shownthat the relationships lie more with Trichobotry sPenz. & Sacc., Sadasivania Subram. and Paraperi-conia M. B. Ellis. The combination of morphologi-cal and developmental characteristics precludes theinclusion of the species in any ofthese genera so thegeneric name Echinocatena is introduced, with asingle species, E. arthrinioides, to accommodate thefungus.

METHODS

For transmission electron microscopy, cultures(IMI 199279) grown on com meal agar (OxoidCM103) were flooded with a mixture of p-for-maldehyde (1 %, w jv) and glutaraldehyde (a's %,v jv ), postfixed in 1 % (w jv) osmium tetroxide,dehydrated in an ethanol: water series andembedded in Spurr's resin, Sections were cutwith an LKB 'Ultrotome' III, stained with leadcitrate and examined in an AEI EM6G electronmicroscope.

Material cultured for scanning electron micro-scopy was fixed in p-formaldehyde (1 %, v jv) andglutaraldehyde (a's %, v jv ) dehydrated in anethanol: water series, placed in amyl acetate beforebeing dehydrated, critically point dried fromcarbon dioxide, coated with gold and observed in

a Cambridge ' Stereoscan ' S 4. Light-microscopeobservations were made from the herbariummaterial, plate and slide cultures.

CONIDIAL ONTOGENY: DESCRIPTION AND

DISCUSSION

Conidiogenous cells arise from aerial hyphae andform an acropetal chain (Fig. 1), by the holoblasticgrowth of the terminal cell (Fig. 2). Short lateralchains of conidiogenous cells may also occur (Fig .1) and these form and develop in the same way asthe main chain except that the original holoblasticoutgrowth occurs at the side of a conidiogenouscell rather than at its apex. The young conidio-genous cell has a two-layered wall (Fig. 3) andforms a perforate septum, with one or moreWoronin bodies, distal to the narrowest part of theisthmus between it and the cell from which itarises. This septum thickens and becomeselectron opaque as the conidiogenous cell matures(Fig. 4) and, in the most mature cells examined,it remains perforate. Septa in the lateral chains areapparently identical with those in the main chain(Fig. 5). An annular thickening develops in theneck of older conidiogenous cells where they giverise to new ones and eventually becomes electronopaque , usually with a striated appearance (Figs.4,5)·

Each conidiogenous cell produces conidia,usually in a lateral position from its distal end, asholoblastic extensions of its wall (Figs. 6, 7). Aperforate septum forms at the conidial base, againdistal to the narrowest point (Fig. 7). Somethickening of the septum occurs and it becomeselectron opaque , but less markedly than theconidiogenous cell junctions. The annular thick-ening at the conidial junction is also not as

Colonies discrete, applanate, dark brown toblack. Mycelium partly immersed, partly super-ficial, composed of branched, septate, pale brown,smooth hyphae. Conidiophores formed from thesuperficial mycelium, micronernatous, mono-nematous, unbranched, straight, pale brown,sparsely echinulate or smooth. Conidiogenous cellsarising in simple or branched acropetal chains fromthe apex of the conidiophore, separated byprominent dark brown, thick septa, pale brownechinulate, cylindrical to doliiform, polyblastic'integrated, indeterminate, distal part fertile witl~up to 7 conidiogenous loci. Conidia solitary, dry,spherical, brown, thick-walled, aseptate, echinulate.

The most closely related genus to Echinocatenais Sadasiuania Subramanian (1957). Sadasivaniaspecies also develop polyblastic catenate conidio-genous cells separated by distinctive thickenedsepta (Ellis, 1971). Sundberg & Wicklow (1973)and Wicklow & Sundberg (1976) demonstrated inS. bhustha Rao & Rao the formation of such septain vivo and in vitro, and moreover, showed thatconidiogenesis is restricted to the unthickened partof the conidiogenous cell. Conidia are thick-walledechinulate and aseptate, Sadasivania, however, canbe distinguished from Echinocatena by thernacronematous rather than micronematous coni-diophores and restriction of the conidiogenouscells to the apical cell of each conidiophore. Withthis arrangement the stout mature conidiophoreseach have a large dark head of conidiogenous cellsand conidia, at maturity. This gives the colonies aquite different appearance from the applanatehabit in Echinocatena resulting from the micro-nematous conidiophores.

126 Conidiogenesis in Echinocatena arthrinioides

pronounced as that of the conidiogenous cell superficiali oriunda, micronematosa, mononematosa,(Fig . 8). As the conidia mature, the walls become non ramosa, recta, pallide brunnea, parce echinulatagreatly thickened and very electron opaque with up vel laevia. Cellulae conidiogenae in catenas simplicesto four layers discernible (Fig. 8). The pore in the vel r.amosas acropetasex apice conidiophoriexorientes,septum remains open throughout maturation sept!s atro brunneis crassis prominentibus separatae,(F'g 9) palhde brunneae, echinulatae, cylindricae vel dolii-

I. •Each conidiogenous cell thus has a cluster of formes, polyblasticae, in conidiophoris incorporatae,

5-7 conidia of various ages (Fig. 10) around its indeterminatae, pane distali fertili cum usque ad 7locis conidiogenis. Conidia solitaria, sicca, sphaerica,

distal end and at maturity there is a densely brunnea, aseptata, parietibus crassis, echinulata.packed column of conidia completely concealing Sp. typ.: E. arthrinioides.the acropetal chain of conidiogenous cells within(Fig. 11). The sequence is summarized in dia-grammatic form (Fig. 12).

The ultrastructure of Echinocatena is similar toother fungi as regards the distribution and appear-ance of the organelles and general wall structure.The septa between the conidiogenous cells are,however, exceptional for their thickness andextreme amount of electron-opaque material, pre-sumably melanin, which they contain. Suchmassive septa could strengthen the entire conidialapparatus, preventing or delaying premature dis-integration, thus allowing the continuous produc-tion of masses of conidia and their maturation tooccur, prior to spore liberation.

The annular thickenings at conidiogenous celland conidial junctions are found in other fungiwhich have acropetal chains, e.g. Alternaria, andprobably are concerned with mechanical supportof the chain.

The factors which determine whether a holoblas-tic outgrowth from a conidiogenous cell becomesa conidium or a branch in the conidiogenous chainare unknown. Initially the developing structures areidentical but they are soon distinguishable on thebasis of septum and wall thickness . It is possiblethat those cells which retain a thin wall are stillable to plasticize it and hence are conidiogenouscells while in potential conidia the wall thickensmore quickly and is not able to extend later.

Echinocatena gen.nov.Coloniae discretae, applanatae, atro brunneae velnigrae. Mycelium partim immersum, partim super-ficiale, ex hyphis rarnosis, septatis, pallide brunneis,laevibus compositum, Conidiophora ex mycelio

Symbols used: C = conidium; CC = conidiogenous cell.

Fig",1. Scanning electron micrograph (SEM) of a young acropetal chain of conidiogenous cells juststarting to produce side chains and conidia. Bar marker = 10 pm.Fig. 2 . ~~nsmission electron micrograph (TEM) of the production, by holoblastic wall extension,of anew conidiogenous cell at the apex of the chain. Bar marker = 1 pm.Fig. 3. TEM of a young conidiogenous cell with the new septum distal to the narrowest pan of thejunction. Bar marker = 1 psn.Fig. 4. TEM of a mature septum between conidiogenous cells. Bar marker = 1 pm.Fig. 5. TEM of a branch region in the chain of conidiogenous cells. Bar marker = 1 pm.

R. Campbell andB. C. Sutton 127

128 Conidiogenesis in Echinocatena arthrinioides

R. Campbell andB. C. Sutton 129

cc

Fig. 12. Diagram of the proposed developmental sequence; the youngest stage is on the left and theoldest on the right. Not to scale.

Somewhat more tenuously related to Echinoca-tena is Parapericonia M. B. Ellis (1976). Conidio-genous cells are again catenate and polyblastic, andproduce thick-walled, brown, aseptate, echinulateconidia. However, the septa separating theconidiogenous cells are not of distinctive structureas in Echinocatena and Sadasivania, further, theyare smooth and develop from uncinate macro-nematous conidiophores aggregated in sporo-dochia.

Trichobotrys Penz. & Sacco (Ellis, 1971) may alsobe compared with Echinocatena, but can bedistinguished by the setiform macronematousconidiophores and catenate conidia. Although theconidiogenous cells are catenate, they are deeply

pigmented and lack the distinctive separatingsepta of either Echinocatena or Sadasivania.

Despite the great superficial similarity withArthrinium, the conidial ontogeny of Echinocatenais completely different. The conidiogenous appara-tus in Arthrinium (Campbell, 1975) consists of aconidiophore mother cell from which a fertilebasauxic conidiophore is produced. Holoblasticconidia develop although their relationship to theconidiophore mother cell is enteroblastic. Thetransverse septa of the fertile conidiophore arerefractive (by light microscopy) but ultrastruc-turally are apparently similar to normal septa ofDeuteromycotina, In Echinocatena there is noconidiophore mother cell and the septa are dark

Fig. 6. TEM of a young conidium produced by holoblastic extension of the wall of the conidiogenouscell. Bar marker = 1 pm.Fig. 7. TEM of a young conidium; note the initiation of the thickening and electron opaque regions inthe wall of the conidiogenous cell. Bar marker = 1 pm.Fig. 8. TEM of a conidiogenous cell with conidia of different ages. The thickening in the wall at thejunction between conidiogenous cells is greater than that at the junction of the conidiogenous cell andthe conidium. Bar marker = 1 pm.

Fig. 9. TEM of the junction between a nearly mature conidium and the conidiogenous cell. Barmarker = 1 pm.

Fig. 10. TEM of a cross-section of a conidiogenous cell surrounded by spores of various ages. Barmarker == 1 pm.Fig. 11. SEM of a column of mature conidia packed around and obscuring the chain of conidiogenouscells. Bar marker = 10 pm.

MYC 69

Conidiogenesis in Echinocatena arthrinioides

c

Fig. 13. Echinocatena arthrinioides. (A) Conidiophore, chain of conidiogenous cells and column ofconidia; (B) immature conidiogenous cells and conidia; (C) conidia.

coloured and of quite different structure. Inaddition, conidia and the whole conidiogenousapparatus are distinctly echinulate in Echinocatena.

Echinocatena arthrinioides sp.nov. (Fig. 13)

Coloniae discretae, applanatae, atro brunneae velnigrae, margine irregulari , usque ad 1 mm diam.Mycelium immersum et superficiale, ex hyphis ramo-sis, septatis, pallide brunneis, laevibus, 2-2 '5 pmcrassis compositum. Conidiophora ex mycelio super-

ficiali oriunda, 10-23 x 2-2'5 pm, micronematosa,mononematosa, nonramosa, recta, pallide brunnea,parce echinulata vel laevia. Cellulae conidiogenae incatenas simplices vel ramosas acropetas ex apiceconidiophori exorientes, 5-10'5 x 3'5-5 usn, septis atrobrunneis crassis prorninentibus separatae, pallidebrunneae, echinulatae, cylindricae vel doliiformes,polyblasticae, in conidiophoris incorporatae, indeter-minatae, parte distali fertili cum usque ad 7 locisconidiogenis. Conidia 3'5-4'5 pm diam, solitaria,sicca, sphaerica, brunnea , aseptata, parietibus crassis,

R. Campbell andB. C. Suttonechinulata, in columnis verticalibus, cylindricis, usquead 35pm longis evoluta.

In foliis ignotis emortuis, Jodhpur, India, K. S.Panwar Ju/Bot/619, 25 Nov. 1975, IMI 199279,holot ypus.

Growth on PDA and MA very slow, 2'5 cm in6 weeks. Immersed mycel ium composed of darkbrown, septate hyphae. Aerial mycelium abundant,denser and more compact towards the centre,becoming floccose, sparse and finally absent fromthe advancing edge, varying from sepia to isabel-line; reverse brown vinaceous (Rayner, 1970).Sporulation abundant.

On the natural substrate colonies are discrete,applanate, dark brown to black, irregular in out-line, up to 1 mm diam. Mycelium immersed andsuperficial, formed of branched, septate, palebrown, smooth hyphae 2-2'5 pm wide. Conidio-phores formed from the superficial mycelium,10-23 x 2-2 '5 pm,micronematous, mononematous,unbranched, straight, pale brown, sparsely echinu-late or smooth. Conidiogenous cells arising insimple or branched acropetal chains from theconidiophore apex, 5-10'5 x 3'5-5 usn, separatedby prominent, thick, dark brown septa, palebrown, echinulate, cylindrical to doliiform,constricted at the septa, polyblastic, integrated,indeterminate, distal part fertile with 5-7 non-pro-tuberant conidiogenous loci. Con idia 3'5-4'5 pm

diam, solitary, dry, spherical, brown, thick-walled,aseptate, echinulate, appearing as vertical cylindri-cal columns up to 35 pm in length, sometimesdistorted by being packed around the conidio-genous cells.

The Science Research Council is thanked forgrants B/SR/90718 and B/RG/1408 to theDepartment of Botany, Bristol University.

REFERENCES

CAMPBELL, R. (1975). The ultrastructure of theArthrinium state of Apiospora montagnei SaccoProtoplasma 83, 51-60.

ELLIS, M. B. (1971 ). Dematia ceous hyphomycetes.Commonwealth Mycological Institute, Kew.

ELLIS, M. B. (1976). More dematiaceous hyphomy cetes.Commonwealth Mycological Institute, Kew.

RAYNER, R. W. (1970). A mycological colour chart.Commonwealth Mycological Institute, Kew.

SUBRAMANIAN, C. V. (1957). Hyphomycetes. III. Twonew genera - Dwayaloma and Sadasivania.Journal ofthe Indian Botanical Society 36,61-67.

SUNDBERG, W. J. & WICKLOW, M. C. (1973). Observa-tions on Sadasivania (fungi imperfecti). Mycologia65, 925-929.

WICKLOW, M. C. & SUNDBERG, W. J. (1976). Culturaland morphological variation in Sadasioania bhustha(fungi imperfecti). My cologia 68, 891-901.

(Accepted for publication 30 January 1977)

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