International Biodeterioration 24 (1988) 271-276

Fungai Adhesion in Aquatic Hyphomycetes

S. J. H a r r i s o n , S. T. Moss & E. B. G. Jones

School of Biological Sciences, Portsmouth Polytechnic, King Henry Building, King Henry I Street, Portsmouth POI 2DY, UK

ABSTRACT

The attachment of propagules of aquatic fungi often takes place under turbulent conditions, i.e. spores of marine lignicolous fungi and conidia of freshwater Hyphomycetes. It has been demonstrated that attachment in Lemonniera aquatica is effected by three arms of the conidium making contact with the surface and forming appressoria. In this study the mechanisms of attachment of conidia of 10 aquatic llyphomycetes to a variety of natural and artificial substrata were followed at the light microscope and scanning electron microscope levels.

Five species produced appressoria, while, in the remaining species attachment and subsequent retention to the substratum was effected solely by the production of mucilage. Examination of selected appressorium-forming species in the transmission electron microscope showed that conidium attachment involved the production of mucilage in the regions of initial contact and only then were appressoria formed. The ecological significance of appressoria to the aquatic Hyphomycetes is discussed.

I N T R O D U C T I O N

Species of aquatic Hyphomycetes have been implicated in the utilization of ligninolytic substrates and are considered to be more important than bacteria in the early colonization of substrata in the aquatic environment forming an initial biofilm community. Of paramount importance in fast

271 International Biodeterioration 0265-3036/88/$03.50© 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain.

272 S. J. Harrison. S T. Mo~ F, R G. Jones

flowing water is the initial attachment of dispersive propagules, conidia in Hyphomycetes, to potential substrates.

Rees & Jones (1984) distinguished four attachment responses of ascospores in the marine environment; none (Amylocarpus encephaloides Currey), entrapment only (Halosphaeria appendiculata Linder), true attachment (Lulworthia medusa (Ell. & Ev.) Cribb & Cribb) and finally entrapment plus attachment (Halosarpheia retorquens Shearer & Crane). Entrapment was described as a reversible process in which spores could be easily dislodged, whereas in attachment (adhesion) the spores were held firmly by mucilage.

Species of aquatic Hyphomycetes show morphological and physio- logical adaptations to the aquatic environment and the degradation of leaf litter and wood. Their conidia are often large (up to 300~m) with branches or appendages. When a tetraradiate spore contacts a surface it does so with three of its four arms (Webster, 1959). In comparison, a sigmoid conidium only makes contact at two points and a spherical or ovoid conidium at only a single point.

In this paper the mechanisms by which different shaped conidia entrap or attach to, and subsequently form a relationship with, a substrate are reported.

MATERIALS AND METHODS

Conidia of the following aquatic hyphomycete species were examined: Anguillospora crassa Ingold; Articulospora tetracladia lngold; Mycocentrospora filiformis (Greathead) lqbal; Clavariopsis aquatica de Wild; Dactylella aquatica (lngold) Ranzoni; Dimorphospora foliicola Tubaki; Heliscus lugdunensis Sacc. & Therry ex lngold; Lemonniera aquatica de Wild; Tetracladium marchalianum de Wild; Tricladium splendens lngold; Varicosporium eiodeae Kegel.

Conidial suspensions of each organism were prepared and settled for a series of time periods on a variety of natural and artificial surfaces in order to determine a sequence of developmental events.

Material was either examined immediately at the light microscope level, fixed, dehydrated in a graded ethanol series and critical point dried for scanning electron microscopy (SEM) or prepared for transmission electron microscopy (TEM).

RESULTS AND DISCUSSION

The percentage germination and, where applicable, appressorium formation were calculated for each species on nucleopore membranes

Fungal adhesion in aquatic Hyphomycetes 273

TABLE 1 Percentage of Germinated Conidia of Aquatic Hyphomycete Species Producing

Appressoria" on Different Surfaces at Different Time Periods After Settlement

Species Substrate and settlement time

2h 6h 12h NM ~ Glass' NM Glass NM Glass

M. fil([ormis 0 0 2,9 6, 7 6.3 33.3 C aquatica 0 0 0 4.2 I.q 25.9 L aquatica 0 4.4 56.7 9S.3 65-3 10~) T marchalianum (l 0 0 5,q 7.4 25.q I~ Hodt'ac 0 0 4.8 7. l 29-7 8,R.5

"mature apprcss~ria only. i.e. those delimited from the gem1 tube by a scptum. ~'NM = nuclcopore nlcnlbrzlnc. 'Glass = glass coverslip.

and ghtss covcrslips, Apprcssor iutu p roduc t iou was uot obse~ 'cd in A. cra.s'xa, A. tetracladia, D. aquatica, D.fidiicola or H. lugdttnensis but occurred in the o ther species examined . It was found that the type of surface had no effect on ge rmina t ion but did inf luence apprcssor ium format ion (Table 1). Apprcssoria produced on the glass wcrc complex, often multiple, structures. 1 lowcvcr, on m e m b r a n e s the apprcssoria wcrc fewer arid s impler with the germ tubes often growing through the holes to aid attachmctat. It is specttlatcd that the format ion o f more complex apprcssoria on glass was a response to thc htck of any other aicl to a t tachmcnt . This indicates that the factors affecting apprcssor ium

Fig. I. Clavariopsis aquatica, l)istal region of conidial arm, 2 h after settlement on nuclcoporc mcmbrune, showing naucilagc layer {ML). SEM. bar scale = ll),um,

274 S.J. Harrison. S. T Moss, E. B. G. Jones"

Fig. 2. Clavariopsis aquatica. TEM of the distal region ofa conidial arm at the same stage of development as shown in trig. I. Note the convoluted plasma membrane (P). two wall layers (Wl, WO). and a zone of mucilage comprising an inner (MLI) and an outer less

compact region (MLO). Bar scale = 2IX) nm.

. - 1

> , , /

- ,,%..~ ,~

Fig. 3. Lemonniera aquatica. Conid ium on nucleopore membrane, 6 h aflcr settlement, showing apprcssorial initials (arrowed). SEM, bar scale = lOOum.

development may include surface energy, hardness and topography and that one of the functions ofappressoria in species of aquatic Hyphomycetes is adhesion of the conidit, m or germ tube to a potential substratc.

Subsequent to settlement but prior to germination the tips of the arms of the tetraradiate conidia of Clavariopsis aquatica produce mucilage (Figs 1 & 2). It is concluded that the mucilage produced by the matt, re conidium prior to settlement aids initial at tachment.

Fungal adhesion in aquatic Hyphomycetes 275

Germinated conidia ofLemonniera aquatica (Fig. 3) and other species with branched conidia produce germ tubes and appressoria at the tips of the arms which are in contact with the substratum. Mature appressoria possess a laver of mucilage and are delimited from the germ tube by a septum (Fig. 4). The appressoria formed on the nucleopore membrane do not appear to be morphologically different from those formed on wood.

At the TEM level the appressorium of Lemonniera aquatica contains electron luscent re~ons closely adpressed to the plasma membrane (Fig. 5)

Fig. 4. Myc+,ccPttrov,orafil~Jrmis. Apprcssorium on a birch veneer 12 h after settlement. Nt>tc the delimiting scptum (S) and st trroundiug nluci[:agc (MI+). Si-M, bar scale =

lOu m.

L

Fig. 5. Lemonniera aquatica. Appress¢~rium showing delimiting scptum (S), surrounding mucilage (ML) and electron luscent wlcuoles (V) associated with the plasma membrane

and numerous mitochondria . TEM, bar scale = 5,urn.

276 S. J. flarrison, S T. Moss. E. B. G, Jones

Fig. 6. ( 7avariot~sis aquatica. Appressorium with Iomasomc-like body (I,) a,,sociatcd with the cell wall. TEM, bar scale = 2~1~ nm.

and associated with cndoplasmic reticuh, m. The apprcssorium of (Tavariopsis aquatica contains multivesicular bodies within the cytoplasm and associated with the cndoplasmic rcticulum and iomasomc-likc structures adjacent to the cell wall (Fig. 6) at sitcs of wall growth and mucilage secretion. "l'hcsc cytoplasmic inclusions may indicate the presence of cxocytotic pathways involved in the formation of new wall layers, lyric enzymes and/or the deposition of cxtracellular mucilage.

Apprcssoria have evolved in species from a rangc of fungal taxa found in tliffcrelat environments. !!ach ecological niche imposes temporal, nutritional and physical slrcsscs on a fungus and this applies equally to the conidia of aqm, tic l lyphomycctcs. Apprcssoria may provide protection at a vulnerable stage in the life cycle of these species and aid retention prior to colonization.

REFERENCES

Rots. (J. & Jones, E. B. G. (1984). Observations on the attachment of spores of m~,rinc fungi. Botanica &laritla 27, 145-N).

Webster. J. (1959). Experiments with spores of aquatic tlyphomycctcs. I. Scdi,ncntation and impaction on smooth surt';.lccs. Attttals of Botany 23, 595-611.