Synnema induction in Penicillium funiculosum by tributyltin compounds

[ 381 ] Trans. Br. mycol. Soc. 89 (3), 381-422 (1987)

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NOTES AND BRIEF ARTICLES SYNNEMA INDUCTION IN PENICILLIUM FUNICULOSUM BY TRIBUTYL TIN COMPOUNDS BY P. J. NEWBY AND G. M. GADD* Department of Biological Sciences, University of Dundee, Dundee, DDr 4HN

Synnema development was induced in Penicillium funiculosum when grown in the presence of sub-lethal concentrations of tributyltin oxide (TBTO) or tributyltin chloride (TBTC). The possible role of such structures in evasion of toxicity is discussed. Heavy-metal compounds of e.g, copper, mercury and tin have been used as fungicides for many years (Dekker, 1976) and tributyltin compounds such as tributyltin oxide (TBTO) and tributyltin chloride (TBTC) receive widespread use as wood preservatives, in antifouling paints and as mildew control agents in household paints and textiles (Peller, 1970). Fungi can exhibit a variety of morphological and physiological responses to heavy metal compounds which may enable tolerance and, in many cases, detoxification (see Gadd, 1986). Fungal responses to organotin compounds have received scant attention, in contrast to those of bacteria, which is surprising in view of the great significance of fungi in the biodegradation of both natural and man-made materials and the widespread use of fungitoxic tin compounds. As part of a wider study on the fungitoxicity of tin compounds, it was observed that both TBTO and TBTC were capable of inducing synnema development in Penicillium funiculosum Thorn and this article reports and discusses this phenomenon. P. funiculosum (IMI 114933) was maintained at ~S °C, in the dark, on potato dextrose agar (PDA) of composition (g 1-1): dried potato (Cadburys), 18'S; D-glucose, 16'S; agar (Lab M, No.2), 1S·0. TBTO and TBTC (obtained from Fluorochem Ltd, Glossop, Derbyshire, SKI3 9XE) were diluted in ethanol and added to molten agar at soo to final concentrations of up to 37 JlM. Other tin compounds tested were tinuv) chloride pentahydrate and dimethyltin dichloride which were incorporated in PDA to a final concentration of 10 mM. Where necessary, the pH of the agar medium was adjusted by the addition of either 0'1 M-HCl or 0'1 M-KOH to molten agar prior to pouring. For the diffusion well assay, P. funiculosum was grown on PDA for approximately I week and portions of the mycelial mat were gently

*

To whom all correspondence should be sent. 14

homogenized in sterile distilled water using a Jencons homogenizer (S strokes) and then 10 ml of solid PDA was inoculated by spreading with 0'1 ml of this suspension adjusted to O.D' 660 ~ O'S and a 6 mm diam well cut in the agar using a sterile cork borer. Aliquots (20 JlI) of solutions of TBTO, TBTC or ethanol were added to the wells and plates were incubated at 2So for 2 weeks. For scanning electron microscopy, 4 mm diam discs were excized from colonies of P. funiculosum growing in the presence of TBTO or TBTC and were fixed in 2'S % (v/v) glutaraldehyde in S roMpiperazine-N,N'-bisjz-ethane sulphonic acid] (PIPES) buffer, pH 6'S, for 2 hat 4°. After washing the discs with buffer, dehydration was carried out using an ascending ethanol in water series (40100% v/v) followed by a Freon/ethanol series (So-IOO% v/v) with IS min immersion at each step. All preparations were dried by the critical point method, attached to aluminium stubs with silver paint and coated with gold using an SEM coating unit (Polaron Equipment Ltd, Model ESloo). Specimens were examined in a Joel JSM scanning electron microscope. During growth of P. funiculosum on concentrations ofTBTO or TBTC between 0'37 and 37 JlM, synnema production was evident and both TBTO and TBTC induced identical morphological changes (Figs I, 3). Synnema production did not occur below 0'37 JlM. Such production appeared independent of pH over the range pH 4'2-7'4 and synnema production was not observed in a laboratory strain of Penicillium italicum Wehmer or Penicillium ochro-chloron Biourge (IMI 39806) grown in the presence ofTBTO or TBTC. Neither tinuv) chloride or dimethyltin dichloride induced synnema development in P. funiculosum at concentrations up to 10 roM. The diffusion well experiments confirmed the inductive effect of TBTO and TBTC with synnema development occurring nearest the wells (Figs 2, 4) and showed that MYC89

Notes and brief articles

Fig.

I.

Growth and synnema production by P . [uni culosum grown for 2 weeks at 25° on PDA containing

37 pM TBTC.

Fig. 2 . Induction of synnema production in P. fun iculosum by TBTC when added to a well cut in a PDA plate. 20 pI of 37 pM TBTC was added to a 6 mID diam well cut in the agar plate which was incubated for 2 weeks at 25°. Synnernatal development occur s ad jacent to the well. Fig . 3. Growth and synnema production by P. funiculosum grown for 2 weeks at 25° on PDA containing 19'6 pM TBTO. Fig. 4. Induction of synnema production in P. funi culosum by TBTO added to a diffusion well ( 2 0 pI of 19'6 pM TBTO) as described for Fig. 2. The right-hand well is an ethanol control. Typical examples are shown from one of ten experiments. Bar markers = I em.

ethanol alone did not induce any morphological effect in P. funiculosum (F ig. 4). .Figures 5-8 show some of the different stages leading to synnema formation by P. funiculosum in the presence of TBTe. Initial overlapping of hyphae forms a centre for potential synnema development (Fig. 5) and further aggregation results in formation of primordia which rise above the colony (Fig. 6). The primordia are small synnemata which further develop by thickening and apical growth and eventually extensive production of conidiophores and conidia (Figs 7, 8). This sequence of events agrees closely with patterns Trans. Br . mycol. Soc. 89 (3), ( 1987)

of synnema formation observed in a variety of other fungal species (H iratsuka & Takai, 1978; Watkinson, 1979). Synnemata are defined as aerial, multihyphal structures in which the apices of the component hyphae advance together and which ultimately form spores (Watkinson, 1979). Thus, they are concerned with spread and survival of a given species and their formation can be triggered by a variety of external factors, e.g, light-dark cycles, low temperature, alcohols, detergents, carbon dioxide and amino acids (Watkinson, 1979). Synnemata of the penicillia have received most atten-

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Notes and brief articles

;t,

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8

.

Figs 5-8. Scanning electron micrographs of synnema development in P. funiculosum induced by 37 pM TBTC in PDA. Fig.S· Overlapping of hyphae forming a potential site for synnema development (near centre of I4-day-old colony), bar = 10 pm. Fig. 6. Close alignment of hyphae and unidirectional growth leading colony), bar = 10 pm.

to

stalk formation (edge of 34-day-old

Fig. 7· Thickened stalk formation with few conidiophores and conidia (near centre of 34-day-old colony), bar = 100 jlm. Fig. 8. Mature synnema with extensive conidiophore and conidium production (centre of 34-day-old colony), bar = lOoltm.

tion and there are some accounts of cation involvement in their formation, e.g. Penicillium clasnforme Bain. and Penicillium clavigerum Demelius required manganese for synnema development (Tinnell et al., 1977). In Sphaerostilbe repens Berkeley & Broome, both synnematal and rhizomorph development only occurred in the presence of calcium, though strontium was also capable of inducing some morphological development (Botton, 1978). Several theories have been emTrans. Br. mycol. Soc. 89 (3), (1987)

ployed to explain the effect of divalent cations and it has been suggested that such cations may reduce repulsive electrostatic forces between cells and/or act as 'bridges' between cell surfaces thus facilitating hyphal aggregation (Botton, 1978). However, interactions of cations with metabolic controls are also likely to be important though this is still an area of uncertainity (Botton, 1978; Watkinson, 1979). To our knowledge, this is the first observation

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Notes and brief articles

that tributyltin compounds can induce apparently true synnematal development in P. funiculosum which is generally regarded as non-synnernatal (Onions et al., 1981). Penicillium funiculosum is known to produce funicles which are defined as ropes of fertile hyphae and are regarded as rudimentary synnemata (Pitt, 1979). However, the structures we observed in the presence of the tributyltin compounds used here were fertile along the majority of their length (2-5 mm), conformed to standard definitions of synnemata (Webster, 1980; Onions et al., 1981) and therefore cannot be regarded as rudimentary structures. As in other synnematal fungi, the production of synnemata results in a wider spatial separation between the conidia and the substrate than in non-synnematal colonies, and this will not only aid dispersal but may also ensure conidium formation away from potential toxicants in the substrate if these cannot be translocated along the hyphae. It may be relevant that conidium production on synnemata can proceed by means of endogenous substrates in the absence of further supplies of substrate (Watkinson, 1979). It is interesting to speculate whether such a morphological response is related to avoidance of toxicity. It is evident that if synnema formation, induced here by tributyltin compounds, is a survival strategy then the phenomenon may be of wider significance in fungi exposed to potential toxic agents and further work on the physiological bases of such responses may be profitable. Paul J. Newby gratefully acknowledges financial support by means of the Tayside Region Centenary

Award. Thanks are also due to Chris Harris for expert photographic assistance. REFERENCES

BOTTON, B. (1978). Influence of calcium on the differentiation and growth of aggregated organs in Sph aerostilbe repens. Canadian Journal of M icrobiology z4, 1039- 1047. DEKKER, J. (1976). Acquired resistance to fung icides. Annual Review of Phytopathology 14, 405-428. GADD, G. M. (1986). Fungal responses towards heavy metals. In Microbes in Extreme Environments (ed. R. A. Herbert & G. A. Codd ), pp. 83-IIO. London: Academic Press. HlRATSUKA, Y. & TAKAl, S. (1978). Morphology and morphogenesis of synnemata of Ceratocystis ulmi. Canadian Journal of Botany 56, 1909-1914. ONIONS, A. H . S., ALLSOPP, D. & EGGINS, H . O. W. ( 1981). Smith's Introduction to Industr ial Mycology. London: Edward Arnold. PITT, J. 1. (1979). The Genus Penicillium and its Teleomorphic States EupenicilIium and Talaromyces, London: Academic Press. POLLER, R. C. (1970). The Chemistry of Organotin Compounds. London : Logos Press Ltd. TINNELL, W . H., JEFFERSON, B. L. & BENOIT, R. E. (1977). Manganese mediated morphogenesis in Penicillium claviforme and Penicillium clauigerum. Canadian J ournal of Microbiology z3, 209-212. WATKINSON, S. C. (1979). Growth of rhizomorphs, mycelial strands, coremia and sclerotia. In Fungal Walls and Hyphal Growth (ed. J. H . Burnett & A. P. J. Trinci), pp. 91-II3. London: Cambridge University Press. WEBSTER, J . (1980). Introduction to Fungi. Cambridge : Cambridge University Pre ss.

EFFECT OF EXOGENOUS LIPIDS ON GROWTH AND AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS BY ABIDIN BIN HAMID* AND JOHN E. SMITH

Department of Bioscience and Biotechnology, Applied Microbiology Division, University of Strathclyde, 204 George Street, Glasgow GI IXW, U.K.

Growth of Aspergillus fiavus was enhanced by the addition of crude and purified linoleic acid hydroperoxides and stearic acid. Aflatoxin production was stimulated by crude and purified linoleic acid hydroperoxides, stearic acid, cis-7,8-epoxY-2-methyl octadecane and cerulenin but was inhibited by linoleic acid. Aflatoxins are a group of closely related polyketide secondary metabolites produced by certain strains of Aspergillus fiavus Link : Fr. and A . parasiticus Speare. Recent evidence indicates that aflatoxins are derived from C20 anthraquinone precursors

* Present address: Food Technology Div ision, M.A .R.D.I., 123°1 G .P.O . 50774, Kuala Lumpur, Malaysia. Trans. Br . mycol. Soc. 89 (3), (1987)

and are biosyntheticaUy decaketides (Turner & Aldridge, 1983). Acetyl-CoA is a key branch point for catabolic and anabolic systems of primary metabolism, particularly fatty acid metabolism (T urn er, 1976). Lipid sources isolated from wheat seeds, have been shown to stimulate the growth of A . fiavus; saturated lipids were less effective than unsaturated lipids (Fanelli & Fabbri, 1980). Fur-

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