Effect of falcarindiol on hyphal growth of Mycocentrospora acerina

Effect of falcarindiol on hyphal growth of Mycocentrospora acerina

Notes and brief articles (volume) of hypha . In the mamillate form, branching has occurred at a much lower critical volume. These changes would indica...

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Notes and brief articles (volume) of hypha . In the mamillate form, branching has occurred at a much lower critical volume. These changes would indicate a breakdown in the integration of hyphae probably brought about by poor aeration ofthe medium, as they have primarily occurred at S /V ratios < 1'0 but not at > 4'0 (growth at a higher temperature may also be affecting similar processes). Thus abnormal growth may be due to fundamental changes in wall synthesis as suggested by Jones & Bu'Lock (1977). The latter workers induced effects similar to those obtained with Pilaira, by use of cyclic AMP. A feature of the morphological changes in Mucor spp ., however, was the production of septa. Septa were also formed in the delimitation of arthrospores in Mucor rouxii (Calmett e) Wehmer (Bartnicki-Garcia & Nickerson, 1962) but were absent from all forms of Pilaira. Whether these results have merely demonstrated another example of the plasticity of the mycelium of this fungus in response to the altered external

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environment (F letcher, 1971) or whether altered forms of branching perform a more adaptive role will require further study. The authors wish to thank Mrs Dee Barrett for technical assistance. REFERENCES

BARTNICKI-GARCIA, S . & NICKERSON, W .] . (1962). Induction of yeast-like development in Mucor by carbon dio xide . Journal of Bacter iology 84, 829--840. FLETCHER, H.] . (1971). Some aspects of the biology of Pilaira anomala - an extremely versatile fungus . Journal of B iological Education 5, 229--237. JONES, B. E. & BU'LOCK, ] . D . (1977). The effects of

N ' , 0 " -dibutyryl adenosine-j ',5' -cyclic monophosphate on morphogenesis in Mucorales. Journal of General Microbiology 103, 29--36. RIPPON,]. W. (1980). Dimorphism in pathogenic fungi. Critical Reviews in Microbiology 8, 49--97. TRINCI, A. P. ]. (1974). A study of the kinetics of hyphal extension and branch initiation of fungal mycelia. J ournal of General Microbiology 81, 225-236.

EFFECT OF FALCARINDIOL ON HYPHAL GROWTH OF

MYCOCENTROSPORA ACERINA BY B. GARROD AND B. G. LEWIS

School of Biological Sciences, Univ ersity of Ea st Anglia, Norwich NR4 7TJ

The polyacetylenic antifungal compound falcarindiol, isolated from carrot roots (G arrod, Lewis & Coxon, 1978), appears to have a broad spectrum of antifungal activity at low concentrations when assessed by measuring its effect upon spore germination (Garrod et al ., 1978 ; Kemp, 1978) and acts on the plasma membrane or on some process necessary for membrane function (Garrod, Lea & Lewis, 1979). The ED so for inhibition of germination of chlamydospores of Mycocentrospora acerina (Hartig) Deighton for this compound was 31'8,ug/ml (Garrod et al., 1978). M. acerina, the causal agent of liquorice rot, is one of the most important long-term storage diseases of carrots and celery (Rader, 1952; Mukula, 1957; Arsvoll, 1969; Derbyshire & Crisp, 1971 ; Day, Lewis & Martin, 1972). This investigation studies the effect of falcarindiol on hyphal growth of this pathogen and considers whether levels found in tissue are likely to be sufficient to account for the limitation of lesion spread. A series of 2'0 % ethanol solutions containing a range of falcarindiol concentrations were prepared; a solution containing 2'0 % ethanol only served as a control. Twelve small rectangular pieces of agar, Trans . Br . mycol. Soc. 78 , (3) (1982)

approximately 20 X 5 x 3 mm (thick), were cut from the edge of a 6-day-old colony of M. aeeTina on V8 juice agar (G arrod et al., 1978) grown at 15 ± 1 "C. Each block of agar bore a hypha I front approximately 10 mm from one edge . These blocks were placed on clean glass slides and surrounded by a carefully cut 'wall' of damp glass fibre paper, to support a large cover slip and to help maintain a high humidity (F ig. 1). Each of these systems was located on a Watson Microsystem 70 microscope, in a room at 15 ± 10, and the length of a single hypha on each slide was measured at 10 min intervals. When a constant rate of increase in hyphallength had been recorded (after about 40 min) 10,u1 of each solution was dropped, with the aid of a Hamilton syringe, on to the hypha 1tip under observation. The hyphal growth measurements were continued for a period of about 1 h. Prior to each measurement the cover slip was removed carefully and replaced immediately afterwards to avoid excessive loss of moisture. Microscope lamps were also turned off between readings to avoid heating effects on the hypha under observation and, at approximately 30 min intervals, 2-3 drops of water were added to the glass-fibre

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Fig. 1. Apparatus for measuring the effect offalcarindiol on growing hyphae of M. acerina. (a) Side elevation, (b) Plan view. (1) glass microscope slide; (2) layered, damp glass fibre paper; (3) mycelium of M. acerina; (4) rectangular block of V8 agar; (5) glass cover slip.

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Fig. 2. Graphs showing rate of hyphal growth of M. acerina before and after treatment with various concentrations of falcarindiol compared with a control. (Arrows indicate time of addition of falcarindiol or control solution.) Replicate measurements are represented by the two symbols 0 and •.

paper wall with a Pasteur pipette to maintain the humidity of the chamber. The effect offalcarindiol on hyphal growth of M. acerina is presented in Fig. 2. Control solutions, containing 2'0% ethanol only, checked the growth of hyphae very slightly but recovery was rapid (approximately 5-10 min); this effect has been reported for other fungi (Higgins, 1978). Doses of 58'8,ug/ml, or more, of falcarindiol immediately halted the growth of hyphal tips. Microscopic examination of these hyphal apices at the end of the Trans. Br. my col. Soc. 78, (3) (1982)

experiment (Fig. 3 A) suggested that the hyphal tips had burst and this may be the result of membrane damage (Garrod et al., 1979). At a concentration of 29'4 ,ug/ml the rate of growth was checked severely but recovery occurred after approximately 1 h. Regrowth and branching of the treated apices was common (Fig. 3 B) but the diameter of branches was markedly less than that of the hypha from which they arose and their cytoplasm was usually less opaque than the cytoplasm of pre-treatment hyphae.

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Notes and brief articles

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B Fig. 3. (a) Typical appearance of hyphal tips of M. acerina on V8 agar after treatment with 2'0% ethanolic solutions containing at least 58'8I'g/ml fa1carindiol. (Arrows indicate sites of possible bursting of hyphaI tip). (b) Regrowth from hyphal tip of M. acerina on V8 agar after treatment with 29"4l'g/ml fa1carindiol in a 2'0% ethanolic solution. Regrowth of an apex, a branch and the initial of a possible second branch are visible. Differences in opaqueness and diameter of new hyphae are apparent. Bars represent 20 I'm.

The response of hyphae to low concentrations of falcarindiol, although not explored in detail here, appears to correspond with other reported reactions of apices to damage, as described by Higgins (1978) with the pterocarpanoid phytoalexin maackiain, and by Robertson (1965) with osmotic shock. The total cessation of hyphal growth of M. acerina at levels of falcarindiol of 58'8,ug/ml or higher suggest that hyphae of this fungus are more sensitive than chlamydospores, where the calculated ED 95 for inhibition of germination was 158.8 ,ug/ml (Garrod, unpubl.), possibly because hyphae have thinner walls. These results show that low concentrations of Trans. Br. mycol. Soc. 78, (3) (1982)

falcarindiol, in addition to inhibiting chlamydospore germination (Garrod et al., 1978), are effective in inhibiting mycelial growth of M. acerina in vitro. It would therefore appear that the previously determined levels of falcarindiol in pericydic parenchyma (Garrod & Lewis, 1979) and woundhealed phloem parenchyma (Garrod & Lewis, 1980) of carrot roots, are sufficiently high to explain the resistance expressed by these tissues towards infection by M. acerina. Weare grateful to the Agricultural Research Council for financial support.

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Notes and brief articles REFERENCES ARSVOLL, K. (1969). Pathogens on carrots in Norway. Meidinger fra Norges Landbrukshogskole 48, 22-26. DAY, J. R., LEWIS, B. G. & MARTIN, S. (1972). Infection of stored celery plants by Centrospora acerina. Annals of Applied Biology 71, 201-210. DERBYSHIRE, D. M. & CRISP, A. F. (1971). Vegetable storage diseases in East Anglia. Proceedings of the 6th British Insecticide and Fungicide Conference, pp. 167-172. GARROD, B., LEA,E. J. A. & LEWIS, B. G. (1979). Studies on the mechanism of action of the antifungal compound fa1carindiol. New Phytologist 83, 463-471. GARROD, B. & LEWIS, B. G. (1979). Location of the antifungal compound fa1carindiol in carrot root tissue. Transactions of the British Mycological Society 72, 5 15-5 17. GARROD, B. & LEWIS, B. G. (1980). Probable role of oil ducts in carrot root tissue. Transactions of the British Mycological Society 75, 166-169.

GARROD, B., LEWIS, B. G. & COXON, D. T. (1978). Cis-heptadeca-1, 9-diene-4, 6-diyne-3, 8-diol, an antifungal polyacetylene from carrot root tissue. Physiological Plant Pathology 13, 241-246. HIGGINS, V. J. (1978). The effect of some pterocarpanoid phytoalexins on germ tube elongation of Stemphylium botryosum. Phytopathology 68, 339-345. KEMP, M. S. (1978). Fa1carindiol an antifungal polyacetylene from Aegopodium podagraria. Phytochemistry 17, 1002. MUKULA, J. (1957). On the decay of stored carrots in Finland. Acta Agriculturae Scandinatnca, Supplementum 2. RADER, W. E. (1952). Diseases of stored carrots in New York State. Cornell University Agricultural Station Bulletin, no. 889. ROBERTSON, N. F. (1965). The mechanism of cellular extension and branching. In The Fungi, vol. 1 (ed. G. C. Ainsworth & A. S. Sussman), pp. 613-623. New York: Academic Press.

EFFECTS OF ENVIRONMENTAL STRESS IN RECOVERY MEDIA ON COLONY FORMATION BY SUBLETHALLY HEAT-INJURED SACCHAROMYCES CEREVISIAE BY L. R. BEUCHAT

Department of Food Science, University of Georgia, Agricultural Experiment Station, Experiment, Georgia 30212, USA Metabolic and structural debilitation of yeast cells as a result of exposure to elevated temperatures has attracted the interest of food mycologists in recent years. The observation that heat-stressed (injured) cells may require special conditions for resuscitation and colony formation in enumeration media has promoted considerable research activity to determine specific site(s) of cellular impairment. The results of these investigations might enable researchers to effectively define procedures for detecting yeasts in thermally processed foods. Heat-stressed yeasts exhibit decreased tolerance to several environmental factors. Fries (1963) reported that the maximum temperature for growth of Rhodotorula glutinis (Fres.) Harrison was reduced by pretreating cells for 2 min at 48°C. While cells of many genera of yeasts which have not been stressed by heat are less tolerant to acid compared to neutral pH (Koburger, 1972; Flannigan, 1974), this lack of tolerance is often magnified by heat injury (Nelson, 1972; Menegazzi & Ingledew, 1980). Enhancement of sensitivity of Candida utilis (Henneberg) Lodder & Kreger-van Rij to sodium chloride as a result of heat treatment has been demonstrated (Tsuchido, Nakagawa, Trans. Br. mycol. Soc. 78, (3) (1982)

Okazaki & Shibasaki, 1972; Shibaski & Tsuchido, 1973)· Exposure of yeasts to heat may result in leakage of cellular contents (Nash & Sinclair, 1968; Hagler & Lewis, 1974). Repair of cells following treatment therefore is often accompanied by an increased dependency upon specific nutrients in the recovery medium (Stevenson & Richards, 1976), increased respiration rates (Graumlich & Stevenson, 1979) and synthesis of cellular components (Nash & Grant, 1969; Tsuchido, Okazaki & Shibasaki, 1972; Graumlich & Stevenson, 1978). The present study was designed to investigate the susceptibility of a Chablis wine strain of Saccharomyces cerevisiae Hansen to heat injury. The ability of stressed cells to recover at various pH values and temperatures when surface-plated on various agars was examined. In addition, recovery agar supplemented with sodium chloride, sucrose and two food preservatives, potassium sorbate and sodium benzoate, was evaluated. Treated cells were exposed to selected antimetabolites in an attempt to determine specific sites of injury. Yeast extract - malt extract - peptone - glucose broth (YMPG, pH 7'0) was used as the basal

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