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Stimulation of growth and reproduction of Sphaeronaemella fimicola by other coprophilous fungi
Mycol. Res. 102 (9) : 1055–1061 (1998)
1055
Printed in the United Kingdom
Stimulation of growth and reproduction of Sphaeronaemella fimicola by other coprophilous fungi
R O L A N D W. S. W E B ER1 A N D J O H N W E B S T ER2 " Washington Singer Laboratories, Department of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG, U.K. # 12 Countess Wear Road, Exeter EX2 6LG, U.K.
An in vitro screen of 83 isolates of coprophilous fungi revealed that many of them were capable of stimulating growth of Sphaeronaemella fimicola at a distance. When in direct contact with S. fimicola, many (excluding the Zygomycotina) also stimulated the production of perithecia. Species of Coniochaeta, Ryparobius, Thelebolus and Trichobolus were the strongest inducers. These and related genera were also associated with perithecia of S. fimicola on dung collected from the field. Hyphae of many of the strong perithecium inducers and a few non-inducing species were parasitized by necrotrophic intrahyphal growth of S. fimicola. Perithecium formation was stimulated even when parasitism was prevented by separation of growing mycelia of Ryparobius pachyascus and S. fimicola by Cellophane. In contrast, prior growth and removal of R. pachyascus followed by inoculation with S. fimicola caused enhanced growth and asexual reproduction without concomitant perithecium formation.
The dung of herbivorous animals is a rich substratum for fungi and supports a high species diversity. Fruit bodies of coprophilous fungi often occur in succession, following the sequence Zygomycotina U Ascomycotina U Basidiomycotina (Dix & Webster, 1995). Succession was thought to be determined by differences in the latent period of germination, mycelial growth rate and the sequential degradation of complex carbohydrates by these fungi. It is now considered to be primarily a function of the time required for fruit body production (Harper & Webster, 1964). The fruiting phase of coprophilous fungi may be terminated by any of a range of constraints imposed by competing fungi. These may include the production of antibiotics (Singh & Webster, 1973 ; Wicklow & Hirschfield, 1979 ; Gloer, 1995), death following hyphal contact especially with Basidiomycotina (Ikediugwu & Webster, 1970 a, b), and parasitism e.g. of Pilaira or Pilobolus species by other members of the Mucorales (Berry & Barnett, 1957 ; Jeffries & Young, 1994). Nevertheless, beneficial interactions between different coprophilous fungi also occur, especially in terms of nutrient exchange, and may be important means by which nutrient deficiencies can be complemented. For instance, Pilobolus kleinii Tiegh. displays markedly enhanced growth, asexual and sexual reproduction in culture in the presence of ammonia released by other coprophilous fungi (Page, 1959, 1960). Pilobolus spp. also have a requirement for the organo-iron compound coprogen, supplied by other fungi and bacteria (Hesseltine et al., 1953 ; Keller-Schierlein & Diekmann, 1970). The release of organic phosphate by associated fungi (hosts) has been shown to be required as a trigger for perithecium formation in culture by Melanospora destruens Shear (¯
Sordaria destruens (Shear) Hawker ; Hawker, 1951) and by Chaetomium globosum Kunze (Hawker, 1948 ; Buston & Khan, 1956). These examples, however, have previously been demonstrated only in vitro, and their significance on the normal substratum remains to be tested. Sphaeronaemella fimicola Marchal is a coprophilous ascomycete which produces a droplet of sticky ascospores at the tip of a long-necked perithecium. In culture, perithecium formation has been observed only in the presence of associated fungi such as Aspergillus repens (de Bary) E. Fisch. and Microascus sordidus Zukal (Cain & Weresub, 1957), whereas in axenic culture only the anamorph (Gabarnaudia fimicola Samson & W. Gams) is produced (Cain & Weresub, 1957 ; Samson, 1974). Furthermore, like the closely related but non-coprophilous Table 1. Fungi associated with perithecia of Sphaeronaemella fimicola on dung samples taken from the field Associated fungi Rabbit (Humber Marsh, near Hereford) ; 2 Apr. 1996 Deer (Haugh Wood, Mordiford, near Hereford) ; 2 Apr. 1996 Sheep (Countess Wear, Exeter) ; 7 Nov. 1996
Rabbit (Dawlish Warren, Devon) ; 10 Nov. 1996
Melanospora sp., Thelebolus sp. Sporormiella sp., Trichobolus sp.
Pilobolus kleinii ; Ascobolus immersus, Coprobia granulata, Iodophanus carneus (Pers.) Korf, Lasiobolus ciliatus, Podospora sp. ; Coprinus ephemerus Mucor mucedo, Mucor sp., Pilobolus sp., Pilaira anomala, Piptocephalis sp. ; Podospora vesticola, Saccobolus depauperatus
Stimulation of Sphaeronaemella by other fungi
1056
Table 2. Screening of a range of coprophilous fungi for their capacity to stimulate growth and perithecium formation in S. fimicola (screens 1–3) and their susceptibility to mycoparasitic attack (screen 4) Growth of S. fimicolaa
Zygomycotina Basidiobolus ranarum Eidam Chaetocladium brefeldii Tiegh. & Le Monn. Mucor mucedo Bref. M. mucedo Section M. hiemalis Wehmer Section Pilaira anomala (Ces.) J. Schro$ t. Pilobolus crystallinus (Wigg.) Tode Ascomycotina Ascobolus albidus P. Crouan A. furfuraceus Pers. A. immersus Pers. A. perplexans Massee & E. S. Salmon Ascozonus crouanii (Sacc.) Boud. Chaetomium dolichotrichum Ames C. spirale Zopf C. subspirale Chivers Coniochaeta hansenii (Oudem.) Cain Coniochaeta sp. Coprobia granulata (Bull.) Boud. Delitschia furfuracea Niessl ex Rehm Delitschia sp. Gymnoascus reesii Baran. Lasiobolus ciliatus (J. C. Schmidt) Boud. Melanospora sp. A Melanospora sp. B Microascus trigonosporus C. W. Emmons & B. O. Dodge Orbilia fimicoloides J. Webster & Spooner Podospora curvicolla (Winter) Niessl P. curvula (de Bary ex Winter) Niessl P. decipiens (Winter ex Fuckel) Niessl P. excentrica N. Lundq. P. intestinacea N. Lundq. P. vesticola (Berk. & Broome) Cain & J. H. Mirza isol. A P. vesticola isol. B Podospora sp. Pseudeurotium sp. Ryparobius pachyascus Zukal ex Rehm isol. A R. pachyascus isol. B Saccobolus caesariatus Renny ex W. Phillips Sordaria fimicola (Rob. ex Desm.) Ces. & de Not. Sporormiella intermedia (Auersw.) Ahmed & Cain Sporormiella sp. Thelebolus nanus Heimerl isol. A T. nanus isol. B T. stercoreus Tode Trichobolus zukalii (Heimerl) Kimbr. Wawelia sp. Fungi Imperfecti Alternaria sp. Arthrobotrys sp. A Arthrobotrys sp. B. Aspergillus candidus Link ex Link A. repens (de Bary) E. Fisch. A. terreus Thom Aspergillus sp. Doratomyces nanus (Ehrenb.) F. J. Morton & G. Sm. D. stemonitis (Pers. : Fr.) F. J. Morton & G. Sm. isol. A D. stemonitis isol. B Geotrichum sp. Gliocladium roseum Bainier Series
Perithecium productionb
Dung source
Scr. 1
Scr. 2
Scr. 3
Scr. 1
Scr. 2
Scr. 3
Parasitismc Screen 4
Frog Rabbit Rabbit Rabbit Rabbit Rabbit Deer
® 0 ® ® ® ®
0 ® ® ® ®
® ® ® ® ® ®
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Deer Rabbit
® ® ® ® 0 ® 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ® 0
0 ® ® 0 0 0 0 0 0 0 ® 0 0
® ® ® ® ® ® ® ® 0 ® 0 0 0 ® ® 0 ® ® 0 0 0 ® ® 0 ® ® 0 0 ®
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 * * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 *
0 0 0 0 0 0 0 0 0 * 0 0 0 * 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Pony Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Deer Rabbit Rabbit Rabbit Rabbit
® ® ® 0 0 0 0
0 0 0 0 0
® ® ® 0 ® 0 ® ® ® ® ®
0 0 0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0 0 0 0 * 0
0 0 0 0 0 0 0 0 0 0 0
Sheep Deer Rabbit Mouse Rabbit Rabbit Deer Rabbit Sheep
Rabbit Pony Rabbit Rabbit Mouse Rabbit Sheep Rabbit Rabbit Rabbit Sheep Sheep Deer Rabbit Deer Rabbit Sheep Deer
R. W. S. Weber and J. Webster
1057
Table 2. (cont.) Growth of S. fimicolaa
Gliomastix murorum (Corda) S. Hughes Graphium putredinis (Corda) S. Hughes Isaria sp. Oedocephalum glomerulosum (Bull.) Sacc. Onychophora coprophila W. Gams, P. J. Fisher & J. Webster Penicillium claviforme Bainier P. funiculosum Thom Stachybotrys cylindrospora C. N. Jensen Stilbella erythrocephala (Ditmar) Lindau Trichothecium roseum (Pers.) Link ex Gray Verticillium tenerum Nees ex Link Volutella ciliata Alb. & Schwein. ex Fr. Basidiomycotina Coprinus cinereus Que! l. C. ephemerus (Bull. ex Fr.) Fr. C. filamentifer Ku$ hner C. heptemerus M. Lange & A. H. Sm. C. miser (Karst.) Karst. C. vermiculifer Joss. ex Dennis Cyathus stercoreus (Schwein.) De Toni Panaeolus semiovatus (Sow. ex Fr.) S. Lundell P. sphinctrinus (Fr.) Que! l. Psilocybe merdaria (Fr.) Ricken Sphaerobolus stellatus Tode : Pers. Sporobolomyces roseus Kluyver & C. B. Niel Stropharia semiglobata (Batsch ex Fr.) Que! l.
Perithecium productionb
Dung source
Scr. 1
Scr. 2
Scr. 3
Scr. 1
Scr. 2
Scr. 3
Parasitismc Screen 4
Rabbit Deer Rabbit Sheep Rabbit Deer Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit
0 0 0 0 ® 0 0
0 0 0 0
® ® ® ® ® ® ® ® ® ® ® ®
0 0 0 0 0 0 0 0 0 0 0
0 * 0 * 0 0 * 0 0 0
0 0 * 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
® 0 0 ® 0 0
0 0 0 0 0 0 0
® ® ® ® ® ® ® ® ® ® ® 0
0 0 0 0 0 0 0 0 0 0
0 * 0 0 0 0 0 * 0 0 0 * 0
0 0 0 * 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
Pony Sheep Sheep Sheep Deer Rabbit Cow Cow Sheep Pony Cow
Screens produced similar results between all three isolates of S. fimicola. In this table, the highest-scoring interactions are represented. Growth of S. fimicola against the test fungus. ®, Collapse of S. fimicola hyphae ahead of or upon contact with test species ; 0, intact hyphae of S. fimicola but no enhanced growth ahead of mycelial encounter, relative to S. fimicola control in the absence of test fungus ; , enhanced growth ahead of encounter, relative to control ; , overgrowth of test fungus by S. fimicola. b Production of perithecia by S. fimicola after 35 d incubation. 0, No perithecia produced ; *, only immature perithecia produced ; , less than 25 mature perithecia per colony of S. fimicola ; , more than 25 mature perithecia per colony. c Absence (0) or presence () of mycoparasitism by any of the three isolates of S. fimicola, as intrahyphal growth inside hyphae of test fungus. a
Sphaeronaemella helvellae Karst. (Vakili, 1985), S. fimicola can act as a necrotrophic mycoparasite (Hawksworth, 1981 ; Weber & Webster, 1997). In the present report, the range of coprophilous fungi capable of stimulating sexual reproduction in S. fimicola and the extent to which these are subjected to mycoparasitic attack have been examined. The results obtained are discussed and interpreted in the context of other interactions reported amongst coprophilous fungi.
dung collected at Humber Marsh (near Hereford, U.K.). Isolates were obtained from ascospores streaked onto V-8 juice agar which contained 20 % (w}v) V-8 juice (Campbell Grocery Products Ltd, King’s Lynn, Norfolk) according to Miller (1955). All fungi were maintained at 4 °C on slopes of potato dextrose agar, V-8 juice agar, or yeast extract–sucrose agar containing (l−") 4 g yeast extract, 20 g sucrose, 1 g KH PO , # % 0±5 g MgSO \7H O and 15 g Lab M2 agar. % #
MATERIALS AND METHODS
Screening experiments for perithecium production and susceptibility to mycoparasitism
Occurrence of Sphaeronaemella fimicola on dung Dung samples collected at various locations were incubated at room temperature (r.t.) in a moist chamber, and those containing S. fimicola were monitored for the presence of fruit bodies of associated fungi. Isolation and maintenance of coprophilous fungi Three isolates of S. fimicola used in this work were : A, from rabbit dung collected at Dawlish Warren (Devon, U.K.) ; B, from deer dung collected at Forstgut Wiegersen (near Buxtehude, Lower Saxony, Germany) ; and C, from rabbit
Conidia of S. fimicola readily germinated on a wide range of agar media and were, therefore, used as inoculum except in experiments where plugs of vegetative mycelium were required. Conidia were produced abundantly on yeast extract–sucrose agar plates within 2 wk of inoculation (20° in the dark). A total of 83 isolates representing 79 coprophilous species (Table 2) were examined for their capacity to induce perithecium production in all three S. fimicola isolates. In each plate, the test species was centrally inoculated with the three S. fimicola isolates evenly displaced at a distance of 3 cm from
Stimulation of Sphaeronaemella by other fungi the centre. In order to widen the range of growth conditions, three different screens were performed which varied in the pH of the medium and in the mode of inoculation. In screen 1, V8 agar was adjusted to pH 6 by addition of NaOH (Diener, 1955), and conidia of S. fimicola were inoculated 3 d before the host fungus was added. In screen 2, adjustment to pH 7±2 was by 10 m Tris}maleate buffer and the mode of inoculation was as for screen 1. In screen 3, adjustment to pH 7±2 was as for screen 2, but inoculation was by mycelial plugs (5 mm diam.) of S. fimicola onto established host mycelium which had been pre-grown for 1–3 wk depending on the growth rate of the species tested. Control plates contained the three S. fimicola isolates without a test fungus. In all screens, incubation was at 20° in the dark. Stimulation of growth relative to controls in the absence of host fungi (screens 1 and 2) and number of perithecia produced by each S. fimicola colony (all screens) were monitored. The three isolates of S. fimicola were screened for evidence of parasitism against all 83 fungal isolates. Five-fold diluted yeast extract–sucrose agar plates were inoculated simultaneously with conidia of S. fimicola and a host fungus, 3 cm apart as described above, and incubated in the dark at 20°. Mycoparasitism was established under the microscope by cutting samples of agar from the zone of interaction of colonies and mounting in cotton blue in lactic acid on a microscope slide. Detailed experiments with R. pachyascus and S. fimicola Dual-culture experiments involving S. fimicola (isol. C) and Ryparobius pachyascus Zukal ex Rehm (isol. A) were carried out in order to characterize further the nature of the interactions involved. A detailed growth study of S. fimicola with R. pachyascus was performed at 20° on V-8 agar adjusted to pH 6, in order to quantify its effect on growth and reproduction in S. fimicola. The intensity of sexual reproduction was expressed as the number of perithecia cm−# of colony, and asexual reproduction by haemocytometer counts of conidia cm−# of colony. The area of the colonies of S. fimicola was determined by tracing their outline on pre-weighed oven-dried paper and reweighing the cut-out tracing after drying. In order to determine whether direct hyphal contact was necessary for the induction of perithecium formation, S. fimicola was grown in dual-culture with R. pachyascus, with colonies being separated by a sterile sheet of Cellophane of 25 µm thickness. In another experiment, R. pachyascus was pre-grown on Cellophane placed over a V-8 agar plate for 7 d at 20°. The agar was then inoculated with S. fimicola after removal of the Cellophane with the mycelium of Ryparobius. In dual-culture experiments on fresh rabbit dung sterilized by autoclaving (121° for 15 min), batches of 15 sterile pellets in contact with each other were inoculated with a conidial suspension of S. fimicola (isol. C) at one end, and with or without a plug of mycelium of R. pachyascus (isol. A) at the other. Incubation was at 20° in the dark. Additionally, pellets with or without prior colonization by R. pachyascus (1 wk at 20° in the dark) were re-sterilized, inoculated with S. fimicola and incubated as above. The aim of these experiments was the
1058 determination of suitable conditions for perithecium production by S. fimicola on the natural substratum. RESULTS Fungi associated with Sphaeronaemella fimicola in the field Dung pellets containing perithecia of Sphaeronaemella fimicola were incubated in a moist chamber and associated fungi were identified. In every sample, members of the Ascomycotina were present, discomycetes being the most prominent group. In contrast, fructifications of Zygomycotina were only occasionally found, and those of Basidiomycotina were rare at the time of fruiting of S. fimicola (Table 1). Screens of associated fungi in vitro The capability of 83 coprophilous fungi to promote growth and perithecium formation in, and to be parasitized by, S. fimicola was similar against three different isolates of S. fimicola, but showed variation between the different incubation conditions of the three separate screens performed. The results are summarized in Table 2 which, for simplicity of presentation, shows only the strongest response by any of the three S. fimicola isolates for each screen. The majority of fungi tested stimulated both growth and, subsequently, sexual reproduction in S. fimicola (Fig. 1). Some isolates promoted one activity, e.g. stimulation only of growth by Pilobolus crystallinus, Coprinus filamentifer, C. vermiculifer, Psilocybe merdaria, Melanospora sp. B and Thelebolus stercoreus, while Volutella ciliata triggered perithecium production, but did not stimulate growth. Growth. Relatively few fungi (20 isolates tested) had no apparent growth-promoting effect on S. fimicola (Table 2, denoted by the symbol 0) or caused collapse of its hyphae at a distance or upon contact (®). The majority of test species from all major groups stimulated growth of S. fimicola at a distance () in at least one of the screens, and 14 species were overgrown by S. fimicola (). With the exception of P. crystallinus and S. roseus, all fungi in the latter category were Ascomycotina or Deuteromycotina with ascomycete affinity. This type of interaction was particularly readily detected in screen 3, in which inoculum of S. fimicola had been placed on established colonies of the test fungi. Of 14 isolates susceptible to overgrowth, six were also parasitized by S. fimicola by means of intrahyphal growth. Exceptions included P. crystallinus and the small yeast cells of S. roseus. The interaction with Pilobolus is noteworthy because it was the only observed case of mutual stimulation in the sense that both fungi displayed more vigorous growth in dual-culture than in axenic culture, and because S. fimicola also induced asexual reproduction in P. crystallinus (Fig. 2). On the other hand, in 10 cases the ability of S. fimicola to parasitize hyphae of test fungi was not accompanied by large-scale overgrowth. Mycoparasitic potential was, therefore, not a prerequisite for overgrowth. Perithecium production. A wide range of Asco-, Basidio- and Deuteromycotina, but not the strongly growth-promoting
1059
Colony area (cm2)
R. W. S. Weber and J. Webster
3 10
5
0
1
Number of conidia × 106cm–2
0
7
14
21
28
35
7
14
21
28
35
14
21
28
35
4
3 2 1 0 0
Number of perithecia cm–2
80
5
60 40 20 0
2 Figs 1, 2. Interactions of Sphaeronaemella fimicola (isolate C ; left) with other fungi growing from the right. Scale bars 1 cm. Fig. 1. Dualculture with Ryparobius pachyascus (isol. A) on V-8 agar (pH 6). Vegetative growth of S. fimicola was stimulated, and perithecia were formed after establishment of contact between the two mycelia. Fig. 2. Dual-culture with Pilobolus crystallinus on V-8 agar (pH 7±2). Vegetative growth of both fungi was stimulated, but S. fimicola did not produce perithecia. Instead, P. crystallinus produced sporangiophores within the colony of S. fimicola, but not on its own.
zygomycete P. crystallinus, stimulated perithecium formation in S. fimicola. Among the strongest stimulators were Coniochaeta spp., Ryparobius pachyascus, Thelebolus nanus and Trichobolus zukalii, which were also susceptible to overgrowth or mycoparasitism by S. fimicola. In some cases perithecium initials and even mature perithecia were formed by S. fimicola prior to any mycelial contact. This occurred at 19 mm in dualculture with Microascus trigonosporus in screen 1, and 8 mm with Aspergillus candidus in screen 2. Interactions between R. pachyascus and S. fimicola in vitro The interaction between R. pachyascus (isol. A), the strongest overall inducer of growth and perithecium production, and S. fimicola (isol. C) was further studied on V-8 agar adjusted to pH 6 by NaOH. A simulation of growth (Fig. 3) and perithecium production (Fig. 5) relative to the pure-culture control was observed and asexual reproduction by phialo-
0
7
Incubation period (d)
Figs 3–5. Stimulation of Sphaeronaemella fimicola (isolate C) by dualculture with Ryparobius pachyascus (isolate A) (E) relative to S. fimicola alone (D) on V-8 agar (pH 6). Data are expressed as xa ³.. of four replicates. Fig. 3. Vegetative growth as colony area. Fig. 4. Asexual reproduction represented as number of phialoconidia cm−# of mycelium. Fig. 5. Sexual reproduction represented as number of perithecia cm−# of mycelium.
conidia was, likewise, enhanced by the presence of R. pachyascus (Fig. 4). When mycelia of R. pachyascus and S. fimicola were separated by a Cellophane sheet, induction of perithecium formation still occurred. Removal of R. pachyascus with the Cellophane sheet permitted subsequent formation of perithecial necks and ascospores by the underlying mycelium of S. fimicola. This occurred only in perithecia which had produced at least a rudimentary neck extension at the time of removal, whereas immature (globose) perithecia failed to develop further (Fig. 6). In another experiment, R. pachyascus was grown on Cellophane-covered agar for 7 d and then removed along with the Cellophane. S. fimicola, inoculated onto such pretreated medium, displayed a greatly enhanced growth rate as compared to untreated control plates (Fig. 7), but no development of perithecia or perithecium initials was observed. Conidia, harvested from pre-treated or control plates and inoculated onto fresh V-8 juice agar, gave rise to mycelia growing at a very similar rate (Fig. 8), which was the same as that on the initial untreated control plates (Fig. 7), suggesting that accumulation of the growth-promoting
Stimulation of Sphaeronaemella by other fungi
1060 substance(s) in conidia and utilization upon germination did not occur to any detectable degree. Inoculation of S. fimicola into sterilized rabbit dung When sterilized rabbit pellets were inoculated simultaneously with S. fimicola (isol. C) and R. pachyascus (isol. A), abundant perithecium production ensued, whereas in the absence of R. pachyascus, only the Gabarnaudia anamorph was produced. Inoculation of S. fimicola onto pellets treated by pre-growth of R. pachyascus and subsequent sterilization resulted in vegetative growth of the fungus and abundant production of conidia without perithecium formation. A destruction of the stimulant for perithecium production by autoclaving is unlikely because it was found to remain active in autoclaved cell-free extracts of R. pachyascus (R. W. S. Weber, unpublished results). Hence, our results indicate that induction of perithecium production in S. fimicola required the continuous presence of a suitable stimulatory fungus such as R. pachyascus, on dung as well as in vitro. DISCUSSION
6 Fig. 6. Perithecia of Sphaeronaemella fimicola (isol. C) after 1 wk simultaneous growth with Ryparobius pachyascus (isol. A) separated by Cellophane, followed by removal of the Cellophane and incubation for a further 3 d. Growth of an aerial neck occurred only in the perithecium which had developed rudiments of a neck extension (arrowhead) at the time of Cellophane removal. Immature (globose) perithecia (arrows) failed to develop further. Scale bar, 100 µm.
25
7
20
Colony radius (mm)
15 10 5 0 0
10
5
10
15
8
5 0 0
5 10 Incubation period (d)
15
Figs 7, 8. The effect of pre-growth of R. pachyascus (isol. A) on vegetative growth (colony radius) of S. fimicola (isol. C). Data are expressed as xa ³.. of four replicates. Fig. 7. Growth of S. fimicola on V-8 agar (pH 6) plates treated by pre-growth on Cellophane and removal of R. pachyascus (E) relative to an untreated control (D). Fig. 8. Growth on fresh V-8 agar plates of inoculum harvested from the treatment (E) and control (D) mycelia of the experiment in Fig. 7.
The capability of other fungi to stimulate perithecium formation in S. fimicola, initially observed by Cain & Weresub (1957) with an A. repens contaminant, has been found to be of widespread occurrence among coprophilous fungi (this study and J. Follett, unpublished). This is also likely to occur on dung in the field. Many strong stimulators, e.g. Saccobolus, Sporormiella, Thelebolus and Trichobolus, are associated with perithecia of S. fimicola on dung samples collected from the field. Furthermore, S. fimicola inoculated onto sterilized fresh rabbit pellets produced perithecia only in the presence of a suitable stimulator such as R. pachyascus, but not on its own. The interactions involved are probably complex because many fungi stimulated not only perithecium production but also vegetative growth of S. fimicola, often at some distance prior to hyphal contact. The simplest possibility is that both processes were stimulated by the same compound, but at different concentrations. If so, this substance would be involved in at least two distinct developmental processes, i.e. growth promotion and induction of perithecia followed by their maturation. The alternative is that these two processes are stimulated by two or more different substances which may explain why some fungi stimulated only either growth or perithecium production in S. fimicola. Furthermore, a limited quantity of the stimulatory substance(s) was sufficient for growth promotion, such as that released by pre-growth and removal of R. pachyascus, whereas a continuous supply of metabolite(s) appeared to be required for perithecium production up to the stage of formation of the neck initial. Multiple growth factor requirements are common especially among biotrophic mycoparasites (Barnett, 1970 ; Calderone & Barnett, 1972 ; Barnett & Binder, 1973). Interestingly, such a continuous supply of stimulant could be maintained across a Cellophane membrane separating S. fimicola from a suitable test species such as R. pachyascus. Direct hyphal contact was thus not required for perithecium production in S. fimicola, yet this fungus has been described as
R. W. S. Weber and J. Webster a necrotrophic mycoparasite of R. pachyascus (Weber & Webster, 1997) and other coprophilous fungi (present report). Weber & Webster (1997), however, pointed out that R. pachyascus could be re-isolated even from areas heavily infected by S. fimicola. Mycoparasitism, which has been reported previously among coprophilous fungi in a biotrophic form for zygomycetes such as Piptocephalis or Chaetocladium parasitizing other zygomycetes (Berry & Barnett, 1957 ; Jeffries & Young, 1994), may thus be more widespread than currently appreciated. The best-documented cases of mycoparasitism as a means of obtaining nutrients involve the contact biotrophs, specialized fungi which parasitize a relatively narrow host range (usually Ascomycotina). They obligately depend on an unidentified vitamin (mycotrophein) and other factors for growth and reproduction (Barnett & Lilly, 1958 ; Calderone & Barnett, 1972 ; Jordan & Barnett, 1978). For several reasons, mycotrophein is unlikely to be involved in the coprophilous fungus interactions reported here ; firstly, growth of S. fimicola was merely stimulated by, but not dependent upon, the presence of a host fungus. Secondly, the range of fungi stimulating growth of S. fimicola was wider than that reported for contact biotrophs (Gain & Barnett, 1970 ; Barnett & Binder, 1973) and included even Basidio- and Zygomycotina. Finally, stimulation of growth and, under certain conditions, perithecium formation occurred at a distance without hyphal contact. Further research is currently being directed towards identifying the compound(s) of interest. It is known that other ascomycetes such as Chaetomium globosum (Buston & Khan, 1956) and Melanospora destruens (Hawker, 1948) require exogenous organic phosphomonoesters as a stimulus for perithecium formation. Such exchanges of nutrients may be common and mutual (e.g. Barnett, 1968), as in the case of S. fimicola and P. crystallinus reported here. It is likely that the provision of growth factors is also important among coprophilous fungi, along with other concerted activities such as the breakdown of complex carbohydrates (Wood & Cooke, 1987). Hence, a diversity of fungi on dung may be required for the efficient growth of each species. We are indebted to Mr P. M. Booth, a B.M.S. Associate, for his support of this work, and we thank Dr D. Pitt for the provision of laboratory facilities. REFERENCES Barnett, H. L. (1968). The effects of light, pyridoxine and biotin on the development of the mycoparasite, Gonatobotryum fuscum. Mycologia 60, 244–251. Barnett, H. L. (1970). Nutritional requirements for axenic growth of some haustorial mycoparasites. Mycologia 62, 750–760. Barnett, H. L. & Binder, F. L. (1973). The fungal host–parasite relationship. Annual Review of Phytopathology 11, 273–292. Barnett, H. L. & Lilly, V. G. (1958). Parasitism of Calcarisporium parasiticum on species of Physalospora and related fungi. West Virginia Agricultural Experiment Station Bulletin 420T. (Accepted 31 October 1997 )
1061 Berry, C. R. & Barnett, H. L. (1957). Mode of parasitism and host range of Piptocephalis virginiana. Mycologia 49, 374–386. Buston, H. W. & Khan, A. H. (1956). The influence of certain micro-organisms on the formation of perithecia by Chaetomium globosum. Journal of General Microbiology 14, 655–660. Cain, R. F. & Weresub, L. K. (1957). Studies of coprophilous ascomycetes. V. Sphaeronaemella fimicola. Canadian Journal of Botany 35, 119–131. Calderone, R. A. & Barnett, H. L. (1972). Axenic growth and nutrition of Gonatobotryum fuscum. Mycologia 64, 153–160. Diener, U. L. (1955). Sporulation in pure culture by Stemphylium solani. Phytopathology 45, 141–145. Dix, N. J. & Webster, J. (1995). Fungal Ecology. Chapman & Hall : London. Gain, R. E. & Barnett, H. L. (1970). Parasitism and axenic growth of the mycoparasite Gonatorhodiella highlei. Mycologia 62, 1122–1129. Gloer, J. B. (1995). The chemistry of fungal antagonism and defense. Canadian Journal of Botany 73, S1265–S1274. Harper, J. E. & Webster, J. (1964). An experimental analysis of the coprophilous fungus succession. Transactions of the British Mycological Society 47, 511–530. Hawker, L. E. (1948). Stimulation of the formation of perithecia of Melanospora destruens Shear by small quantities of certain phosphoric esters of glucose and fructose. Annals of Botany (New Series) 12, 77–79. Hawker, L. E. (1951). Morphological and physiological studies on Sordaria destruens (Shear) comb. nov. (syn. Melanospora destruens), Sordaria fimicola and Melanospora zamiae. Transactions of the British Mycological Society 34, 174–186. Hawksworth, D. L. (1981). A survey of the fungicolous conidial fungi. In Biology of Conidial Fungi, Vol. I (ed. G. T. Cole & B. Kendrick), pp. 171–244. Academic Press : New York. Hesseltine, C. W., Whitehill, A. R. & Pidacks, C. (1953). Coprogen, a new growth factor present in dung required by Pilobolus. Mycologia 45, 7–19. Ikediugwu, F. E. O. & Webster, J. (1970 a). Antagonism between Coprinus heptemerus and other coprophilous fungi. Transactions of the British Mycological Society 54, 181–204. Ikediugwu, F. E. O. & Webster, J. (1970 b). Hyphal interference in a range of coprophilous fungi. Transactions of the British Mycological Society 54, 205–210. Jeffries, P. & Young, T. W. K. (1994). Interfungal Parasitic Relationships. CAB International : Wallingford, Oxon. Jordan, E. G. & Barnett, H. L. (1978). Nutrition and parasitism of Melanospora zamiae. Mycologia 70, 300–312. Keller-Schierlein, W. & Diekmann, H. (1970). Stoffwechselprodukte von Mikroorganismen. 85. Zur Konstitution des Coprogens. Helvetica Chimica Acta 53, 2035–2044. Miller, P. M. (1955). V-8 juice as a general purpose medium for fungi and bacteria. Phytopathology 45, 461–462. Page, R. M. (1959). Stimulation of asexual reproduction of Pilobolus by Mucor plumbeus. American Journal of Botany 46, 579–585. Page, R. M. (1960). The effect of ammonia on growth and reproduction of Pilobolus kleinii. Mycologia 52, 480–489. Samson, R. A. (1974). Paecilomyces and some allied hyphomycetes. Studies in Mycology 6, 1–119. Singh, N. & Webster, J (1973). Antagonism between Stilbella erythrocephala and other coprophilous fungi. Transactions of the British Mycological Society 61, 487–495. Vakili, N. G. (1985). Mycoparasitic fungi associated with potential stalk rot pathogens of corn. Phytopathology 75, 1201–1207. Weber, R. W. S. & Webster, J. (1997). The coprophilous fungus Sphaeronaemella fimicola – a facultative mycoparasite. Mycologist 11, 50–51. Wicklow, D. T. & Hirschfield, B. J. (1979). Evidence of a competitive hierarchy among coprophilous populations. Canadian Journal of Microbiology 25, 855–858. Wood, S. N. & Cooke, R. C. (1987). Nutritional competence of Pilaira anomala in relation to exploitation of faecal resource units. Transactions of the British Mycological Society 88, 247–255.
1055
Printed in the United Kingdom
Stimulation of growth and reproduction of Sphaeronaemella fimicola by other coprophilous fungi
R O L A N D W. S. W E B ER1 A N D J O H N W E B S T ER2 " Washington Singer Laboratories, Department of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG, U.K. # 12 Countess Wear Road, Exeter EX2 6LG, U.K.
An in vitro screen of 83 isolates of coprophilous fungi revealed that many of them were capable of stimulating growth of Sphaeronaemella fimicola at a distance. When in direct contact with S. fimicola, many (excluding the Zygomycotina) also stimulated the production of perithecia. Species of Coniochaeta, Ryparobius, Thelebolus and Trichobolus were the strongest inducers. These and related genera were also associated with perithecia of S. fimicola on dung collected from the field. Hyphae of many of the strong perithecium inducers and a few non-inducing species were parasitized by necrotrophic intrahyphal growth of S. fimicola. Perithecium formation was stimulated even when parasitism was prevented by separation of growing mycelia of Ryparobius pachyascus and S. fimicola by Cellophane. In contrast, prior growth and removal of R. pachyascus followed by inoculation with S. fimicola caused enhanced growth and asexual reproduction without concomitant perithecium formation.
The dung of herbivorous animals is a rich substratum for fungi and supports a high species diversity. Fruit bodies of coprophilous fungi often occur in succession, following the sequence Zygomycotina U Ascomycotina U Basidiomycotina (Dix & Webster, 1995). Succession was thought to be determined by differences in the latent period of germination, mycelial growth rate and the sequential degradation of complex carbohydrates by these fungi. It is now considered to be primarily a function of the time required for fruit body production (Harper & Webster, 1964). The fruiting phase of coprophilous fungi may be terminated by any of a range of constraints imposed by competing fungi. These may include the production of antibiotics (Singh & Webster, 1973 ; Wicklow & Hirschfield, 1979 ; Gloer, 1995), death following hyphal contact especially with Basidiomycotina (Ikediugwu & Webster, 1970 a, b), and parasitism e.g. of Pilaira or Pilobolus species by other members of the Mucorales (Berry & Barnett, 1957 ; Jeffries & Young, 1994). Nevertheless, beneficial interactions between different coprophilous fungi also occur, especially in terms of nutrient exchange, and may be important means by which nutrient deficiencies can be complemented. For instance, Pilobolus kleinii Tiegh. displays markedly enhanced growth, asexual and sexual reproduction in culture in the presence of ammonia released by other coprophilous fungi (Page, 1959, 1960). Pilobolus spp. also have a requirement for the organo-iron compound coprogen, supplied by other fungi and bacteria (Hesseltine et al., 1953 ; Keller-Schierlein & Diekmann, 1970). The release of organic phosphate by associated fungi (hosts) has been shown to be required as a trigger for perithecium formation in culture by Melanospora destruens Shear (¯
Sordaria destruens (Shear) Hawker ; Hawker, 1951) and by Chaetomium globosum Kunze (Hawker, 1948 ; Buston & Khan, 1956). These examples, however, have previously been demonstrated only in vitro, and their significance on the normal substratum remains to be tested. Sphaeronaemella fimicola Marchal is a coprophilous ascomycete which produces a droplet of sticky ascospores at the tip of a long-necked perithecium. In culture, perithecium formation has been observed only in the presence of associated fungi such as Aspergillus repens (de Bary) E. Fisch. and Microascus sordidus Zukal (Cain & Weresub, 1957), whereas in axenic culture only the anamorph (Gabarnaudia fimicola Samson & W. Gams) is produced (Cain & Weresub, 1957 ; Samson, 1974). Furthermore, like the closely related but non-coprophilous Table 1. Fungi associated with perithecia of Sphaeronaemella fimicola on dung samples taken from the field Associated fungi Rabbit (Humber Marsh, near Hereford) ; 2 Apr. 1996 Deer (Haugh Wood, Mordiford, near Hereford) ; 2 Apr. 1996 Sheep (Countess Wear, Exeter) ; 7 Nov. 1996
Rabbit (Dawlish Warren, Devon) ; 10 Nov. 1996
Melanospora sp., Thelebolus sp. Sporormiella sp., Trichobolus sp.
Pilobolus kleinii ; Ascobolus immersus, Coprobia granulata, Iodophanus carneus (Pers.) Korf, Lasiobolus ciliatus, Podospora sp. ; Coprinus ephemerus Mucor mucedo, Mucor sp., Pilobolus sp., Pilaira anomala, Piptocephalis sp. ; Podospora vesticola, Saccobolus depauperatus
Stimulation of Sphaeronaemella by other fungi
1056
Table 2. Screening of a range of coprophilous fungi for their capacity to stimulate growth and perithecium formation in S. fimicola (screens 1–3) and their susceptibility to mycoparasitic attack (screen 4) Growth of S. fimicolaa
Zygomycotina Basidiobolus ranarum Eidam Chaetocladium brefeldii Tiegh. & Le Monn. Mucor mucedo Bref. M. mucedo Section M. hiemalis Wehmer Section Pilaira anomala (Ces.) J. Schro$ t. Pilobolus crystallinus (Wigg.) Tode Ascomycotina Ascobolus albidus P. Crouan A. furfuraceus Pers. A. immersus Pers. A. perplexans Massee & E. S. Salmon Ascozonus crouanii (Sacc.) Boud. Chaetomium dolichotrichum Ames C. spirale Zopf C. subspirale Chivers Coniochaeta hansenii (Oudem.) Cain Coniochaeta sp. Coprobia granulata (Bull.) Boud. Delitschia furfuracea Niessl ex Rehm Delitschia sp. Gymnoascus reesii Baran. Lasiobolus ciliatus (J. C. Schmidt) Boud. Melanospora sp. A Melanospora sp. B Microascus trigonosporus C. W. Emmons & B. O. Dodge Orbilia fimicoloides J. Webster & Spooner Podospora curvicolla (Winter) Niessl P. curvula (de Bary ex Winter) Niessl P. decipiens (Winter ex Fuckel) Niessl P. excentrica N. Lundq. P. intestinacea N. Lundq. P. vesticola (Berk. & Broome) Cain & J. H. Mirza isol. A P. vesticola isol. B Podospora sp. Pseudeurotium sp. Ryparobius pachyascus Zukal ex Rehm isol. A R. pachyascus isol. B Saccobolus caesariatus Renny ex W. Phillips Sordaria fimicola (Rob. ex Desm.) Ces. & de Not. Sporormiella intermedia (Auersw.) Ahmed & Cain Sporormiella sp. Thelebolus nanus Heimerl isol. A T. nanus isol. B T. stercoreus Tode Trichobolus zukalii (Heimerl) Kimbr. Wawelia sp. Fungi Imperfecti Alternaria sp. Arthrobotrys sp. A Arthrobotrys sp. B. Aspergillus candidus Link ex Link A. repens (de Bary) E. Fisch. A. terreus Thom Aspergillus sp. Doratomyces nanus (Ehrenb.) F. J. Morton & G. Sm. D. stemonitis (Pers. : Fr.) F. J. Morton & G. Sm. isol. A D. stemonitis isol. B Geotrichum sp. Gliocladium roseum Bainier Series
Perithecium productionb
Dung source
Scr. 1
Scr. 2
Scr. 3
Scr. 1
Scr. 2
Scr. 3
Parasitismc Screen 4
Frog Rabbit Rabbit Rabbit Rabbit Rabbit Deer
® 0 ® ® ® ®
0 ® ® ® ®
® ® ® ® ® ®
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Deer Rabbit
® ® ® ® 0 ® 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ® 0
0 ® ® 0 0 0 0 0 0 0 ® 0 0
® ® ® ® ® ® ® ® 0 ® 0 0 0 ® ® 0 ® ® 0 0 0 ® ® 0 ® ® 0 0 ®
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 * * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 *
0 0 0 0 0 0 0 0 0 * 0 0 0 * 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Pony Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Deer Rabbit Rabbit Rabbit Rabbit
® ® ® 0 0 0 0
0 0 0 0 0
® ® ® 0 ® 0 ® ® ® ® ®
0 0 0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0 0 0 0 * 0
0 0 0 0 0 0 0 0 0 0 0
Sheep Deer Rabbit Mouse Rabbit Rabbit Deer Rabbit Sheep
Rabbit Pony Rabbit Rabbit Mouse Rabbit Sheep Rabbit Rabbit Rabbit Sheep Sheep Deer Rabbit Deer Rabbit Sheep Deer
R. W. S. Weber and J. Webster
1057
Table 2. (cont.) Growth of S. fimicolaa
Gliomastix murorum (Corda) S. Hughes Graphium putredinis (Corda) S. Hughes Isaria sp. Oedocephalum glomerulosum (Bull.) Sacc. Onychophora coprophila W. Gams, P. J. Fisher & J. Webster Penicillium claviforme Bainier P. funiculosum Thom Stachybotrys cylindrospora C. N. Jensen Stilbella erythrocephala (Ditmar) Lindau Trichothecium roseum (Pers.) Link ex Gray Verticillium tenerum Nees ex Link Volutella ciliata Alb. & Schwein. ex Fr. Basidiomycotina Coprinus cinereus Que! l. C. ephemerus (Bull. ex Fr.) Fr. C. filamentifer Ku$ hner C. heptemerus M. Lange & A. H. Sm. C. miser (Karst.) Karst. C. vermiculifer Joss. ex Dennis Cyathus stercoreus (Schwein.) De Toni Panaeolus semiovatus (Sow. ex Fr.) S. Lundell P. sphinctrinus (Fr.) Que! l. Psilocybe merdaria (Fr.) Ricken Sphaerobolus stellatus Tode : Pers. Sporobolomyces roseus Kluyver & C. B. Niel Stropharia semiglobata (Batsch ex Fr.) Que! l.
Perithecium productionb
Dung source
Scr. 1
Scr. 2
Scr. 3
Scr. 1
Scr. 2
Scr. 3
Parasitismc Screen 4
Rabbit Deer Rabbit Sheep Rabbit Deer Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit
0 0 0 0 ® 0 0
0 0 0 0
® ® ® ® ® ® ® ® ® ® ® ®
0 0 0 0 0 0 0 0 0 0 0
0 * 0 * 0 0 * 0 0 0
0 0 * 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
® 0 0 ® 0 0
0 0 0 0 0 0 0
® ® ® ® ® ® ® ® ® ® ® 0
0 0 0 0 0 0 0 0 0 0
0 * 0 0 0 0 0 * 0 0 0 * 0
0 0 0 * 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
Pony Sheep Sheep Sheep Deer Rabbit Cow Cow Sheep Pony Cow
Screens produced similar results between all three isolates of S. fimicola. In this table, the highest-scoring interactions are represented. Growth of S. fimicola against the test fungus. ®, Collapse of S. fimicola hyphae ahead of or upon contact with test species ; 0, intact hyphae of S. fimicola but no enhanced growth ahead of mycelial encounter, relative to S. fimicola control in the absence of test fungus ; , enhanced growth ahead of encounter, relative to control ; , overgrowth of test fungus by S. fimicola. b Production of perithecia by S. fimicola after 35 d incubation. 0, No perithecia produced ; *, only immature perithecia produced ; , less than 25 mature perithecia per colony of S. fimicola ; , more than 25 mature perithecia per colony. c Absence (0) or presence () of mycoparasitism by any of the three isolates of S. fimicola, as intrahyphal growth inside hyphae of test fungus. a
Sphaeronaemella helvellae Karst. (Vakili, 1985), S. fimicola can act as a necrotrophic mycoparasite (Hawksworth, 1981 ; Weber & Webster, 1997). In the present report, the range of coprophilous fungi capable of stimulating sexual reproduction in S. fimicola and the extent to which these are subjected to mycoparasitic attack have been examined. The results obtained are discussed and interpreted in the context of other interactions reported amongst coprophilous fungi.
dung collected at Humber Marsh (near Hereford, U.K.). Isolates were obtained from ascospores streaked onto V-8 juice agar which contained 20 % (w}v) V-8 juice (Campbell Grocery Products Ltd, King’s Lynn, Norfolk) according to Miller (1955). All fungi were maintained at 4 °C on slopes of potato dextrose agar, V-8 juice agar, or yeast extract–sucrose agar containing (l−") 4 g yeast extract, 20 g sucrose, 1 g KH PO , # % 0±5 g MgSO \7H O and 15 g Lab M2 agar. % #
MATERIALS AND METHODS
Screening experiments for perithecium production and susceptibility to mycoparasitism
Occurrence of Sphaeronaemella fimicola on dung Dung samples collected at various locations were incubated at room temperature (r.t.) in a moist chamber, and those containing S. fimicola were monitored for the presence of fruit bodies of associated fungi. Isolation and maintenance of coprophilous fungi Three isolates of S. fimicola used in this work were : A, from rabbit dung collected at Dawlish Warren (Devon, U.K.) ; B, from deer dung collected at Forstgut Wiegersen (near Buxtehude, Lower Saxony, Germany) ; and C, from rabbit
Conidia of S. fimicola readily germinated on a wide range of agar media and were, therefore, used as inoculum except in experiments where plugs of vegetative mycelium were required. Conidia were produced abundantly on yeast extract–sucrose agar plates within 2 wk of inoculation (20° in the dark). A total of 83 isolates representing 79 coprophilous species (Table 2) were examined for their capacity to induce perithecium production in all three S. fimicola isolates. In each plate, the test species was centrally inoculated with the three S. fimicola isolates evenly displaced at a distance of 3 cm from
Stimulation of Sphaeronaemella by other fungi the centre. In order to widen the range of growth conditions, three different screens were performed which varied in the pH of the medium and in the mode of inoculation. In screen 1, V8 agar was adjusted to pH 6 by addition of NaOH (Diener, 1955), and conidia of S. fimicola were inoculated 3 d before the host fungus was added. In screen 2, adjustment to pH 7±2 was by 10 m Tris}maleate buffer and the mode of inoculation was as for screen 1. In screen 3, adjustment to pH 7±2 was as for screen 2, but inoculation was by mycelial plugs (5 mm diam.) of S. fimicola onto established host mycelium which had been pre-grown for 1–3 wk depending on the growth rate of the species tested. Control plates contained the three S. fimicola isolates without a test fungus. In all screens, incubation was at 20° in the dark. Stimulation of growth relative to controls in the absence of host fungi (screens 1 and 2) and number of perithecia produced by each S. fimicola colony (all screens) were monitored. The three isolates of S. fimicola were screened for evidence of parasitism against all 83 fungal isolates. Five-fold diluted yeast extract–sucrose agar plates were inoculated simultaneously with conidia of S. fimicola and a host fungus, 3 cm apart as described above, and incubated in the dark at 20°. Mycoparasitism was established under the microscope by cutting samples of agar from the zone of interaction of colonies and mounting in cotton blue in lactic acid on a microscope slide. Detailed experiments with R. pachyascus and S. fimicola Dual-culture experiments involving S. fimicola (isol. C) and Ryparobius pachyascus Zukal ex Rehm (isol. A) were carried out in order to characterize further the nature of the interactions involved. A detailed growth study of S. fimicola with R. pachyascus was performed at 20° on V-8 agar adjusted to pH 6, in order to quantify its effect on growth and reproduction in S. fimicola. The intensity of sexual reproduction was expressed as the number of perithecia cm−# of colony, and asexual reproduction by haemocytometer counts of conidia cm−# of colony. The area of the colonies of S. fimicola was determined by tracing their outline on pre-weighed oven-dried paper and reweighing the cut-out tracing after drying. In order to determine whether direct hyphal contact was necessary for the induction of perithecium formation, S. fimicola was grown in dual-culture with R. pachyascus, with colonies being separated by a sterile sheet of Cellophane of 25 µm thickness. In another experiment, R. pachyascus was pre-grown on Cellophane placed over a V-8 agar plate for 7 d at 20°. The agar was then inoculated with S. fimicola after removal of the Cellophane with the mycelium of Ryparobius. In dual-culture experiments on fresh rabbit dung sterilized by autoclaving (121° for 15 min), batches of 15 sterile pellets in contact with each other were inoculated with a conidial suspension of S. fimicola (isol. C) at one end, and with or without a plug of mycelium of R. pachyascus (isol. A) at the other. Incubation was at 20° in the dark. Additionally, pellets with or without prior colonization by R. pachyascus (1 wk at 20° in the dark) were re-sterilized, inoculated with S. fimicola and incubated as above. The aim of these experiments was the
1058 determination of suitable conditions for perithecium production by S. fimicola on the natural substratum. RESULTS Fungi associated with Sphaeronaemella fimicola in the field Dung pellets containing perithecia of Sphaeronaemella fimicola were incubated in a moist chamber and associated fungi were identified. In every sample, members of the Ascomycotina were present, discomycetes being the most prominent group. In contrast, fructifications of Zygomycotina were only occasionally found, and those of Basidiomycotina were rare at the time of fruiting of S. fimicola (Table 1). Screens of associated fungi in vitro The capability of 83 coprophilous fungi to promote growth and perithecium formation in, and to be parasitized by, S. fimicola was similar against three different isolates of S. fimicola, but showed variation between the different incubation conditions of the three separate screens performed. The results are summarized in Table 2 which, for simplicity of presentation, shows only the strongest response by any of the three S. fimicola isolates for each screen. The majority of fungi tested stimulated both growth and, subsequently, sexual reproduction in S. fimicola (Fig. 1). Some isolates promoted one activity, e.g. stimulation only of growth by Pilobolus crystallinus, Coprinus filamentifer, C. vermiculifer, Psilocybe merdaria, Melanospora sp. B and Thelebolus stercoreus, while Volutella ciliata triggered perithecium production, but did not stimulate growth. Growth. Relatively few fungi (20 isolates tested) had no apparent growth-promoting effect on S. fimicola (Table 2, denoted by the symbol 0) or caused collapse of its hyphae at a distance or upon contact (®). The majority of test species from all major groups stimulated growth of S. fimicola at a distance () in at least one of the screens, and 14 species were overgrown by S. fimicola (). With the exception of P. crystallinus and S. roseus, all fungi in the latter category were Ascomycotina or Deuteromycotina with ascomycete affinity. This type of interaction was particularly readily detected in screen 3, in which inoculum of S. fimicola had been placed on established colonies of the test fungi. Of 14 isolates susceptible to overgrowth, six were also parasitized by S. fimicola by means of intrahyphal growth. Exceptions included P. crystallinus and the small yeast cells of S. roseus. The interaction with Pilobolus is noteworthy because it was the only observed case of mutual stimulation in the sense that both fungi displayed more vigorous growth in dual-culture than in axenic culture, and because S. fimicola also induced asexual reproduction in P. crystallinus (Fig. 2). On the other hand, in 10 cases the ability of S. fimicola to parasitize hyphae of test fungi was not accompanied by large-scale overgrowth. Mycoparasitic potential was, therefore, not a prerequisite for overgrowth. Perithecium production. A wide range of Asco-, Basidio- and Deuteromycotina, but not the strongly growth-promoting
1059
Colony area (cm2)
R. W. S. Weber and J. Webster
3 10
5
0
1
Number of conidia × 106cm–2
0
7
14
21
28
35
7
14
21
28
35
14
21
28
35
4
3 2 1 0 0
Number of perithecia cm–2
80
5
60 40 20 0
2 Figs 1, 2. Interactions of Sphaeronaemella fimicola (isolate C ; left) with other fungi growing from the right. Scale bars 1 cm. Fig. 1. Dualculture with Ryparobius pachyascus (isol. A) on V-8 agar (pH 6). Vegetative growth of S. fimicola was stimulated, and perithecia were formed after establishment of contact between the two mycelia. Fig. 2. Dual-culture with Pilobolus crystallinus on V-8 agar (pH 7±2). Vegetative growth of both fungi was stimulated, but S. fimicola did not produce perithecia. Instead, P. crystallinus produced sporangiophores within the colony of S. fimicola, but not on its own.
zygomycete P. crystallinus, stimulated perithecium formation in S. fimicola. Among the strongest stimulators were Coniochaeta spp., Ryparobius pachyascus, Thelebolus nanus and Trichobolus zukalii, which were also susceptible to overgrowth or mycoparasitism by S. fimicola. In some cases perithecium initials and even mature perithecia were formed by S. fimicola prior to any mycelial contact. This occurred at 19 mm in dualculture with Microascus trigonosporus in screen 1, and 8 mm with Aspergillus candidus in screen 2. Interactions between R. pachyascus and S. fimicola in vitro The interaction between R. pachyascus (isol. A), the strongest overall inducer of growth and perithecium production, and S. fimicola (isol. C) was further studied on V-8 agar adjusted to pH 6 by NaOH. A simulation of growth (Fig. 3) and perithecium production (Fig. 5) relative to the pure-culture control was observed and asexual reproduction by phialo-
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Figs 3–5. Stimulation of Sphaeronaemella fimicola (isolate C) by dualculture with Ryparobius pachyascus (isolate A) (E) relative to S. fimicola alone (D) on V-8 agar (pH 6). Data are expressed as xa ³.. of four replicates. Fig. 3. Vegetative growth as colony area. Fig. 4. Asexual reproduction represented as number of phialoconidia cm−# of mycelium. Fig. 5. Sexual reproduction represented as number of perithecia cm−# of mycelium.
conidia was, likewise, enhanced by the presence of R. pachyascus (Fig. 4). When mycelia of R. pachyascus and S. fimicola were separated by a Cellophane sheet, induction of perithecium formation still occurred. Removal of R. pachyascus with the Cellophane sheet permitted subsequent formation of perithecial necks and ascospores by the underlying mycelium of S. fimicola. This occurred only in perithecia which had produced at least a rudimentary neck extension at the time of removal, whereas immature (globose) perithecia failed to develop further (Fig. 6). In another experiment, R. pachyascus was grown on Cellophane-covered agar for 7 d and then removed along with the Cellophane. S. fimicola, inoculated onto such pretreated medium, displayed a greatly enhanced growth rate as compared to untreated control plates (Fig. 7), but no development of perithecia or perithecium initials was observed. Conidia, harvested from pre-treated or control plates and inoculated onto fresh V-8 juice agar, gave rise to mycelia growing at a very similar rate (Fig. 8), which was the same as that on the initial untreated control plates (Fig. 7), suggesting that accumulation of the growth-promoting
Stimulation of Sphaeronaemella by other fungi
1060 substance(s) in conidia and utilization upon germination did not occur to any detectable degree. Inoculation of S. fimicola into sterilized rabbit dung When sterilized rabbit pellets were inoculated simultaneously with S. fimicola (isol. C) and R. pachyascus (isol. A), abundant perithecium production ensued, whereas in the absence of R. pachyascus, only the Gabarnaudia anamorph was produced. Inoculation of S. fimicola onto pellets treated by pre-growth of R. pachyascus and subsequent sterilization resulted in vegetative growth of the fungus and abundant production of conidia without perithecium formation. A destruction of the stimulant for perithecium production by autoclaving is unlikely because it was found to remain active in autoclaved cell-free extracts of R. pachyascus (R. W. S. Weber, unpublished results). Hence, our results indicate that induction of perithecium production in S. fimicola required the continuous presence of a suitable stimulatory fungus such as R. pachyascus, on dung as well as in vitro. DISCUSSION
6 Fig. 6. Perithecia of Sphaeronaemella fimicola (isol. C) after 1 wk simultaneous growth with Ryparobius pachyascus (isol. A) separated by Cellophane, followed by removal of the Cellophane and incubation for a further 3 d. Growth of an aerial neck occurred only in the perithecium which had developed rudiments of a neck extension (arrowhead) at the time of Cellophane removal. Immature (globose) perithecia (arrows) failed to develop further. Scale bar, 100 µm.
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Figs 7, 8. The effect of pre-growth of R. pachyascus (isol. A) on vegetative growth (colony radius) of S. fimicola (isol. C). Data are expressed as xa ³.. of four replicates. Fig. 7. Growth of S. fimicola on V-8 agar (pH 6) plates treated by pre-growth on Cellophane and removal of R. pachyascus (E) relative to an untreated control (D). Fig. 8. Growth on fresh V-8 agar plates of inoculum harvested from the treatment (E) and control (D) mycelia of the experiment in Fig. 7.
The capability of other fungi to stimulate perithecium formation in S. fimicola, initially observed by Cain & Weresub (1957) with an A. repens contaminant, has been found to be of widespread occurrence among coprophilous fungi (this study and J. Follett, unpublished). This is also likely to occur on dung in the field. Many strong stimulators, e.g. Saccobolus, Sporormiella, Thelebolus and Trichobolus, are associated with perithecia of S. fimicola on dung samples collected from the field. Furthermore, S. fimicola inoculated onto sterilized fresh rabbit pellets produced perithecia only in the presence of a suitable stimulator such as R. pachyascus, but not on its own. The interactions involved are probably complex because many fungi stimulated not only perithecium production but also vegetative growth of S. fimicola, often at some distance prior to hyphal contact. The simplest possibility is that both processes were stimulated by the same compound, but at different concentrations. If so, this substance would be involved in at least two distinct developmental processes, i.e. growth promotion and induction of perithecia followed by their maturation. The alternative is that these two processes are stimulated by two or more different substances which may explain why some fungi stimulated only either growth or perithecium production in S. fimicola. Furthermore, a limited quantity of the stimulatory substance(s) was sufficient for growth promotion, such as that released by pre-growth and removal of R. pachyascus, whereas a continuous supply of metabolite(s) appeared to be required for perithecium production up to the stage of formation of the neck initial. Multiple growth factor requirements are common especially among biotrophic mycoparasites (Barnett, 1970 ; Calderone & Barnett, 1972 ; Barnett & Binder, 1973). Interestingly, such a continuous supply of stimulant could be maintained across a Cellophane membrane separating S. fimicola from a suitable test species such as R. pachyascus. Direct hyphal contact was thus not required for perithecium production in S. fimicola, yet this fungus has been described as
R. W. S. Weber and J. Webster a necrotrophic mycoparasite of R. pachyascus (Weber & Webster, 1997) and other coprophilous fungi (present report). Weber & Webster (1997), however, pointed out that R. pachyascus could be re-isolated even from areas heavily infected by S. fimicola. Mycoparasitism, which has been reported previously among coprophilous fungi in a biotrophic form for zygomycetes such as Piptocephalis or Chaetocladium parasitizing other zygomycetes (Berry & Barnett, 1957 ; Jeffries & Young, 1994), may thus be more widespread than currently appreciated. The best-documented cases of mycoparasitism as a means of obtaining nutrients involve the contact biotrophs, specialized fungi which parasitize a relatively narrow host range (usually Ascomycotina). They obligately depend on an unidentified vitamin (mycotrophein) and other factors for growth and reproduction (Barnett & Lilly, 1958 ; Calderone & Barnett, 1972 ; Jordan & Barnett, 1978). For several reasons, mycotrophein is unlikely to be involved in the coprophilous fungus interactions reported here ; firstly, growth of S. fimicola was merely stimulated by, but not dependent upon, the presence of a host fungus. Secondly, the range of fungi stimulating growth of S. fimicola was wider than that reported for contact biotrophs (Gain & Barnett, 1970 ; Barnett & Binder, 1973) and included even Basidio- and Zygomycotina. Finally, stimulation of growth and, under certain conditions, perithecium formation occurred at a distance without hyphal contact. Further research is currently being directed towards identifying the compound(s) of interest. It is known that other ascomycetes such as Chaetomium globosum (Buston & Khan, 1956) and Melanospora destruens (Hawker, 1948) require exogenous organic phosphomonoesters as a stimulus for perithecium formation. Such exchanges of nutrients may be common and mutual (e.g. Barnett, 1968), as in the case of S. fimicola and P. crystallinus reported here. It is likely that the provision of growth factors is also important among coprophilous fungi, along with other concerted activities such as the breakdown of complex carbohydrates (Wood & Cooke, 1987). Hence, a diversity of fungi on dung may be required for the efficient growth of each species. We are indebted to Mr P. M. Booth, a B.M.S. Associate, for his support of this work, and we thank Dr D. Pitt for the provision of laboratory facilities. REFERENCES Barnett, H. L. (1968). The effects of light, pyridoxine and biotin on the development of the mycoparasite, Gonatobotryum fuscum. Mycologia 60, 244–251. Barnett, H. L. (1970). Nutritional requirements for axenic growth of some haustorial mycoparasites. Mycologia 62, 750–760. Barnett, H. L. & Binder, F. L. (1973). The fungal host–parasite relationship. Annual Review of Phytopathology 11, 273–292. Barnett, H. L. & Lilly, V. G. (1958). Parasitism of Calcarisporium parasiticum on species of Physalospora and related fungi. West Virginia Agricultural Experiment Station Bulletin 420T. (Accepted 31 October 1997 )
1061 Berry, C. R. & Barnett, H. L. (1957). Mode of parasitism and host range of Piptocephalis virginiana. Mycologia 49, 374–386. Buston, H. W. & Khan, A. H. (1956). The influence of certain micro-organisms on the formation of perithecia by Chaetomium globosum. Journal of General Microbiology 14, 655–660. Cain, R. F. & Weresub, L. K. (1957). Studies of coprophilous ascomycetes. V. Sphaeronaemella fimicola. Canadian Journal of Botany 35, 119–131. Calderone, R. A. & Barnett, H. L. (1972). Axenic growth and nutrition of Gonatobotryum fuscum. Mycologia 64, 153–160. Diener, U. L. (1955). Sporulation in pure culture by Stemphylium solani. Phytopathology 45, 141–145. Dix, N. J. & Webster, J. (1995). Fungal Ecology. Chapman & Hall : London. Gain, R. E. & Barnett, H. L. (1970). Parasitism and axenic growth of the mycoparasite Gonatorhodiella highlei. Mycologia 62, 1122–1129. Gloer, J. B. (1995). The chemistry of fungal antagonism and defense. Canadian Journal of Botany 73, S1265–S1274. Harper, J. E. & Webster, J. (1964). An experimental analysis of the coprophilous fungus succession. Transactions of the British Mycological Society 47, 511–530. Hawker, L. E. (1948). Stimulation of the formation of perithecia of Melanospora destruens Shear by small quantities of certain phosphoric esters of glucose and fructose. Annals of Botany (New Series) 12, 77–79. Hawker, L. E. (1951). Morphological and physiological studies on Sordaria destruens (Shear) comb. nov. (syn. Melanospora destruens), Sordaria fimicola and Melanospora zamiae. Transactions of the British Mycological Society 34, 174–186. Hawksworth, D. L. (1981). A survey of the fungicolous conidial fungi. In Biology of Conidial Fungi, Vol. I (ed. G. T. Cole & B. Kendrick), pp. 171–244. Academic Press : New York. Hesseltine, C. W., Whitehill, A. R. & Pidacks, C. (1953). Coprogen, a new growth factor present in dung required by Pilobolus. Mycologia 45, 7–19. Ikediugwu, F. E. O. & Webster, J. (1970 a). Antagonism between Coprinus heptemerus and other coprophilous fungi. Transactions of the British Mycological Society 54, 181–204. Ikediugwu, F. E. O. & Webster, J. (1970 b). Hyphal interference in a range of coprophilous fungi. Transactions of the British Mycological Society 54, 205–210. Jeffries, P. & Young, T. W. K. (1994). Interfungal Parasitic Relationships. CAB International : Wallingford, Oxon. Jordan, E. G. & Barnett, H. L. (1978). Nutrition and parasitism of Melanospora zamiae. Mycologia 70, 300–312. Keller-Schierlein, W. & Diekmann, H. (1970). Stoffwechselprodukte von Mikroorganismen. 85. Zur Konstitution des Coprogens. Helvetica Chimica Acta 53, 2035–2044. Miller, P. M. (1955). V-8 juice as a general purpose medium for fungi and bacteria. Phytopathology 45, 461–462. Page, R. M. (1959). Stimulation of asexual reproduction of Pilobolus by Mucor plumbeus. American Journal of Botany 46, 579–585. Page, R. M. (1960). The effect of ammonia on growth and reproduction of Pilobolus kleinii. Mycologia 52, 480–489. Samson, R. A. (1974). Paecilomyces and some allied hyphomycetes. Studies in Mycology 6, 1–119. Singh, N. & Webster, J (1973). Antagonism between Stilbella erythrocephala and other coprophilous fungi. Transactions of the British Mycological Society 61, 487–495. Vakili, N. G. (1985). Mycoparasitic fungi associated with potential stalk rot pathogens of corn. Phytopathology 75, 1201–1207. Weber, R. W. S. & Webster, J. (1997). The coprophilous fungus Sphaeronaemella fimicola – a facultative mycoparasite. Mycologist 11, 50–51. Wicklow, D. T. & Hirschfield, B. J. (1979). Evidence of a competitive hierarchy among coprophilous populations. Canadian Journal of Microbiology 25, 855–858. Wood, S. N. & Cooke, R. C. (1987). Nutritional competence of Pilaira anomala in relation to exploitation of faecal resource units. Transactions of the British Mycological Society 88, 247–255.