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Alternative repetitional conidia in Conidiobolus adiaeretus: development and germination
Mycol. Res. 104 (10) : 1270–1275 (October 2000). Printed in the United Kingdom.
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Alternative repetitional conidia in Conidiobolus adiaeretus : development and germination
Arthur A. CALLAGHAN1*, Steven D. WATERS2 and Robert J. MANNING1 " Biology Division, School of Sciences, Staffordshire University, Stoke-on-Trent, ST4 2DE, UK. # Science Department, Stoke-on-Trent College, Shelton, Stoke-on-Trent, ST4 2DG, UK. E-mail : a.a.callaghan!staffs.ac.uk Received 28 May 1999 ; accepted 21 December 1999.
We report the ability of Conidiobolus adiaeretus to form repetitional microconidia and capilliconidia. Detailed descriptions of development and germination are given. This is the first report of any entomophthoralean fungus producing both these alternative conidial types. This cuts across current definitions of subgenera in Conidiobolus which are based on the absence of these conidial types or on the presence of either microconidia or capilliconidia but not both. Co-occurrence of both these spore types, along with the unusual mode of formation of the microconidia, and the ability to derive capilliconidia directly from the latter, reinforce other features which set C. adiaeretus apart. Taxonomic, evolutionary and ecological implications are briefly discussed.
INTRODUCTION In Conidiobolus, as in most entomophthoralean fungi, primary (1y) conidia can directly develop secondary (2y) conidia and these in turn can yield tertiary (3y) spores and so on, potentially, to a fifth generation. These ‘ repetitional ’ conidia can be closely similar to the actively discharged globose parent spore (‘ replicative ’ conidia) or be of different type (‘ alternative ’ conidia). Their development can replace vegetative germination if nutrients are not available. Depending on the Conidiobolus species, alternative conidia can include microconidia (multiple tiny conidia developed and discharged by one parent conidium), capilliconidia (elongate, passively detached and borne singly on a slender stalk), and, for three species, elongate discharged conidia (King 1977, Waters & Callaghan 1989). Currently no Conidiobolus species has been reported to develop both microconidia and capilliconidia and delineation of three subgenera is based on the presence of one or the other of these forms, or the absence of both types (Humber 1989). Exploration of the effect of conditions on the formation of alternative conidia (Waters & Callaghan 1999) has emphasised how an observer could fail to see the latter if conditions, especially reduced water activity (aw), are nonoptimal. Similarly, over-emphasis on 1y conidia could decrease the chance of seeing alternative types because 2y and 3y globose conidia often form them more readily. It was during the screening of strains of Conidiobolus species and Basidiobolus species in relation to water activity that Conidiobolus
adiaeretus was found to form both microconidia and capilliconidia. This paper describes the unusual development of both of these alternative types of conidia and gives data for their germination on agar substrates of contrasting nutritional status. Taxonomic, evolutionary and ecological implications are briefly discussed.
MATERIALS AND METHODS Strains and media The strain of C. adiaeretus used was 04 : 041 (Staffordshire University collection), isolated from larch litter. Limited tests, not reported in detail, on two other strains from the same habitat, 04 : 020 (IMI 313575) and 04 : 201, and on an ex-type strain (CBS 136.57) also disclosed the developmental patterns reported. (1) Sources of 1y conidia were grown on malt extract agar, MEA (Oxoid, 2 % w\v malt extract L39, 1n2 % w\v agar no. 3) at 20p1 mC. Dishes were inverted over fluorescent lights (irradiance c. 1n5 Wm−#) to direct conidiophores downwards and thus minimise retention of 1y conidia and stray discharge of 2y conidia. (2) Secondary conidia were obtained by incubating inverted dishes containing deposits of 1y conidia on RWA (Oxoid agar no. 1, 1 % w\v ; de-ionised water). The 2y globose conidia then developed and discharged.
A. A. Callaghan, S. D. Waters and R. J. Manning (3) To enhance induction of microconidia, parent 2y conidia were showered (step 2) onto RWA with aw reduced by addition of sorbitol (Harris 1981, Waters & Callaghan 1999). The sorbitol agars (SAs) had an aw l 0n998 (sorbitol 1n8 % w\v) or aw l 0n995 (sorbitol 5n5 % w\v). It was on these non-nutrient SA substrates that microconidium formation was mainly monitored. Thus, to obtain deposits of any particular generation of repetitional conidia, a dish source of the previous generation was inverted over the chosen catch substrate. Preliminary trials established suitable durations of spore discharge and of incubation to optimise numbers of spores and views of particular stages.
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Repeated microscopic observations were made at intervals of minutes or hours according to the speed of development. Between observations the low-heat lamp used was switched off ; general room lighting in the constant temperature room stayed on. Recording was by photography or by freehand drawing and associated measurements. When repeated drawings of several spores in a deposit were required, individual spores were re-located using an England Finder (Philip Harris). Numbers of nuclei per conidium were determined for all conidium types using epi-fluorescence microscopy of spores were re-located using an England Finder (Philip Harris). Numbers of nuclei per conidium were determined for all conidium types using epi-fluorescence microscopy of spores stained with bisbenzimide (Kangatharalingham & Ferguson 1984).
(1) Within 1 h of discharge onto the catch substrate (RWA or SA), those spores not forming replicative globose conidia developed (4) 5–12 (18) outgrowths in one or more polar whorls (Fig. 6, 10A data). (2) Each growth swelled to 9–10 µm diameter and a crowded (‘ stage-1 ’) cluster was completed c. 2 h from discharge (Figs 2, 5A and 6). (3) Within the next 2–3 h, most or all of the stage-1 spheres developed a bud which swelled to, again, 9–10 µm. These outer ‘ stage-2 ’ cluster units are the microconidia (Figs 4, 5C, 7, 11). (4) Microconidia were discharged to 1 mm from the parent cluster. Often all microconidia were discharged and left the initial whorl (stage-1 cluster) as a completely spent, flat, empty rosette of launching sites ; a ‘ cluster residue ’ (Fig. 7). When viewed from above it usually obscured the original parent spore wall (Figs 7, 8B, 12). The development described occurred on 70–80 % of 2y globose conidia on SA of aw 0n998. The frequency was much less on RWA or on SA aw 0n995. However the developmental patterns were consistent irrespective of conditioning substance used (sucrose, glycerol, sorbitol) or water activity value (down to 0n995). The number of stage-1 outgrowths (and subsequent number of microconidia at stage-2) was greater for larger parent spores. Regression of N [number of microconidia per spore] on [diam. of parent conidium] is significant, r# l 0n53, P 0n01, n l 41. The frequency distributions of total numbers of spherical outgrowths per spore determined at times corresponding to stages-1 and -2 reflect a general and consistent doubling process (Fig. 10).
Germination of microconidia and capilliconidia
Development of capilliconidia
Substrates for monitoring germination included RWA and SAs as above. Additionally, in some experiments, several concentrations of malt extract were incorporated into RWA at 0–0n25 % w\v and in other experiments glucose or N-acetyl glucosamine were separately incorporated at similar concentrations. Microconidia readily showered onto test substrates but capilliconidia usually remained on their conidiophores on the SA. To observe their germination, discs were cut from the SA canopy and gently touched, spore-side down on to test substrates. Many capilliconidia adhered to the latter and could be scored for mode of germination.
Quite often, one or more of the stage-1 spherical outgrowths failed to develop a microconidium, or, some of the latter were not ejected. From such units capilliconidia could develop (Figs 5D–E, 8A–B, 12). This happened on up to 60 % of cluster residues on SA aw 0n998. Capilliconidia also formed readily from discharged microconidia (Figs 8C) and from other capilliconidia (Fig. 9B, C). From the originating spherical unit, whether on a cluster residue or a discharged microconidium, a slender conidiophore, basal diam. c. 3 µm, tapering to 1–2 µm, grew away from the substratum to a length of c. 45–55 (68) µm. Typically there is a kink (angle 40–50 m) some 5–8 µm from the tip, then, terminally, an asymmetrically attached (heterotropic) capilliconidium differentiated (Figs 5E, 8). These were 16–24 (often 20) µm long and c. 5–6 µm diam, elongate, and gradually increasing in diameter from the proximal end to greatest width in the distal half ; sometimes there was an abrupt rim present (Fig. 8E). Distally, the spore tapers to a blunt or rounded end. No evidence was obtained for the presence of a hapteron or adhesive layer on the surface (Fig. 12). Nevertheless, after passive release, they readily attached to, for example, a fine needle brought near to them and became oriented normal to the new surface. Both microconidia and capilliconidia were found to contain 5–6 nuclei. Numbers in 1y and 2y (and perhaps some 3y)
Observation and recording
RESULTS Development of microconidia Formation of microconidia by C. adiaeretus is unusual and involves a two-stage process. Two generations of multiple spherical units form on the parent globose conidium. Only the second set are microconidia which are discharged. The overall process is illustrated by Figs 1–4. These show 3 different spores on RWA (Fig. 4 is a later stage of Fig. 3). From numerous other observations, including repeated ones on individual spores, the typical development was seen as follows :
Conidiobolus adiaeretus repetitional conidia
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Figs 1–4. Conidiobolus adiaeretus. Two-stage development of microconidia from globose conidia on RWA. Fig. 1. Globose 2y conidium with papilla. Fig. 2. A stage-1 cluster of (8) buds on a parent conidium. Fig. 3. A stage-2 cluster with variable rates of development of microconidia. Fig. 4. Later stage of Fig. 3. Symmetrical formation of 6 nearly full-size microconidia. Bar l 20 µm.
A
1h
B
C
D
E
2h 3h 4h 40m
6h 30m
Fig. 5. Conidiobolus adiaeretus. Repeated observation on a 2y globose conidium on SA (aw 0n998). Times are approximate periods from arrival of the parent spore on agar surface during showering (see text). A–B, start of stage-2 cluster with one (arrowed) microconidium initiated and 2 extra central buds ; C, 6 radial microconidia developing (these were fully grown 30 m later) ; D, residue of cluster after discharge of 3 microconidia and 5 still present. A slender conidiophore is growing away from the residue ; E, a feebly discharged microconidium has fallen close by (it replicated c. 2 h later). A terminal capilliconidium has now formed. Bar l 20 µm.
6
7
Figs. 6–7. Conidiobolus adiaeretus. Range of examples of development stages in deposits of 2y globose conidia on SA (aw 0n998). Bar l 20 µm. Fig. 6. Early and late stage-1 cluster formation ; showing minimum (4) and maximum (18) outgrowths per spore. Fig. 7. Stage-2 clusters and cluster residues ; contrasting sizes.
globose conidia, taken together, varied widely, increasing with spore size, from 12 to 130 nuclei per spore. Germination In the absence of added nutrients (e.g. on RWA) both
microconidia and capilliconidia can replicate their own spore type (Figs 8A, 9) or sometimes sprout germ tubes which abort (Table 1). On a relatively rich medium, such as 1 % MEA, germination of all conidial forms is by germ tube. With low nutrient concentrations both repetitional conidium formation and vegetative germination can occur ; however, above a
A. A. Callaghan, S. D. Waters and R. J. Manning
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9 A B
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D B
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Frequency
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E
22 20 18 16 14 12 10 8 6 4 2 0
A Incubation 2h
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8 10 12 14 16 18 20 22 24
No. outgrowths per parent conidium
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22 20 18 16 14 12 10 8 6 4 2 0
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No. outgrowths per parent conidium
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Figs 8–12. Capilliconidia originating from undischarged spherical units on residues and from an individual discharged microconidium : A, profile view showing flat cluster residue and erect capilliconidiophores ; B, cluster residue showing post-discharge holes at conidiogenic loci ; C, capilliconidium from discharged microconidium ; D, capilliconidium from tiny two-locus cluster ; E, Common alternative shape showing prominent rim (slightly larger scale), adjacent bar l 20 µm. Fig. 9. Conidiobolus adiaeretus. Repetitional germination of microconidia and capilliconidia on RWA. A, 4y capilliconidium forming a spherical 5y conidium ; B, sequence of two capilliconidia from an initial 4y microconidium ; C, profile view of capilliconidium replicating ; D, replicative microconidia and two of the smallest products (diam. 3 µm). Bar l 20 µm. Fig. 10. Total number of spherical outgrowths per parent 2y globose conidium of Conidiobolus adiaeretus at times corresponding approximately to stage-1 (A) and stage-2 (B) of microconidium cluster formation. Spore numbers 103 (A) and 194 (B). Fig. 11. Conidiobolus adiaeretus (04 : 041). SEM of a stage-1 cluster forming microconidia. During conidium showering and incubation of a collembolan cadaver, the parent conidium, now an empty husk (H), was caught on the animal’s bristles and held just above its surface. Cluster formation then occurred. (JEOL JSM 840A microscope ; gold-coated). Fig. 12. Conidiobolus adiaeretus (04 : 041). SEM of a cluster residue on a polycarbonate membrane filter with 5 loci (arrowed) from which microconidia have been discharged. Three of the detached microconidia (MC) are adjacent. A microconidium retained on the residue has given rise to a capilliconidium (CC) borne terminally on a slender conidiophore (processing as for Fig. 11).
particular nutrient level ( 0n05 % for microconidia and 0n05 % for capilliconidia), there is a marked emphasis on the latter. Table 1 shows that all conidium types increased the proportion of vegetative germination relative to repetitional spore formation as nutrition levels increased ; but, for
microconidia, the % vegetative germination was consistently and markedly higher than that for any generation of globose conidia, and for any given nutrient level. In four other repeat experiments, not given here in detail, levels of vegetative germination for 1y globose conidia on 0n05 % MEA ranged
Conidiobolus adiaeretus repetitional conidia
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Table 1. Vegetative germination of C. adiaeretus conidial types on RWA incorporating low levels of malt extract (MEA). Proportion (mean %p1 ; n l 3 replicates) of conidia forming normal branching germ tubes instead of repetitional conidia. Sample size 200–300 spores per replicate, except for 3y\4y globose conidia (30–50 spores per replicate). Very few ( 0n1 %) spores failed to germinate ; they are excluded. Conidia on RWA and capilliconidia on all test substrates were monitored alive ; all other spores were killed by formaldehyde vapour for scoring. Substrate % w\v MEA
Microconidia (diam. 9–10 µm)
0 (RWA) 0n05 0n1 0n15 0n25
(0) 19n4p0n6 83n4p2n6 82n1p1n7 91n4p0n8
1y globose conidia 1 0n5 10n3p3 15n1p3n9 32n7p2
2y globose conidia
3y\4y globose conidiaa
Capilliconidia
0 0n5p0n03 1n9p0n8 6n6p1n5 23n1p1n2
(0) 3n2p0n8 3n0p0n1 9n4p1n3 ND
(0) 90b nr 100b ND ND
Five separate experiments with capilliconidia. were made ; in each, a deposit of 100 spores was scanned. (0) l zero viable vegetative germination ; some germ tubes and germlings grew then aborted. ND, not determined. a Difficult to determine hierarchical status of conidia in mixed deposits of 3y\4y conidia : globose conidia, diam. 11 µm and replicating microconidia, 8 µm present. b Germlings eventually sporulated.
from 6–18 % while for microconidia, the range was 44–99 %. Germination of capilliconidia in relation to increasing nutrient level showed a pattern similar to that in microconidia. Thus, typically, there is a preponderance of replicative germination (next-generation capilliconidia) on RWA and a marked swing to vegetative germination ( 90 %) on 0n05 % and 0n1 % MEA. In the absence of nutrients, 5y capilliconidia formed germ tubes which, eventually, aborted (Table 1). In limited tests using only glucose or N-acetylglucosamine in RWA, no evidence of germ tube induction was obtained. DISCUSSION In Drechsler’s original (1953) description of C. adiaeretus and in the summary redescription by King (1977), there is no mention of any conidial forms as alternatives to the replicative globose conidia which closely resemble the primary conidia. The present work establishes the ability of this fungus to develop both microconidia and capilliconidia. The mode of development of microconidia of C. adiaeretus contrasts with that in the 10 other species of Conidiobolus reported as forming microconidia (King 1977). In these, as described in detail by Prasertphon (1963) for C. coronatus, the initial outgrowths from the parent spore do not swell and collectively form a first stage cluster. Instead, each parent spore directly develops numerous conidiogenous loci, at each of which a microconidium forms at its tip. Discharge leaves an emptied parent spore with the positions of the conidiogenous loci showing as tiny raised outgrowths of the crumpled parent spore wall. Prasertphon also describes fluid droplets exuded from the residual sterigmata after microspore discharge. No droplets were seen by us. In C. adiaeretus, discharge of microconidia is probably by their own turgor causing them to bounce off the initially firm base ; a mechanism in line with that for globose conidia in the genus (Ingold 1971). The presence of a papilla on each microconidium suggests this is the mechanism. However, there may be involvement of the basal units ; sometimes after discharge tiny holes can be seen in the latter (Fig. 8B). In our experiments most microconidia of C. adiaeretus were of diameter 9–10 µm. This is within the ranges cited by King (1977) for all other Conidiobolus species forming
such spores. For C. adiaeretus, Drechsler (1953) figures the smallest repetitional globose conidia with diameter 13 µm, which is larger than any microconidia observed by us. Benjamin (1962) describes a very different development of the non-discharged microconidia in Basidiobolus microsporus. In this species, the parent globose conidium cleaves into multiple uninucleate segments. Each sterigma originates from one segment of the parent spore. In C. adiaeretus no such internal division was detected. Capilliconidia are reported from only four other Conidiobolus species (King 1977), although they have been observed (Callaghan, unpubl.) from C. bangalorensis (ARSEF 449, extype culture ; Srinivasan & Thirumalachar 1967), which, incidently, also has long, wide conidiophores. Re-examination of other species may disclose hitherto unnoticed accessory conidia. The capilliconidia of C. adiaeretus somewhat resemble those of Zoophthora and Basidiobolus and, as in these genera and in Neozygites, insertion on the conidiophore is heterotropic (Humber 1981, 1984). This contrasts with the orthotropic orientation in other Conidiobolus species. Absence of a hapteron perhaps suggests that most similarity is with capilliconidia of Zoophthora. The formation of capilliconidia from microconidia seems unique. The taxonomic revision of Entomophthorales (by Humber (1989, 1997) recognized three subgenera in Conidiobolus : Delacroixia for species forming microconidia ; Capillidium, for species forming capilliconidia ; Conidiobolus for species producing neither of these types of accessory spore. Clearly, the position of C. adiaeretus is problematic. Several features of C. adiaeretus have been considered unusual by previous authors (Drechsler 1953, King 1977, Ingold 1992). These include : the very slender ramifying hyphae with few septa ; the hugely inflated conidiophores which seem to form more by local growth with rather little associated mass migration of the contents of basal hyphae ; the formation of a primary conidium without associated emptying of the conidiophore ; and the marked induction of chlamydospores by low temperature. The mode of development and features of the accessory conidia described here, further set this species apart. From a phylogenetic aspect, one view of Conidiobolus species is that they exhibit features close to those of saprotrophic ancestors
A. A. Callaghan, S. D. Waters and R. J. Manning of the Entomophthorales (Humber 1984, Evans 1988) and Basidiobolus, although having many similar features by convergence, perhaps had a more distant and different origin (Cavalier-Smith 1987, Nagahama et al. 1995). If Conidiobolus is indeed a paraphyletic genus (Jensen et al. 1998), C. adiaeretus could be regarded as one of the species which has retained a wide range of (ancestral) features some of which have been lost by other species as they evolved. A different view would be that its current ‘ eco-nutritional versatility ’ (sensu Cooke & Rayner 1984) as a (presumptive) saprotroph represents a quite specialised, more recently evolved, suite of adaptive features rather than reflecting a primitive nutritional mode. C. adiaeretus is quite different from other Conidiobolus species and the new findings here do not necessarily prompt the immediate removal of the subgenus groupings, although a more precise specification of the microconidium mode of development (sensu Prasertphon 1963) and a stipulation that the capilliconidia must be orthotropic would leave each of the two groups consisting of very similar fungi. Further revision of the genus should perhaps wait until a more thorough search has been made for alternative conidial types and until a data base exists for inferring the molecular phylogeny of the genus. The microconidia of C. adiaeretus are implicated in its occurrence in larch litter (Callaghan, unpubl.). Of total colonyforming-units (cfu) from homogenised litter sieved through successively smaller mesh sizes, some 50 % cfu (in summer) or 8 % (in winter) were from the fraction passed through mesh size 15 µm. The colonies probably originated from microconidia or late generation replicative globose conidia. Current work focuses on the adaptive significance of accessory conidia especially in terms of avoidance of competition, specialisation to target invertebrates and thus niche differentiation. Conceivably, for C. adiaeretus the combination of microconidia being locally scattered and the ability of each to generate a capilliconidium able to contact a mobile arthropod (or other invertebrate, e.g. nematodes), would be particularly effective where target animals (such as collembola) were aggregating. Also, the ability of microconidia and capilliconidia to germinate at relatively low nutrient levels, perhaps coupled with the response to lowered aw, could be advantageous on arthropod integuments (Waters & Callaghan 1999). These alternative conidia may be infective for particular invertebrate live host(s), or, be the initiating units for opportunistic colonisation of exuvia or eventual cadaver. The fungus has been isolated from a collembolan cadaver and has been shown experimentally to colonise collembola and mite cadavers (Waters & Manning, unpubl.) but we have no proof of pathogenicity or of the actual targets for saprotrophy (or necrotrophy) in its habitat.
1275 Ithaca, NY) for a culture of C. bangalorensis. We also thank two anonymous referees for very useful comments.
REFERENCES
A C K N O W L E D G E M E N TS
Benjamin, R. K. (1962) A new Basidiobolus that forms microspores. Aliso 5 : 223–233. Cavalier-Smith, T. (1987) The origin of fungi and pseudofungi. In Evolutionary Biology of the Fungi (A. D. M. Rayner, C. M. Brazier & D. Moore, eds) : 1–15. Cambridge University Press, Cambridge, UK. Cooke, R. C. & Rayner, A. D. M. (1984) Ecology of Saprotrophic Fungi. Longman, London. Drechsler, C. (1953) Three new species of Conidiobolus. Journal of the Washington Academy of Sciences 43 : 29–43. Evans, H. C. (1988) Coevolution of entomogenous fungi and their hosts. In Coevolution of Fungi with Plants and Animals (K. A. Pirozinski & D. L. Hawksworth, eds) : 150–171. Academic Press, London. Harris, R. F. (1981) The effect of water potential on microbial growth and activity. In Water Potential Relations in Soil Micro-organisms (J. F. Parr, W. R. Gardner & L. F. Elliot, eds) : 23–84. [Special Publication No. 9.] Soil Society of America, Madison WS. Humber, R. A. (1981) An alternative view of certain taxonomic criteria used in the Entomophthorales (Zygomycetes). Mycotaxon 13 : 191–240. Humber, R. A. (1984) Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In Fungus\Insect Relationships : perspectives in ecology and evolution (Q. Wheeler & M. Blackwell, eds) : 166–183. Columbia University Press, New York. Humber, R. A. (1989) Synopsis of a revised classification for the Entomophthorales (Zygomycotina). Mycotaxon 34 : 441–460. Humber, R. A. (1997) Fungi-Identification. In Manual of Techniques in Insect Pathology (L. A. Lacey, ed.) : 153–185. Academic Press, London. Ingold, C. T. (1971) Fungal Spores and their Dispersal. Clarendon Press, Oxford. Ingold, C. T. (1992) Conidiobolus as a genus to explore. The Mycologist 6 : 82–83. Jensen, A. B., Gargas, A., Eilenberg, J. & Rosendahl, S. (1998) Relationships of the insect-pathogenic order Entomophthorales (Zygomycotina, Fungi) based on phylogenetic analysis of nuclear small sub-unit ribosomal DNA sequences (SSU rDNA). Fungal Genetics and Biology 24 : 325–334. Kangatharalingham, N. & Ferguson, M. W. (1984) A simple and rapid technique for fluorescent staining of fungal nuclei. Current Mycology 10 : 99–104. King, D. S. (1977) Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy III. Descriptions of recognised species. Canadian Journal of Botany 55 : 718–729. Nagahama, T., Sato, H., Shimazu, J. & Sigiyama, J. (1995) Molecular phylogeny of entomophthoralean fungi – evidence from 18S ribosomalRNA gene-sequences. Mycologia 87 : 203–209. Prasertphon, S. (1963) Conidial formation in Entomophthora coronata. Journal of Insect Pathology 5 : 318–335. Srinivasan, M. C. & Thirumalachar, M. J. (1967) Evaluation of taxonomic characters in the genus Conidiobolus with a key to known species. Mycologia 59 : 698–713. Waters, S. D. & Callaghan, A. A. (1989) Conidiobolus iuxtagenitus, a new species with discharged elongate repetitional conidia and conjugation tubes. Mycological Research 93 : 223–226. Waters, S. D. & Callaghan, A. A. (1999) Conidium germination in cooccurring Conidiobolus and Basidiobolus in relation to their ecology. Mycological Research 103 : 1259–1268.
We thank G. W. Barlow for photographic processing, J. J. Ranford for digitising and adding inserts to Figs 11–12, and R. A. Humber (ARSEF ;
Corresponding Editor : R. S. Currah
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Alternative repetitional conidia in Conidiobolus adiaeretus : development and germination
Arthur A. CALLAGHAN1*, Steven D. WATERS2 and Robert J. MANNING1 " Biology Division, School of Sciences, Staffordshire University, Stoke-on-Trent, ST4 2DE, UK. # Science Department, Stoke-on-Trent College, Shelton, Stoke-on-Trent, ST4 2DG, UK. E-mail : a.a.callaghan!staffs.ac.uk Received 28 May 1999 ; accepted 21 December 1999.
We report the ability of Conidiobolus adiaeretus to form repetitional microconidia and capilliconidia. Detailed descriptions of development and germination are given. This is the first report of any entomophthoralean fungus producing both these alternative conidial types. This cuts across current definitions of subgenera in Conidiobolus which are based on the absence of these conidial types or on the presence of either microconidia or capilliconidia but not both. Co-occurrence of both these spore types, along with the unusual mode of formation of the microconidia, and the ability to derive capilliconidia directly from the latter, reinforce other features which set C. adiaeretus apart. Taxonomic, evolutionary and ecological implications are briefly discussed.
INTRODUCTION In Conidiobolus, as in most entomophthoralean fungi, primary (1y) conidia can directly develop secondary (2y) conidia and these in turn can yield tertiary (3y) spores and so on, potentially, to a fifth generation. These ‘ repetitional ’ conidia can be closely similar to the actively discharged globose parent spore (‘ replicative ’ conidia) or be of different type (‘ alternative ’ conidia). Their development can replace vegetative germination if nutrients are not available. Depending on the Conidiobolus species, alternative conidia can include microconidia (multiple tiny conidia developed and discharged by one parent conidium), capilliconidia (elongate, passively detached and borne singly on a slender stalk), and, for three species, elongate discharged conidia (King 1977, Waters & Callaghan 1989). Currently no Conidiobolus species has been reported to develop both microconidia and capilliconidia and delineation of three subgenera is based on the presence of one or the other of these forms, or the absence of both types (Humber 1989). Exploration of the effect of conditions on the formation of alternative conidia (Waters & Callaghan 1999) has emphasised how an observer could fail to see the latter if conditions, especially reduced water activity (aw), are nonoptimal. Similarly, over-emphasis on 1y conidia could decrease the chance of seeing alternative types because 2y and 3y globose conidia often form them more readily. It was during the screening of strains of Conidiobolus species and Basidiobolus species in relation to water activity that Conidiobolus
adiaeretus was found to form both microconidia and capilliconidia. This paper describes the unusual development of both of these alternative types of conidia and gives data for their germination on agar substrates of contrasting nutritional status. Taxonomic, evolutionary and ecological implications are briefly discussed.
MATERIALS AND METHODS Strains and media The strain of C. adiaeretus used was 04 : 041 (Staffordshire University collection), isolated from larch litter. Limited tests, not reported in detail, on two other strains from the same habitat, 04 : 020 (IMI 313575) and 04 : 201, and on an ex-type strain (CBS 136.57) also disclosed the developmental patterns reported. (1) Sources of 1y conidia were grown on malt extract agar, MEA (Oxoid, 2 % w\v malt extract L39, 1n2 % w\v agar no. 3) at 20p1 mC. Dishes were inverted over fluorescent lights (irradiance c. 1n5 Wm−#) to direct conidiophores downwards and thus minimise retention of 1y conidia and stray discharge of 2y conidia. (2) Secondary conidia were obtained by incubating inverted dishes containing deposits of 1y conidia on RWA (Oxoid agar no. 1, 1 % w\v ; de-ionised water). The 2y globose conidia then developed and discharged.
A. A. Callaghan, S. D. Waters and R. J. Manning (3) To enhance induction of microconidia, parent 2y conidia were showered (step 2) onto RWA with aw reduced by addition of sorbitol (Harris 1981, Waters & Callaghan 1999). The sorbitol agars (SAs) had an aw l 0n998 (sorbitol 1n8 % w\v) or aw l 0n995 (sorbitol 5n5 % w\v). It was on these non-nutrient SA substrates that microconidium formation was mainly monitored. Thus, to obtain deposits of any particular generation of repetitional conidia, a dish source of the previous generation was inverted over the chosen catch substrate. Preliminary trials established suitable durations of spore discharge and of incubation to optimise numbers of spores and views of particular stages.
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Repeated microscopic observations were made at intervals of minutes or hours according to the speed of development. Between observations the low-heat lamp used was switched off ; general room lighting in the constant temperature room stayed on. Recording was by photography or by freehand drawing and associated measurements. When repeated drawings of several spores in a deposit were required, individual spores were re-located using an England Finder (Philip Harris). Numbers of nuclei per conidium were determined for all conidium types using epi-fluorescence microscopy of spores were re-located using an England Finder (Philip Harris). Numbers of nuclei per conidium were determined for all conidium types using epi-fluorescence microscopy of spores stained with bisbenzimide (Kangatharalingham & Ferguson 1984).
(1) Within 1 h of discharge onto the catch substrate (RWA or SA), those spores not forming replicative globose conidia developed (4) 5–12 (18) outgrowths in one or more polar whorls (Fig. 6, 10A data). (2) Each growth swelled to 9–10 µm diameter and a crowded (‘ stage-1 ’) cluster was completed c. 2 h from discharge (Figs 2, 5A and 6). (3) Within the next 2–3 h, most or all of the stage-1 spheres developed a bud which swelled to, again, 9–10 µm. These outer ‘ stage-2 ’ cluster units are the microconidia (Figs 4, 5C, 7, 11). (4) Microconidia were discharged to 1 mm from the parent cluster. Often all microconidia were discharged and left the initial whorl (stage-1 cluster) as a completely spent, flat, empty rosette of launching sites ; a ‘ cluster residue ’ (Fig. 7). When viewed from above it usually obscured the original parent spore wall (Figs 7, 8B, 12). The development described occurred on 70–80 % of 2y globose conidia on SA of aw 0n998. The frequency was much less on RWA or on SA aw 0n995. However the developmental patterns were consistent irrespective of conditioning substance used (sucrose, glycerol, sorbitol) or water activity value (down to 0n995). The number of stage-1 outgrowths (and subsequent number of microconidia at stage-2) was greater for larger parent spores. Regression of N [number of microconidia per spore] on [diam. of parent conidium] is significant, r# l 0n53, P 0n01, n l 41. The frequency distributions of total numbers of spherical outgrowths per spore determined at times corresponding to stages-1 and -2 reflect a general and consistent doubling process (Fig. 10).
Germination of microconidia and capilliconidia
Development of capilliconidia
Substrates for monitoring germination included RWA and SAs as above. Additionally, in some experiments, several concentrations of malt extract were incorporated into RWA at 0–0n25 % w\v and in other experiments glucose or N-acetyl glucosamine were separately incorporated at similar concentrations. Microconidia readily showered onto test substrates but capilliconidia usually remained on their conidiophores on the SA. To observe their germination, discs were cut from the SA canopy and gently touched, spore-side down on to test substrates. Many capilliconidia adhered to the latter and could be scored for mode of germination.
Quite often, one or more of the stage-1 spherical outgrowths failed to develop a microconidium, or, some of the latter were not ejected. From such units capilliconidia could develop (Figs 5D–E, 8A–B, 12). This happened on up to 60 % of cluster residues on SA aw 0n998. Capilliconidia also formed readily from discharged microconidia (Figs 8C) and from other capilliconidia (Fig. 9B, C). From the originating spherical unit, whether on a cluster residue or a discharged microconidium, a slender conidiophore, basal diam. c. 3 µm, tapering to 1–2 µm, grew away from the substratum to a length of c. 45–55 (68) µm. Typically there is a kink (angle 40–50 m) some 5–8 µm from the tip, then, terminally, an asymmetrically attached (heterotropic) capilliconidium differentiated (Figs 5E, 8). These were 16–24 (often 20) µm long and c. 5–6 µm diam, elongate, and gradually increasing in diameter from the proximal end to greatest width in the distal half ; sometimes there was an abrupt rim present (Fig. 8E). Distally, the spore tapers to a blunt or rounded end. No evidence was obtained for the presence of a hapteron or adhesive layer on the surface (Fig. 12). Nevertheless, after passive release, they readily attached to, for example, a fine needle brought near to them and became oriented normal to the new surface. Both microconidia and capilliconidia were found to contain 5–6 nuclei. Numbers in 1y and 2y (and perhaps some 3y)
Observation and recording
RESULTS Development of microconidia Formation of microconidia by C. adiaeretus is unusual and involves a two-stage process. Two generations of multiple spherical units form on the parent globose conidium. Only the second set are microconidia which are discharged. The overall process is illustrated by Figs 1–4. These show 3 different spores on RWA (Fig. 4 is a later stage of Fig. 3). From numerous other observations, including repeated ones on individual spores, the typical development was seen as follows :
Conidiobolus adiaeretus repetitional conidia
1
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2
3
4
Figs 1–4. Conidiobolus adiaeretus. Two-stage development of microconidia from globose conidia on RWA. Fig. 1. Globose 2y conidium with papilla. Fig. 2. A stage-1 cluster of (8) buds on a parent conidium. Fig. 3. A stage-2 cluster with variable rates of development of microconidia. Fig. 4. Later stage of Fig. 3. Symmetrical formation of 6 nearly full-size microconidia. Bar l 20 µm.
A
1h
B
C
D
E
2h 3h 4h 40m
6h 30m
Fig. 5. Conidiobolus adiaeretus. Repeated observation on a 2y globose conidium on SA (aw 0n998). Times are approximate periods from arrival of the parent spore on agar surface during showering (see text). A–B, start of stage-2 cluster with one (arrowed) microconidium initiated and 2 extra central buds ; C, 6 radial microconidia developing (these were fully grown 30 m later) ; D, residue of cluster after discharge of 3 microconidia and 5 still present. A slender conidiophore is growing away from the residue ; E, a feebly discharged microconidium has fallen close by (it replicated c. 2 h later). A terminal capilliconidium has now formed. Bar l 20 µm.
6
7
Figs. 6–7. Conidiobolus adiaeretus. Range of examples of development stages in deposits of 2y globose conidia on SA (aw 0n998). Bar l 20 µm. Fig. 6. Early and late stage-1 cluster formation ; showing minimum (4) and maximum (18) outgrowths per spore. Fig. 7. Stage-2 clusters and cluster residues ; contrasting sizes.
globose conidia, taken together, varied widely, increasing with spore size, from 12 to 130 nuclei per spore. Germination In the absence of added nutrients (e.g. on RWA) both
microconidia and capilliconidia can replicate their own spore type (Figs 8A, 9) or sometimes sprout germ tubes which abort (Table 1). On a relatively rich medium, such as 1 % MEA, germination of all conidial forms is by germ tube. With low nutrient concentrations both repetitional conidium formation and vegetative germination can occur ; however, above a
A. A. Callaghan, S. D. Waters and R. J. Manning
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8
9 A B
C C
D B
D
10
Frequency
A
E
22 20 18 16 14 12 10 8 6 4 2 0
A Incubation 2h
0
2
4
6
8 10 12 14 16 18 20 22 24
No. outgrowths per parent conidium
11
22 20 18 16 14 12 10 8 6 4 2 0
B Incubation 6–7h
0
2
4
6
8 10 12 14 16 18 20 22 24
No. outgrowths per parent conidium
12
Figs 8–12. Capilliconidia originating from undischarged spherical units on residues and from an individual discharged microconidium : A, profile view showing flat cluster residue and erect capilliconidiophores ; B, cluster residue showing post-discharge holes at conidiogenic loci ; C, capilliconidium from discharged microconidium ; D, capilliconidium from tiny two-locus cluster ; E, Common alternative shape showing prominent rim (slightly larger scale), adjacent bar l 20 µm. Fig. 9. Conidiobolus adiaeretus. Repetitional germination of microconidia and capilliconidia on RWA. A, 4y capilliconidium forming a spherical 5y conidium ; B, sequence of two capilliconidia from an initial 4y microconidium ; C, profile view of capilliconidium replicating ; D, replicative microconidia and two of the smallest products (diam. 3 µm). Bar l 20 µm. Fig. 10. Total number of spherical outgrowths per parent 2y globose conidium of Conidiobolus adiaeretus at times corresponding approximately to stage-1 (A) and stage-2 (B) of microconidium cluster formation. Spore numbers 103 (A) and 194 (B). Fig. 11. Conidiobolus adiaeretus (04 : 041). SEM of a stage-1 cluster forming microconidia. During conidium showering and incubation of a collembolan cadaver, the parent conidium, now an empty husk (H), was caught on the animal’s bristles and held just above its surface. Cluster formation then occurred. (JEOL JSM 840A microscope ; gold-coated). Fig. 12. Conidiobolus adiaeretus (04 : 041). SEM of a cluster residue on a polycarbonate membrane filter with 5 loci (arrowed) from which microconidia have been discharged. Three of the detached microconidia (MC) are adjacent. A microconidium retained on the residue has given rise to a capilliconidium (CC) borne terminally on a slender conidiophore (processing as for Fig. 11).
particular nutrient level ( 0n05 % for microconidia and 0n05 % for capilliconidia), there is a marked emphasis on the latter. Table 1 shows that all conidium types increased the proportion of vegetative germination relative to repetitional spore formation as nutrition levels increased ; but, for
microconidia, the % vegetative germination was consistently and markedly higher than that for any generation of globose conidia, and for any given nutrient level. In four other repeat experiments, not given here in detail, levels of vegetative germination for 1y globose conidia on 0n05 % MEA ranged
Conidiobolus adiaeretus repetitional conidia
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Table 1. Vegetative germination of C. adiaeretus conidial types on RWA incorporating low levels of malt extract (MEA). Proportion (mean %p1 ; n l 3 replicates) of conidia forming normal branching germ tubes instead of repetitional conidia. Sample size 200–300 spores per replicate, except for 3y\4y globose conidia (30–50 spores per replicate). Very few ( 0n1 %) spores failed to germinate ; they are excluded. Conidia on RWA and capilliconidia on all test substrates were monitored alive ; all other spores were killed by formaldehyde vapour for scoring. Substrate % w\v MEA
Microconidia (diam. 9–10 µm)
0 (RWA) 0n05 0n1 0n15 0n25
(0) 19n4p0n6 83n4p2n6 82n1p1n7 91n4p0n8
1y globose conidia 1 0n5 10n3p3 15n1p3n9 32n7p2
2y globose conidia
3y\4y globose conidiaa
Capilliconidia
0 0n5p0n03 1n9p0n8 6n6p1n5 23n1p1n2
(0) 3n2p0n8 3n0p0n1 9n4p1n3 ND
(0) 90b nr 100b ND ND
Five separate experiments with capilliconidia. were made ; in each, a deposit of 100 spores was scanned. (0) l zero viable vegetative germination ; some germ tubes and germlings grew then aborted. ND, not determined. a Difficult to determine hierarchical status of conidia in mixed deposits of 3y\4y conidia : globose conidia, diam. 11 µm and replicating microconidia, 8 µm present. b Germlings eventually sporulated.
from 6–18 % while for microconidia, the range was 44–99 %. Germination of capilliconidia in relation to increasing nutrient level showed a pattern similar to that in microconidia. Thus, typically, there is a preponderance of replicative germination (next-generation capilliconidia) on RWA and a marked swing to vegetative germination ( 90 %) on 0n05 % and 0n1 % MEA. In the absence of nutrients, 5y capilliconidia formed germ tubes which, eventually, aborted (Table 1). In limited tests using only glucose or N-acetylglucosamine in RWA, no evidence of germ tube induction was obtained. DISCUSSION In Drechsler’s original (1953) description of C. adiaeretus and in the summary redescription by King (1977), there is no mention of any conidial forms as alternatives to the replicative globose conidia which closely resemble the primary conidia. The present work establishes the ability of this fungus to develop both microconidia and capilliconidia. The mode of development of microconidia of C. adiaeretus contrasts with that in the 10 other species of Conidiobolus reported as forming microconidia (King 1977). In these, as described in detail by Prasertphon (1963) for C. coronatus, the initial outgrowths from the parent spore do not swell and collectively form a first stage cluster. Instead, each parent spore directly develops numerous conidiogenous loci, at each of which a microconidium forms at its tip. Discharge leaves an emptied parent spore with the positions of the conidiogenous loci showing as tiny raised outgrowths of the crumpled parent spore wall. Prasertphon also describes fluid droplets exuded from the residual sterigmata after microspore discharge. No droplets were seen by us. In C. adiaeretus, discharge of microconidia is probably by their own turgor causing them to bounce off the initially firm base ; a mechanism in line with that for globose conidia in the genus (Ingold 1971). The presence of a papilla on each microconidium suggests this is the mechanism. However, there may be involvement of the basal units ; sometimes after discharge tiny holes can be seen in the latter (Fig. 8B). In our experiments most microconidia of C. adiaeretus were of diameter 9–10 µm. This is within the ranges cited by King (1977) for all other Conidiobolus species forming
such spores. For C. adiaeretus, Drechsler (1953) figures the smallest repetitional globose conidia with diameter 13 µm, which is larger than any microconidia observed by us. Benjamin (1962) describes a very different development of the non-discharged microconidia in Basidiobolus microsporus. In this species, the parent globose conidium cleaves into multiple uninucleate segments. Each sterigma originates from one segment of the parent spore. In C. adiaeretus no such internal division was detected. Capilliconidia are reported from only four other Conidiobolus species (King 1977), although they have been observed (Callaghan, unpubl.) from C. bangalorensis (ARSEF 449, extype culture ; Srinivasan & Thirumalachar 1967), which, incidently, also has long, wide conidiophores. Re-examination of other species may disclose hitherto unnoticed accessory conidia. The capilliconidia of C. adiaeretus somewhat resemble those of Zoophthora and Basidiobolus and, as in these genera and in Neozygites, insertion on the conidiophore is heterotropic (Humber 1981, 1984). This contrasts with the orthotropic orientation in other Conidiobolus species. Absence of a hapteron perhaps suggests that most similarity is with capilliconidia of Zoophthora. The formation of capilliconidia from microconidia seems unique. The taxonomic revision of Entomophthorales (by Humber (1989, 1997) recognized three subgenera in Conidiobolus : Delacroixia for species forming microconidia ; Capillidium, for species forming capilliconidia ; Conidiobolus for species producing neither of these types of accessory spore. Clearly, the position of C. adiaeretus is problematic. Several features of C. adiaeretus have been considered unusual by previous authors (Drechsler 1953, King 1977, Ingold 1992). These include : the very slender ramifying hyphae with few septa ; the hugely inflated conidiophores which seem to form more by local growth with rather little associated mass migration of the contents of basal hyphae ; the formation of a primary conidium without associated emptying of the conidiophore ; and the marked induction of chlamydospores by low temperature. The mode of development and features of the accessory conidia described here, further set this species apart. From a phylogenetic aspect, one view of Conidiobolus species is that they exhibit features close to those of saprotrophic ancestors
A. A. Callaghan, S. D. Waters and R. J. Manning of the Entomophthorales (Humber 1984, Evans 1988) and Basidiobolus, although having many similar features by convergence, perhaps had a more distant and different origin (Cavalier-Smith 1987, Nagahama et al. 1995). If Conidiobolus is indeed a paraphyletic genus (Jensen et al. 1998), C. adiaeretus could be regarded as one of the species which has retained a wide range of (ancestral) features some of which have been lost by other species as they evolved. A different view would be that its current ‘ eco-nutritional versatility ’ (sensu Cooke & Rayner 1984) as a (presumptive) saprotroph represents a quite specialised, more recently evolved, suite of adaptive features rather than reflecting a primitive nutritional mode. C. adiaeretus is quite different from other Conidiobolus species and the new findings here do not necessarily prompt the immediate removal of the subgenus groupings, although a more precise specification of the microconidium mode of development (sensu Prasertphon 1963) and a stipulation that the capilliconidia must be orthotropic would leave each of the two groups consisting of very similar fungi. Further revision of the genus should perhaps wait until a more thorough search has been made for alternative conidial types and until a data base exists for inferring the molecular phylogeny of the genus. The microconidia of C. adiaeretus are implicated in its occurrence in larch litter (Callaghan, unpubl.). Of total colonyforming-units (cfu) from homogenised litter sieved through successively smaller mesh sizes, some 50 % cfu (in summer) or 8 % (in winter) were from the fraction passed through mesh size 15 µm. The colonies probably originated from microconidia or late generation replicative globose conidia. Current work focuses on the adaptive significance of accessory conidia especially in terms of avoidance of competition, specialisation to target invertebrates and thus niche differentiation. Conceivably, for C. adiaeretus the combination of microconidia being locally scattered and the ability of each to generate a capilliconidium able to contact a mobile arthropod (or other invertebrate, e.g. nematodes), would be particularly effective where target animals (such as collembola) were aggregating. Also, the ability of microconidia and capilliconidia to germinate at relatively low nutrient levels, perhaps coupled with the response to lowered aw, could be advantageous on arthropod integuments (Waters & Callaghan 1999). These alternative conidia may be infective for particular invertebrate live host(s), or, be the initiating units for opportunistic colonisation of exuvia or eventual cadaver. The fungus has been isolated from a collembolan cadaver and has been shown experimentally to colonise collembola and mite cadavers (Waters & Manning, unpubl.) but we have no proof of pathogenicity or of the actual targets for saprotrophy (or necrotrophy) in its habitat.
1275 Ithaca, NY) for a culture of C. bangalorensis. We also thank two anonymous referees for very useful comments.
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Benjamin, R. K. (1962) A new Basidiobolus that forms microspores. Aliso 5 : 223–233. Cavalier-Smith, T. (1987) The origin of fungi and pseudofungi. In Evolutionary Biology of the Fungi (A. D. M. Rayner, C. M. Brazier & D. Moore, eds) : 1–15. Cambridge University Press, Cambridge, UK. Cooke, R. C. & Rayner, A. D. M. (1984) Ecology of Saprotrophic Fungi. Longman, London. Drechsler, C. (1953) Three new species of Conidiobolus. Journal of the Washington Academy of Sciences 43 : 29–43. Evans, H. C. (1988) Coevolution of entomogenous fungi and their hosts. In Coevolution of Fungi with Plants and Animals (K. A. Pirozinski & D. L. Hawksworth, eds) : 150–171. Academic Press, London. Harris, R. F. (1981) The effect of water potential on microbial growth and activity. In Water Potential Relations in Soil Micro-organisms (J. F. Parr, W. R. Gardner & L. F. Elliot, eds) : 23–84. [Special Publication No. 9.] Soil Society of America, Madison WS. Humber, R. A. (1981) An alternative view of certain taxonomic criteria used in the Entomophthorales (Zygomycetes). Mycotaxon 13 : 191–240. Humber, R. A. (1984) Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In Fungus\Insect Relationships : perspectives in ecology and evolution (Q. Wheeler & M. Blackwell, eds) : 166–183. Columbia University Press, New York. Humber, R. A. (1989) Synopsis of a revised classification for the Entomophthorales (Zygomycotina). Mycotaxon 34 : 441–460. Humber, R. A. (1997) Fungi-Identification. In Manual of Techniques in Insect Pathology (L. A. Lacey, ed.) : 153–185. Academic Press, London. Ingold, C. T. (1971) Fungal Spores and their Dispersal. Clarendon Press, Oxford. Ingold, C. T. (1992) Conidiobolus as a genus to explore. The Mycologist 6 : 82–83. Jensen, A. B., Gargas, A., Eilenberg, J. & Rosendahl, S. (1998) Relationships of the insect-pathogenic order Entomophthorales (Zygomycotina, Fungi) based on phylogenetic analysis of nuclear small sub-unit ribosomal DNA sequences (SSU rDNA). Fungal Genetics and Biology 24 : 325–334. Kangatharalingham, N. & Ferguson, M. W. (1984) A simple and rapid technique for fluorescent staining of fungal nuclei. Current Mycology 10 : 99–104. King, D. S. (1977) Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy III. Descriptions of recognised species. Canadian Journal of Botany 55 : 718–729. Nagahama, T., Sato, H., Shimazu, J. & Sigiyama, J. (1995) Molecular phylogeny of entomophthoralean fungi – evidence from 18S ribosomalRNA gene-sequences. Mycologia 87 : 203–209. Prasertphon, S. (1963) Conidial formation in Entomophthora coronata. Journal of Insect Pathology 5 : 318–335. Srinivasan, M. C. & Thirumalachar, M. J. (1967) Evaluation of taxonomic characters in the genus Conidiobolus with a key to known species. Mycologia 59 : 698–713. Waters, S. D. & Callaghan, A. A. (1989) Conidiobolus iuxtagenitus, a new species with discharged elongate repetitional conidia and conjugation tubes. Mycological Research 93 : 223–226. Waters, S. D. & Callaghan, A. A. (1999) Conidium germination in cooccurring Conidiobolus and Basidiobolus in relation to their ecology. Mycological Research 103 : 1259–1268.
We thank G. W. Barlow for photographic processing, J. J. Ranford for digitising and adding inserts to Figs 11–12, and R. A. Humber (ARSEF ;
Corresponding Editor : R. S. Currah