Conidium germination in co-occurring Conidiobolus and Basidiobolus in relation to their ecology

Mycol. Res. 103 (10) : 1259–1269 (1999)

1259

Printed in the United Kingdom

Conidium germination in co-occurring Conidiobolus and Basidiobolus in relation to their ecology

S. D. W A T E RS1 A N D A. A. C A L L A G H A N2* " Science Department, Stoke-on-Trent College, Stoke Road, Shelton, Stoke-on-Trent ST4 2DG, U.K. # School of Sciences, Biology Division, Staffordshire University, Stoke-on-Trent ST4 2DE, U.K.

Germination of primary and secondary conidia of 33 strains (12 spp.) of co-occurring Conidiobolus and Basidiobolus from three closely adjacent managed habitats was studied in relation to general nutrient level, water activity of substratrum, light and temperature. A further 26 strains (16 spp.) derived from culture collections, and including insect pathogens and saprotrophs, were also compared. Nutrient levels triggering germ-tube formation differed widely amongst strains ; capilliconidial strains and\or presumptive pathogens usually had a low threshold. Successive generations of globose replicative conidia formed alternative conidial forms in increasing proportions. Water activity (aw) strong influenced development of non-globose conidial forms (microconidia, capilliconidia and actively discharged elongate conidia). Profiles of response to nutrients, aw, temperature and light differed between strains (and species) and the comparison represents a first stage in determining niche differentiation, including the putative interactions of these common fungi with litter invertebrates.

For most fungi of the Entomophthorales there exist developmental alternatives to vegetative germination of primary conidia (King & Humber, 1981). Instead of germ tubes, secondary conidia may form. The repetitional conidial type may be similar to or distinct from the parent conidium and, when the secondary (or higher order) conidia germinate, the possibility of alternative development remains until the energy reserves of the conidia are inadequate (Prasertphon, 1963). For proven arthropod pathogens, a majority of the order, there have been many studies of the influence of environmental factors such as temperature, relative humidity (water activity), pH and light on conidium germination (Newman & Carner, 1975 ; van Roermund, Perry & Tyrrell, 1984 ; Magalha4 es et al., 1991 ; Morgan et al., 1995 ; Oduor et al., 1996 a). Other studies have shown that it is often the secondary conidia which are infective for potential insect targets (Brobyn & Wilding, 1977 ; Glare, Chilvers & Milner, 1985 ; Bellini, Mullens & Jesperson, 1992 ; Oduor et al., 1996 b). Specific induction (and inhibition) of vegetative germination on external surfaces of live arthropods has been linked with particular lipids, hydrocarbons or other integument substances (Latge! et al., 1987 ; St Leger, 1991 ; Hajek & St Leger, 1994). Other factors affecting pre-penetration events for spores on insect cuticles, including appressorium formation, have also been studied (Magalha4 es et al., 1991 ; Nadeau, Dunphy & Boisvert, 1996). Conidiobolus (sensu King, 1976 ; Humber, 1989) and Basidiobolus include one true known insect pathogen (C. obscurus), and opportunistic pathogens such as C. coronatus, C. thromboides and C. osmodes (Papierok, 1986). Some aspects of conidium germination of these species have

been studied (Kevorkian, 1937 ; Prasertphon, 1963 ; Yendol, 1968 ; Callaghan, 1974, 1978), with most detailed attention being given to C. obscurus by Latge! and his co-workers (e.g. Sampedro, Uziel & Latge! , 1984). Clearly, conidium germination is of crucial relevance to the success of the insect pathogens. Most Conidiobolus and Basidiobolus live in soils and litter, however, and are not known to be pathogens. Very little is known about their nutrient substrates, dispersal, duration and manner of persistence, or about their interaction with other soil organisms. As a group they show a diversity of repetitional conidia which includes forms not reported for most other entomophthoralean genera (e.g. microconidia) as well as those in common with other genera (e.g. capilliconidia and actively discharged elongate conidia). Considering the possible roles of the various conidial types there is a strong presumption, but little direct evidence, that they are adapted to enhance the contact with animals in the soil and litter. Capilliconidia can adhere to the bodies of passing animals (Drechsler, 1956 ; Glare et al., 1985) and the active discharge of globose or elongate conidia and of microconidia could well be effective mechanisms for spreading these fungi, whether they are saprotrophs or pathogens, from one animal-associated substrate to others. Substrates themselves could be exuviae, faeces and cadavers or, indeed, live animals. These presumptive saprotrophs could have fairly broad substrate requirements or be quite target-specific. They could be necrotrophs or, less likely given the ease with which they grow on agar media, biotrophic pathogens of mites, collembola or other invertebrates. Groups of co-occurring Conidiobolus and Basidiobolus species, perhaps characteristic of particular litter

Germination in Conidiobolus and Basidiobolus or soil habitats (Drechsler, in King, 1976 ; Smith & Callaghan, 1987 ; I. J. Hopkins, unpublished), can be considered as guilds exploiting invertebrate substrates. To aid understanding of their ability to co-exist (i.e. their niche differentiation) we compare, for conidium germination, the requirements and tolerances of a range of strains isolated from three contrasting habitats (see materials and methods), and from a wider range of representative strains. First, the general level of nutrients which switches germination from formation of repetitional conidia, on non-nutrient substrates, to vegetative germination, was determined for 59 strains (ca 20 species). Then, for subsets of those species isolated most frequently we estimated changing proportions of alternative conidial forms which develop from the germination of primary, secondary and tertiary globose conidia (‘ hierarchical pattern ’) and the influence of water activity, temperature and light on the diversion to alternative morphogenesis.

MATERIALS AND METHODS Fungus strains and spore sources Thirty-three isolates (12 spp.) from the top 3 cm of litter and soil in three managed habitats (Table 1) were used. These habitats, at Keele, Staffordshire, U.K. comprise a larch plantation (map ref. SJ 821441), an arable field (SJ 822441) and a hayfield (SJ 820447). They have been monitored for several years by Smith (Smith & Callaghan, 1987) and, later in much more detail with improved techniques (I. J. Hopkins, unpublished). Another 26 strains (16 spp.) were from collections (Table 2). Of these, 14 were originally isolated from insects and represent proven or presumptive pathogens. These totals include 11 strains used in an earlier study (Callaghan, 1978). Isolates were routinely stored in liquid nitrogen and maintained by infrequent subculturing on 2 % malt extract agar, MEA (Oxoid Ltd, agar no. 2, malt extract L39). All primary conidia showered onto test substrates were discharged, from mycelium 2–3 d old from cultures on MEA, either by inverting the source dish or by canopying cut discs from the source, over the substrate. Source dishes were, in earlier experiments, Petri

1260 dishes (diam. 5 cm) or, later, multiple-well dishes (Sterilin Ltd) with groups of wells assigned to particular species. Sources were grown inverted over fluorescent lighting which gave an irradiance ca 1n5 W m−# ; at 20p1 mC. This procedure reduced the occurrence of primary conidia adhering to sources and thus minimized the contamination of primary conidium showers by secondary spores. Duration of showering was 30 min to 1 h annd usually gave a density of deposit of ca 40–80 conidia mm−#. When a shower of secondary globose conidia was required, an initial heavy deposit of primary conidia on refined water agar, RWA (Oxoid agar no. 1, 1 % w\v, de-ionized water) was canopied over a particular test substrate by inversion of the RWA container. This was done in the same standard conditions as source growth and primary showering, but exact timing differed according to species and required monitoring for each experiment. After an incubation of conidial deposits in the particular conditions of each experiment (see below) conidia were killed by vapour from drops of 15 % methanal solution (formaldehyde) placed in lids of the inverted dishes. Deposits were eventually scanned and germinating conidia in spaced fields of view were categorized as forming a germ-tube, globose repetitional conidium or alternative conidial form. Total number of conidia scored per sample (replicate) was usually 200–500. In most experimental conditions incubation was able to be extended until most conidia had germinated. The period depended on species, as well as on the particular conditions. Thus, when scored, most conidia were committed to a particular development mode. All experiments were repeated at least once (for nutrient threshold experiments) or twice (other experiments). Nutrient threshold A range of agars (Oxoid no. 1) of eight different malt extract concentrations (0–1 % w\v) was deployed in Petri dishes (initial experiments) or in 25-well dishes which allowed several species to be processed simultaneously. Incubation of showered substrate dishes for 4 h in light at 20m allowed germination of almost all conidia. Conidia were then killed and scored. Initially, each experiment included six replicate dishes

Table 1. Strains isolated from litter of target habitats and code numbers" assigned for the present study

B. ranarum Eidam C. nr rhysosporus Drechsler C. heterosporus Dreschler C. pumillus Drechsler Conidiobolus sp.# C. thromboides Drechsler C. coronatus (Costantin) Batko C. osmodes Drechsler C. lamprauges Drechsler C. firmipilleus Drechsler C. iuxtagenitus S. D. Waters and Callaghan% C. adiaeretus Drechsler " # $ %

Isolation data available on request. Multiple capilliconidia formed per parent conidium. From colonized collembolan cadaver, R. Manning. Waters & Callaghan, 1989.

Arable field

Larch plantation

Pasture

29 32 33 35 — 41 44 47 49, 50 53 54 —

30 — — — 40 42 45 — 51 — 55, 56 61$, 62, 63

31 — 34 36, 37 — 43 46 48 52 — 57, 58, 59, 60 —

S. D. Waters and A. A. Callaghan

1261

Table 2. Strains received from culture collections and other sources

Basidiobolus ranarum Conidiobolus rhysosporus C. brefeldianus Couch C. stromoideus Sriniv. & Thirum. C. obscurus (I. M. Hall & P. H. Dunn) Remaud. & S. Keller C. thromboides C. thromboides C. thromboides C. coronatus C. coronatus C. coronatus B. haptosporus Drechsler C. rhysosporus C. heterosporus C. megalotocus Drechsler C. lamprauges C. polytocus Drechsler C. firmipilleus C. incongruus Drechsler C. eurymitus Drechsler C. thromboides C. thromboides C. thromboides C. thromboides C. obscurus C. obscurus " # $ %

No.

Code\source

Frog dung Litter Litter Musca% Aphid Aphid Aphid Aphid Aphid Aphid Aphid Litter ? Litter Litter Decaying leaves Litter Plant material Litter Litter Aphid Aphid Aphid Aphid Aphid Aphid ?

1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 19 20 21 23 24 25 26 27 28

John Webster" CBS 157.56 CBS 180.62 CBS 220.64 CBS 181.60 CBS 183.60 NW 3# NW 1 IMI 70229 NWCC3 NWCC1 IMI 108126 RS448$ CBS 138.57 CBS 139.57 CBS 154.56 CBS 168.55 CBS 167.55 CBS 178.61 ATCC 16017 NW 16 NW 15 NW 8 NW 7 NW 92 RS 133

John Webster, University of Exeter. NW l Neil Wilding, Rothamsted Experimental Station. RS l Richard Soper isolates courtesy of Richard Humber (Humber, 1986). Musca domestica with a primary infection of another entomophthoraceous fungus.

100 Vegetative germination (%)

Origin

80

From probit plot: 50 % Germination at 0·093% With 95 % CL 0·08–0·1 %

60 40 20 0 –4 (nom. 0 %)

–3 ( 0·001)

–2 ( 0·01)

–1 (0·1)

0 (1·0)

Concentration of  (%); log scale and (% w/v)

Fig. 1. Germination of primary conidia of Conidiobolus adiaeretus 62 on agars with different concentrations of malt extract (ME). Proportions (%) of germinating conidia forming germ tubes instead of repetitional conidia are plotted as means of six countsp... Estimated % ME associated with 50 % vegetative germination (and 95 % confidence limits) are given to exemplify the initial use of a simple probit method.

per strain per MEA concentration. Thus, for 10 strains, detailed scores were obtained for each substrate dilution. The plot of per cent vegetative germination against log (malt "! extract concentration) gave response curves. Fig. 1 shows a typical example of the sigmoid curves obtained. A simple graphical method using probit-transformed data (Wardlaw,

1985) allowed estimation of the concentration of malt extract associated with 50 % of germination by germ-tube (and an estimate of 95 % confidence limits). These values represent the ‘ nutrient threshold ’ (Callaghan, 1974, 1978). In practice, the trend in conidial behaviour as nutrient concentration increased was sufficiently consistent, and confidence limits were so narrow that a modified simpler method was adopted. This enabled the relatively rapid screening of more strains from habitat monitoring, and from other sources. Thus for 49 strains, instead of full counts, successive conidium deposits along the substrate wells representing a concentration series were scanned. By inspection, malt extract concentrations associated with scores immediately above and below ca 50 % germination were located. The approximate concentration associated with 50 % germination was calculated by interpolation. For each strain two replicate series were scanned ; with consistent result. Fig. 2 displays threshold values for all strains tested. Hierarchical germination patterns for successive conidium generations RWA was used as substrate. Primary and secondary deposits were obtained by procedures described above. A tertiary deposit of discharged globose conidia was produced by canopying a secondary deposit over substrate RWA. Precise timings of showering and changes of sources had to be determined afresh for each species. Fig. 3 shows patterns of repetitional conidium formation for 12 of the 16 strains tested.

Germination in Conidiobolus and Basidiobolus

1262

63 adiaer 62 61 57 iux 56 iux 59 iux 54 iux 55 iux 53 iux 52 lam 51 lam 48 osm 47 osm 46 cor 45 cor 44 cor 41 thr 42 thr 43 thr

Strains deployed in numbered sequence

63

60 iux

From target habitats (non-capilliconidial isolates)

40

40 beta 37 pum 35 pum

36 pum

16 meg 11

26 thr From culture collection sources

8 thr 7 thr

31 Br

30 Br

28 obs 27 obs

6 thr

23 thr 19 firm 17 lam 18 poly 21 cur 14 het 12 Bh 13 rhy 11 cor 10 cor From culture collections 9 cor

–2 (0·01)

20 incon

5 E thax 4 strom 3 bref

2 rhy 1 Br

Callaghan 1978 –2·5

29 Br

25 thr 24 thr

1 –3 (0·001%)

50 lam 49 lam

From target habitats (capilliconidial isolates)

33 het

34 het 28

32 pum

58 iux

–1·5

–1 (0·1)

0 (1·0)

– 0·5

0·5

Concentration of  (%); log scale and (% w/v)

Fig. 2. Transition to vegetative germination (nutrient thresholds) in a range of isolates of Conidiobolus and Basidiobolus spp. from diverse sources. Estimates of the concentration of malt extract associated with 50 % of primary conidia germinating vegetatively and 50 % forming repetitional conidia. Point numbers and abbreviated species names correspond to strain numbers and names in Tables 1 and 2.

As previously reported (King, 1976) the strains of C. thromboides and C. lamprauges formed no conidium type other than globose repetitional conidia.

A range of refined agars with eight different water activities (aw) 1n00–0n95 was prepared by incorporating sorbitol (glucitol, Aldrich Chemical Co.) at appropriate concentrations (Harris, 1981) in RWA. Trials also separately tested the effects of sucrose (Anagnostopoulos & Dhavises, 1980) and glycerol (Dallyn & Fox, 1980) as conditioning substances. Germination patterns (i.e. concentration preventing germination and changing proportion of alternative conidial forms) along the sequence of aw agars were closely similar for each substance. Sorbitol was preferred for main experiments, being least likely to be metabolized ; Basidiobolus, for example, has been shown to utilize sucrose (Latge! , 1975). Sorbitol agars (autoclaved at 115m for 10 min) were either deployed singly in Petri dishes (5 cm diam.) or in triplicate in 25-well dishes. Before use, substrates were stored at 20m for ca 24 h to allow the evaporation of free liquid on the agar surface. Behaviour of primary and secondary conidia was studied in separate experiments. Duration of incubation was 6 h for aw 1n000– 0n995 and 7 d for aw 0n99–0n95. In most experiments germination was entirely by repetitional conidium-formation

Alternative conidium type (%)

Effect of water activity

100

C. heter C. iux 34 56

C. firm C. firm 53 19

0

0

0

0

C. adia (*)

C. cor 46

C. cor 45

C. cor 44

0 0 nd 0 12 3 12 3 1 2 3 12 3 Successive spore generations 1, 2, 3

12 3

B. ran 31

C. pum 37

nd

nd

C. adia 62

C. adia nr 63

50

0 100

50

0

0 nd 12 3

Fig. 3. Globose conidia of Conidiobolus and Basidiobolus forming alternative conidium types : Proportions (%) of alternative types formed by parent globose conidia of successive generations on RWA. Results of one group of experiments are displayed. Each estimate is based on one conidial deposit of usually
500 conidia (for primary and secondary generations) and 200–300 for tertiary deposits. nd l not determined ; spore numbers were too low. 0 l 0 % alternative conidia. Repeat experiments gave similar overall profiles. Abbreviated species names, and numbers correspond to strains in Tables 1 and 2, with nr (near) 63 replacing 63 and (*) which is CBS 136n57.

S. D. Waters and A. A. Callaghan

1263

Table 3. Primary conidia of Conidiobolus and Basidiobolus on sorbitol-agars of different water activities : Proportion (%) forming a conidium type alternative to secondary globose conidia. Sample size
300 conidia Water activity and approx. water potential, ψ (kMPa)*

Species, strain number

0 ψ

1n000 0

0n999 0n138

0n998 0n28

0n995 0n70

B. ranarum C. lamprauges C. firmipilleus C. adiaeretus C. iuxtagenitus C. pumillus C. heterosporus C. coronatus C. thromboides

31 49 53 62 56 37 34 45 43

1n0 0 0 0 0 52 24 0 0

0n3 0 0 0 0 36 31 0 0

0 0 0 0 0 51 40 0 0

42 0 0 0 0 82 20 0 0

Incubation period

4–6 h

0n990 1n38

0n985 2n12

0n980 2n78

0n950 7n06

Conidium type

99 0 0 0 0 14 ab (0) 0

(100) 0 — (0) 0 — — — 0

— — — — — — — — (0)

— — — — — — — — —

cc None mc mc adec cc cc mc None

1d

7d

* After Magan, 1997. k No germination. () 40 % overall germination ; per cent forming alternative spore type in parentheses. In all other cases overall germination was 100 %. ab abnormal irregular growth. Conidium types ; cc l capilliconidia ; mc l microconidia ; adec l actively discharged elongate conidia. Formation of germ tubes was rare and is not represented in the table.

Table 4. Secondary conidia of Conidiobolus and Basidiobolus on sorbitol-agars of different water activities : Proportion (%) forming a conidium type alternative to tertiary globose conidia. Sample size
250 conidia Water activity

B. ranarum C. lamprauges C. firmipilleus C. adiaeretus C. iuxtagenitus C. pumillus C. heterosporus C. coronatus C. thromboides Incubation period

31 49 53 62 56 37 34 45 43

1n000

0n999

0n998

0n995

0n990

0n985

0n980

0n950

Conidium type

0 0 15 0 60 76 31 45 0

62 0 14 0n7 77 44 30 80 0

53 0 3n8 35 89 57 39 54 0

95 0 2n7 43 95 64 ab 81 0

97 0 0 69 97 ab ab 95 0

— — — 0 — — — 0 0

— — — — — — — — 0

— — — — — — — — —

cc None mc mc adecs cc cc mc None

4–6 h

1d

7d

k No germination. In all other cases germination was 100 %. ab abnormal irregular growth. Formation of germ tubes was rare and is not represented in the table.

and almost all conidia germinated except below the aw ‘ cutoff ’ values (Tables 3, 4). Effects of temperature and light For six species, the proportion of germinating conidia forming an alternative to globose replicative conidia was determined for primary deposits and, in separate experiments, for secondary deposits. First, RWA was used as substrate then, to enhance the general levels of alternative development, the whole set of experiments was repeated using a sorbitolconditioned agar judged from Tables 3 and 4 to be optimal for the formation of its particular alternative conidia. To represent the usual temperature range (ca 5–30m) associated with growth and sporulation in Entomophthorales of the temperate zone (Hall & Papierok, 1982), contrasting temperatures 5m, 15m and

25m were selected. Dishes of the appropriate substrate were inverted, over fluorescent lights, individually enclosed in transparent polythene bags and duplicate sets were enclosed in black light-proof polythene bags. Temperature in the bags did not differ from ambient values. Monitoring of spare illuminated conidium deposits informed the decision to remove test dishes when most conidia had germinated. Incubation periods varied from several days (5m), to ca 8 h (15m) and ca 3 h (25m). Killing and scoring were as stated earlier. Typically four replicate deposits were used for each treatment. Some failure of conidium discharge reduced the replicate numbers for particular strains. Results for RWA are displayed (Fig. 4) only for those species with levels of alternative conidium formation
10 % in any one set of conditions. Thus generally, in agreement with Tables 3 and 4, there are no data to show for any primary deposit except for C. pumillus and none for

Alternative conidium type (%)

Germination in Conidiobolus and Basidiobolus 100

100

100 C. pum 37 cc

C. pum 37 cc

50

50

5

15

25

0

5

15

100

25

C. iux 56 adec

100

Secondary deposit

C. heter 34 cc

Secondary deposit

Primary deposit

0

1264 C. firm 53 mc Secondary deposit

Secondary deposit 50

50

0

0

5 15 25 Incubation temeperature (°C)

50

5

15

25

0 5

15

25

Alternative conidium type (%)

Fig. 4. Effect of light ( ), dark () and temperature on the proportion (%) of alternative conidium types formed by primary and secondary globose conidia on RWA. Only those species which gave
10 % values are plotted (see text). Meansp1 ... (n l 4, except C. iuxtagenitus and C. firmipilleus based on single deposits of
500 conidia). Incubation periods depended on species and temperature (see text).

B. ran 31 cc a w = 0·990 100

100

50

50

0

5

15

25

0

C. heter 34 cc a w = 0·998 C. pum 37 cc a w = 0.995 C. lux 56 adec a w = 0.998 C. adi 63 mc a w = 0.995 100 100 100

5

15

25

50

50

0

0

5 15 25 Incubation temperature (°C)

50

5

15

25

0

5

15

25

Fig. 5. Effect of light ( ), dark () and temperature on the proportion (%) of alternative spore types formed by primary conidia of Conidiobolus and Basidiobolus. Substrates were sorbitol\RWA of aw optimal (at 20m) for each species to form its non-globose conidia (Table 3). Results of one group of experiments are represented. Meansp1 .. for n l 4 (n l 2 for C. pumillus and C. iuxtagenitus). Incubation periods depended on species and temperature. For B. ranarum, overall germination 50 % at 5m and, in dark at 15m\25m (see text).

secondary deposits of B. ranarum, C. adiaeretus or C. firmipilleus. Fig. 5 shows results for primary deposits (except for C. firmipilleus with zero alternative conidia) and Fig. 6 for secondary deposits on optimal sorbitol-agars. For each strain, per cent data were analysed by one-way, nested ANOVA after arcsin Nx transformation. Follow-up was by an S-N-K multiple comparison of means. Where appropriate the possibility of trend was tested by a linear regression. All graphics and statistical calculations were done using UNISTAT software. Time course of repetitional conidial-formation in light and dark During the previous sets of experiments it became apparent that, for B. ranarum, C. adiaeretus and C. iuxtagenitus, the overall rate of germination yielding globose conidia in dark seemed often to lag behind that in light. Simultaneous timecourses for germination of primary conidia on RWA in light and dark were determined by deploying the substrate in 96well dishes of the type dissected into 6i24-well strips. Each species’ conidium deposit was present in four wells per strip. After showering from inverted 96-well source dishes, strips were sealed and deployed in transparent or opaque bags by a rapid standardized procedure. Removal of one strip from each bag type at intervals allowed killing and subsequent scoring

of deposits in a standard manner. Figs 7–9 show the timecourses of overall germination.

RESULTS Induction of germ-tubes Inspection of Fig. 2 suggests several generalizations. The wide differences in nutrient thresholds (encompassing a range of three orders of magnitude) reflect large differences in sensitivity to the induction of germ tubes. Most strains tested (ca 60 %) required a malt extract concentration of 0n03–0n2 % to germinate vegetatively. Perhaps unsurprisingly, threshold values for several strains of any one species can be closely similar when from the same habitat (e.g. C. adiaeretus and C. iuxtagenitus from the larch plantation) or differ greatly when from widely different sources (e.g. C. coronatus and C. thromboides). Of particular interest are those strains, ca 30 %, which have a markedly low threshold, ca 0n001–0n03 % malt extract. Many of these are from collections (Table 2, Fig. 2) but were originally isolated from aphids. Most other ‘ low threshold ’ strains are the isolates from our target habitats which can, on non-nutrient substrates form capilliconidia (Table 1, Fig. 2). The correlation is not perfect ; in total some 15 out of 22 proven or presumptive pathogens and\or capilliconidial strains have especially low thresholds.

S. D. Waters and A. A. Callaghan 100

1265

B. ran 31 cc a w = 0·990

50

0 5 100

15

25

C. het 34 cc a w = 0·998

50

0 5 100

15

25

C. pum 37 cc a w = 0·995

Alternative conidium type (%)

50

0 5 100

15

25

Replicative conidium generations developing alternative conidial types For 12 strains (seven spp.), Fig. 3 displays the proportion of alternative conidial forms produced by three generations of globose conidia on RWA and incubated at 20m in light. For 10 of these strains primary conidia formed no (or very few) nonglobose conidia. Conidiobolus pumillus no. 37 (very high) and C. coronatus no. 46 (ca 10 %) consistently yielded their alternative conidial type. For all strains tested, secondary globose conidia formed alternative types, sometimes in very high proportions (e.g. C. iuxtagenitus, actively discharged elongate conidia ; C. pumillus, capilliconidia). Tertiary globose conidia also formed the alternative spores, often in even greater proportion than from secondary globose parents (e.g. C. coronatus no. 46, microconidia). From primary to tertiary globose conidia, increasing propensity to form an alternative conidial type does seem a general pattern. Actual proportions vary between strains of the same species (Fig. 3) and between experiments. We tested several strains of species reported as not forming alternative conidial types. Indeed, none was seen for C. lamprauges and C. thromboides, but, for C. adiaeretus, microconidia, hitherto undescribed, were disclosed even in the type strain (CBS 136.57). The microconidia themselves (as tertiary or quaternary conidia) were able to germinate by formation of a germ tube, globose conidium or, by a minute capilliconidium. Formation of capilliconidia and microconidia by the same species seems to cut across current taxonomic groupings in Conidiobolus (Humber, 1989).

C. iux 56 adecs a w = 0·998

Effect of water activity 50

0 5 100

15

25

C. adi 63 mc a w = 0·995

50

0 100

5

15

25

C. firm 53 mc a w = 0·998

50

0

15 25 5 Incubation temperature (°C)

For primary conidia of 6 species tested (Table 3), manipulation of substrate aw did not induce alternative conidium-formation. In B. ranarum, however, there is a marked rise in formation of capilliconidia as aw drops to 0n995. The optimum aw of 0n99 (water potential, Ψ, of k1n38 MPa) yielded 100 % of capilliconidia from total germination of the primary conidia. The other capilliconidial strains tested, both formed their alternative conidium type at aw from 1n00 down to 0n99 but C. heterosporus seemed to be more sensitive to lower aw (reduced per cent at 0n995) ; C. pumillus yielded a high per cent even at this value. Repeat experiments gave slightly different values but the differences between strains were consistent. After 7 d incubation, on sorbitol-agar of aw 0n98 (Ψ of k2n78 MPa), no germination had occurred in any strain tested (except C. thromboides). Secondary conidium deposits of all strains (except C. lamprauges and C. thromboides) formed their alternative conidial type over some part of the Fig. 6. Effect of light ( ), dark () and temperature on the proportion (%) of alternative conidial types formed by secondary globose conidia of Conidiobolus and Basidiobolus. Substrates were sorbitol\RWA of aw optimal (at 20m) for each species to form its nonglobose conidia (Table 4). Results of one group of experiments are represented. Meansp1 ... for n l 4 (n l 2 for C. iuxtagenitus and C. firmipilleus). Incubation periods depended on species and temperature. For B. ranarum overall germination 50 % at 5m (see text).

Germination in Conidiobolus and Basidiobolus

1266

Table 5. Summary of temperature effects on proportion (%) of alternative conidium types Significantly different means (! temp [m]) Fig. 4 (mainly 2y) C. pumillus 37 (1y) C. pumillus 37 (2y) C. heterosporus 34 C. iuxtagenitus 56 C. firmipilleus 53 Fig. 5 (1y) B. ranarum 31 C. heterosporus 34 C. pumillus 37 C. iuxtagenitus 56 C. adiaeretus 63 Fig. 6 (2y) B. ranarum 31 C. heterosporus 34 C. pumillus 47 C. iuxtagenitus 56 C. adiaeretus 63 C. firmipilleus 53

Low ns Low Low Low

ANOVA (P)

! 25

0n0009 ns

!5 !5 !5

0n008 nt nt

High ! all Low ! 5 Low ! 25 ns Low ! 5

nt

High ! all ns ns Low ! 5 Low ! ns

nt ns ns

0n0002 0n012 ns 0n0001

0n030 0n0001 ns

Regression kve — jve — —

(P)

0n0006 ns 0n009 nt nt

— jve kve jve jve

nt

— — — — jve —

nt ns ns ns

0n0003 0n002 0n034 0n0001

0n0001 ns

ns l not significant ; nt l not tested. Data of Figs 4–6 analysed, after arcsin Ni transformation by one-way, nested ANOVA and S-N-K multiple comparison of means (P l 0n05). Means for light and dark treatments were not significantly different and replicates per temperature were pooled. Trends tested by linear regression of replicates on temperature, ‘ trend ’ implies ‘ from 5m to 25m ’. Means significantly different given as low (or high)!stated temperature. Parent conidium types : 1y l primary globose conidia ; 2y l secondary globose conidia.

range of agars offered (Fig. 4). The profiles for B. ranarum, C. heterosporus and C. pumillus were very similar to those for their primary conidia. Conidiobolus adiaeretus formed most microconidia at aw 0n995 ; C. coronatus yielded high proportions of microconidia from aw values 0n999 down to 0n990. In interesting contrast, C. firmipilleus formed its microconidia only at the highest values of water activity (to 0n999). The characteristic actively discharged elongate conidia of C. iuxtagenitus were produced in increasingly high per cent as aw values decreased from 1n00 to 0n99. Thus, formation of alternative conidial types is influenced by water stress in a manner differing according to the strain and species. As for primary conidia, for all strains except C. thromboides, germination by formation of repetitional conidia seemed to cease at aw 0n98 (ψ of k2n78 MPa) and none occurred at aw 0n950 in 7 d of incubation.

Vegetative germination and growth In experiments not reported here in detail, involving the same strains as in Tables 3 and 4, primary conidia germinated vegetatively on sorbitol-conditioned 1 % MEA down to aw ca 0n975 (Ψ of ca k3n48 MPa). Conidiobolus adiaeretus, C. iuxtagenitus, C. lamprauges and C. pumillus showed only low levels ( 25 %) after 7 d incubation and C. heterosporus had abnormal germ tubes. No germ tubes developed at aw ca 0n945 (Ψ of ca k7n76 MPa) after 7 d. In other experiments, hyphal extension from agar\mycelium block inocula continued slowly for 7 d at aw of 0n975 for all species except for C. iuxtagenitus, C. lamprauges and C. pumillus and at aw ca 0n945 no hyphae sprouted from inocula in 7 d.

Temperature, light and alternative conidial forms Experiments in which temperature and presence or absence of light were manipulated had as a general aim the disclosure of any marked differences in response between species. Table 5 summarizes possible temperature effects displayed in Figs 4–6 and associated statistics. In no case was light shown to exert any influence. For several species, irrespective of the kind of alternative conidium, a relatively low proportion of globose conidia developed the alternative form at 5m. This was particularly marked for secondary conidia on RWA for C. heterosporus, C. iuxtagenitus and C. firmipilleus (Fig. 4). It also occurred for primary conidia of C. heterosporus and C. iuxtagenitus and for microconidial formation by primary and secondary globose conidia of C. adiaeretus, on sorbitolconditioned agar. Use of agars with optimal aw predictably enhanced proportions of alternative conidial types but tended to even out the levels across the temperature range. Water activity seems a powerful influence ; although C. adiaeretus at its optimal aw still displayed the ‘ low temperature effect ’ (Fig. 6). Inadvertently the sorbitol-agar used for C. firmipilleus (aw l 0n998) was actually sub-optimal compared with RWA (Table 4). Consistent with this, levels of microconidia were somewhat lower (Fig. 6). Contrary to the apparently common effect of low temperature discussed above, C. pumillus seemed to form more capilliconidia at lower temperatures (Figs 4, 5 ; Table 5). Light and globose conidium replication Light does seem to be involved in the formation of actively discharged repetitional conidia by three of the species tested.

S. D. Waters and A. A. Callaghan

1267

7

8

9

Overall germination (%)

100 light

light

light

80 60

dark

dark

40

dark

20 0 0

10

20

30

Incubation time (h)

40

50 0

2

4 6 8 Incubation time (h)

10

12 0

2

4 6 8 Incubation time (h)

10

12

Figs 7–9. Time-course of germination of primary conidia on RWA in light and dark. Meansp1 ... (n l 4). Fig. 7. B. ranarum 31. Fig. 8. C. adiaeretus 62. Fig. 9. C. iuxtagenitus 58.

The time-courses of primary conidia germinating to produce replicative globose conidia (B. ranarum and C. adiaeretus) or elongate alternative conidia (in C. iuxtagenitus), show (Figs 7–9) that, in the absence of light, development of the repetitional conidia is delayed by many hours. DISCUSSION The long evolution of present-day Conidiobolus and Basidiobolus from ancestral saprotrophs (Humber, 1984 ; Evans, 1988) can be expected to have diversified the species retaining a saprotrophic mode. Developments could include specialization for particular target invertebrates ; features aiding the avoidance of competition from non-entomophthoralean saprotrophs ; and acquisition of tolerances and requirements for abiotic conditions which allow Conidiobolus and Basidiobolus species to evade competition with each other. The present results are relevant to the latter. The triggering of vegetative germination is clearly as crucial for a saprotroph as for a pathogen. Given the lack of knowledge of specific invertebrate targets (substrates), and of the nature of their interaction with Basidiobolus and Conidiobolus, the current study has concentrated on a rather general determinant of vegetative germination, malt extract. Earlier work on nutrient threshold (Callaghan, unpublished), failed to demonstrate single substances like glucose, glucosamine or amino acids inducing germ-tubes. Only the complete malt extract, or unknown component(s) of it, seemed effective. Tested strains from the target habitats showed wide differences in sensitivity to nutrients. The capilliconidial species (C. heterosporus, C. pumillus, a Conidiobolus sp. with multiple capilliconidiophores and some Basidiobolus strains), together with C. megalotocus and C. eurymitus, may be pathogens judged by analogy with the low threshold C. coronatus, C. thromboides and C. obscurus from aphids. Detailed work on C. obscurus shows virulent strains as having a greater tendency to form germ-tubes (lower threshold) than avirulent ones, on aphid cuticles (Latge! , Papierok & Sampedro, 1982). Strain 5 in the present study has a rather high threshold (Callaghan, 1978) but, as Sampedro et al. (1984) showed, C. obscurus strains can vary in their germination responses according to conditions. Cuticular extracts from aphids were particularly implicated in germination of conidia in virulent

strains (Latge! et al., 1987). No simplistic extrapolation of the current nutrient threshold results to field conditions is intended, but the widely differing sensitivities to a general trigger for germ-tube induction may link with the response of strains to leakage of potential nutrients from faeces, exuviae, animal cadavers or from live animals (stressed and unstressed). Results may thus reflect strain differences in target specificity and in trophic mode. Nutrient deficiency for Conidiobolus and Basidiobolus seems to lead to an ‘ escape ’ or ‘ dissemination ’ phase involving discharge and\or mobile animals as vectors. In contrast, at least for the spores of some non-entomophthoralean entomopathogens on target integuments, nutrient deficiency is a signal to switch from saprotrophic to pathogenic mode (Clarkson & Charnley, 1997). The ability of Basidiobolus and Conidiobolus conidia to form repetitional globose conidia and sometimes alternative forms is well known (Eidam, 1886 ; King, 1976). For C. coronatus Prasertphon (1963) has described how several generations of repetitional conidium formation lead to globose conidia and to microconidia. There is, however, little quantitative information about the proportions of alternative types formed in repetitional conidia generations. The increasing tendency to switch development to an alternative conidial type as globose conidia deplete their energy supplies is shown in Fig. 3. Presumably this is the evolved ‘ escape ’ mechanism mentioned above operating at a nutrient-deficient microsite. The development of alternatives to globose conidia are affected by water availability. Page & Humber (1973) remark, for C. coronatus on mannitol-conditioned agars, that slight reductions in the turgor pressure of the conidium can have important developmental implications. We extend this to ecological considerations. We have, however, used osmotic conditioning with no involvement of matric potential. Furthermore, no measurements of actual substrate water potential were able to be made. The consistently similar results with conditioning substances other than sorbitol, and with repeat experiments, suggests a data set useful for cautious extrapolation to field conditions. Tables 3 and 4 show clear contrasts between strains (and probably species). Of capilliconidial strains, B. ranarum has marked preference for aw at levels close to those precluding germination (0n99, ψ of k1n38 MPa), C. pumillus annd C. heterosporus both form capilliconidia from aw nr 1n0 to 0n998 ; on substratum of

Germination in Conidiobolus and Basidiobolus greater water stress the latter is adversely affected. For strains producing microconidia, similar interesting differences show. C. adiaeretus has preference for relatively high stress, C. coronatus (2y globose conidia) is relatively unaffected down to aw of ca 0n99, but C. firmipilleus (isolated from the arable field soil) is intolerant of water stress and forms microconidia mainly at aw 1n0–0n999. The formation of tertiary actively discharged elongate conidia by C. iuxtagenitus was at high levels at all aw values down to ‘ germination cut-off ’ at 0n99. As with the scatter of microconidia by C. coronatus, this may be effective dissemination under water stress ; the smaller ballistospore may need slightly less turgor pressure. Our (limited) results on vegetative germination and hyphal growth (zero or low levels at aw of ca 0n975 and no germ-tubes formed or hyphae growing at aw ca 0n945 after 7 d) suggests a sensitivity approximately in line with that of common mitosporic entomophathogens with a cut-off level for germination reported as aw 0n93 (Gillespie & Crawford, 1986). In their detailed study of B. haptosporus isolates from vertebrates and litter, Zahari & Shipton (1988) found very slow growth at Ψ of k5n6 MPa (aw 0n960). More generally, for vegetative germination and growth, the Conidiobolus and Basidiobolus species tested seem more sensitive than many terrestrial fungi and hardly differ from the relatively sensitive basidiomycetes of soil and litter (Dix & Webster, 1995). In the experiments at different temperatures, a temperatureassociated alteration of the actual values of aw for substrates would be expected. At 5m the aw would be slightly raised compared with that at 25m. In the range of values used (0n99–1n0), however, the alteration is likely to be small (Dix & Webster, 1995). Furthermore, behaviour of the fungi (e.g. ‘ cold effect ’, see below) is not consistent with any significant distortion of water activity values. Table 5 data do suggest that for several species, low litter temperatures would not favour alternative conidium formation. Again, individual differences are interesting ; C. pumillus for example, shows the opposite with maximum formation of capilliconidia at 5m. For deposits of secondary conidia put on ‘ optimal ’ sorbitolconditioned agars at different temperatures it was clear that water activity dominated ; only C. adiaeretus showed the ‘ low-temperature ’ effect. For C. obscurus (Latteur, 1980) temperatures below 10m favoured secondary conidia resembling the primary spore ; higher temperatures induced a variant spore form with a very different papilla. Other temperature influences have been studied for a range of arthropod pathogens in other entomophthoraceous genera (e.g. Magalha4 es et al., 1991 ; Morgan et al., 1995 ; Oduor et al., 1996 a, b). The detailed findings, often including study of factors interacting, tend to be specific to the biology of each pathogen and its targets ; generalizations are difficult to make. Only those spore forms which are actively and directionally discharged seem to have an associated involvement with light. For B. ranarum no. 31 from a pasture in Staffordshire the result is closely similar to that reported for a quite different strain (no. 1, Table 2) from frog dung near Exeter, U.K. The light involvement for C. adiaeretus and C. iuxtagenitus parallels earlier findings for C. obscurus (no. 5), C. thromboides (no. 6) and for C. stromoideus (no. 4), (Callaghan, 1974, 1978). In contrast, from the same study, C. coronatus, C. brefeldianus and

1268 C. rhysosporus seemed unaffected by light. Although in the present study light did not influence the formation of secondary or tertiary capilliconidia or microconidia (Table 5), we did not follow up possible effects on the subsequent germination of these spores. In their detailed study of germination of capilliconidia of Neozygites floridana, Oduor et al. (1996 b) exemplify inhibitory effects of light. Germination was delayed and subsequent levels of germination reached in light were distinctly lower than in dark. Furthermore, production of primary conidia and capilliconidia was reduced by light. The data presented in the current study represent a first stage in attempts to define the detailed biology of Conidiobolus and Basidiobolus in a limited habitat, a larch plantation and its immediate surroundings. Our current and future work is focused on the invertebrates and entomophthoralean fungi of a relatively small area (ca 20i20 m). The species of Conidiobolus and Basidiobolus given most attention are those most frequently disclosed from the habitat. If the natural substrates, living or dead, of any of these fungi can be identified then study of fungal requirements and tolerances can start to match the more focused research on known entomopathogens in other genera of the Entomophthorales. REFERENCES Anagnostopoulos, G. O. & Dhavises, G. (1980). The role of proline and other amino acids in osmoregulation of Escherichia coli. In Microbial Growth and Survival in Extremes of Environment (ed. G. W. Gould & J. E. L. Corry), pp. 141–147. Academic Press : New York, U.S.A. Bellini, R., Mullen, B. A. & Jespersen, J. B. (1992). Infectivity of two members of the Entomophthora musci complex (Zygomycotina : Entomophthorales) for Musca domestica (Diptera : Muscidae). Entomophaga 37, 11–19. Brobyn, P. J. & Wilding, N. (1977). Invasive and developmental processes of Entomophthora species infecting aphids. Transactions of the British Mycological Society 69, 349–366. Callaghan, A. A. (1974). Effect of pH and light on conidium germination in Basidiobolus ranarum. Transactions of the British Mycological Society 63, 13–18. Callaghan, A. A. (1978). Effect of nutrient level, pH and light on conidial germination in entomophthoraceous fungi. Transactions of the British Mycological Society 70, 271–275. Clarkson, T. M. & Charnley, A. K. (1997). New insights into the mechanisms of fungal pathogenesis in insects. Trends in Microbiology 4, 197–203. Dallyn, H. & Fox, A. (1980). Spoilage of materials of reduced water activity by xerophilic fungi. In Microbial Growth and Survival in Extremes of Environment (ed. G. W. Gould & J. E. L. Corry), pp. 129–139. Academic Press : New York, U.S.A. Dix, N. J. & Webster, J. (1995). Fungal Ecology. Chapman & Hall : London. Drechsler, C. (1956). Supplementary developmental stages of Basidiobolus ranarum and B. haptosporus. Mycologia 48, 655–676. Eidam, E. (1886). Basidiobolus eine neue gattung der entomophthoraceen. Beitrage zur Biologie der Pflanzen 4, 183–251. Evans, H. C. (1988). Co-evolution of entomogenous fungi and their insect hosts. In Co-evolution of Fungi with Plants and Animals (ed. K. A. Pirozynski & D. L. Hawksworth), pp. 149–171. Academic Press : London. Gillespie, A. T. & Crawford, E. (1986). Effect of water activity on conidial germination and mycelial growth of Beauveria bassiana, Metarhizium anisopliae, Paecilomyces spp. and Verticillium lecanii. In Fundamental and Applied Aspects of Invertebrate Pathology (ed. R. A. Samson, J. M. Vlak & D. Peters), p. 254. Society of Invertebrate Pathology, Wageningen, Netherlands. Glare, T. R., Chilvers, G. A. & Milner, R. J. (1985). Capilliconidia as infective spores in Zoophthora phalloides (Entomophthorales). Transactions of the British Mycological Society 85, 463–470.

S. D. Waters and A. A. Callaghan Hajek, A. E. & St Leger, R. J. (1994). Interactions between fungal pathogens and insect hosts. Annual Review of Entomology 39, 293–322. Hall, R. A. & Papierok, B. (1982). Fungi as biological control agents of agricultural and medical importance. Parasitology 84, 205–240. Harris, R. F. (1981). The effect of water potential on microbial growth and activity. In Water Potential Relations in Soil Micro-organisms (ed. J. F. Parr, W. R. Gardner & L. F. Elliot), pp. 23–84. Special Publication No. 9. Soil Society of America : Madison, U.S.A. Humber, R. A. (1984). Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In Fungus-Insect Relationships : Perspectives in Ecology and Evolution (ed. Q. Wheeler & M. Blackwell), pp. 166–183. Columbia University : N.Y. Humber, R. A. (1986). Catalog of Strains. USDA – ARS Collection of Entomopathogic Fungal Cultures : Ithaca, New York. Humber, R.A. (1989). Synopsis of a revised classification for the Entomophthorales (Zygomycotina). Mycotaxon 34, 441–460. Kevorkian, A. G. (1937). Observations on the genus Conidiobolus. Journal of the Agricultural University of Porto Rico 21, 191–200. King, D. S. (1976). Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy. I. Biology and cluster analysis. Canadian Journal of Botany 54, 45–65. King, D. S. & Humber, R. A. (1981). Identification of entomophthorales. In Microbial Control of Pests and Plant Diseases 1970–1980 (ed. H. D. Burges), pp. 107–127. Academic Press : New York, U.S.A. Latge! , J.-P. (1975). Croissance et sporulaton de 6 especes d’Entomophthorales. 1. Influence de la nutrition carbonee. Entomophaga 20, 201–207. Latge! , J.-P., Papierok, B. & Sampedro, L. (1982). Aggressivite! de Conidiobolus obscurus vis-a' -vis du puceron du pois, Acyrthosiphon pisum (Hemiptera : Aphididae). 1. Comportement des conidies sur la cuticule avant la penetration du tube germinatif dans l’insecte. Entomophaga 27, 323–330. Latge! , J.-P., Sampedro, L., Brey, P. & Diaquin, M. (1987). Aggressiveness of Conidiobolus obscurus against pea aphid : Influences of cuticular extracts on ballistospore germination of aggressive and non-aggressive strains. Journal of General Microbiology 133, 1987–1997. Latteur, G. (1980). The persistence of infectivity of conidia of Entomophthora obscura at different temperatures on the surface of an unsterilised soil. Acta Oecologica\Oecologica Applicata 1, 29–34. Magalha4 es, B. P., Humber, R. A., Shields, E. J. & Roberts, D. W. (1991). Effects of environment and nutrition on conidium germination and appressorium formation by Zoophthora radicans (Zygomycetes, Entomophthorales) : a pathogen of the potato leafhopper (Homoptera ; Cicadellidae). Environmental Entomology 20, 1460–1468. Magan, N. (1997). Fungi in extreme environments. In The Mycota, IV. Environmental and Microbial Relationships (ed. D. T. Wicklow & B. E. Soderstrom), pp. 99–114. Springer-Verlag : Berlin. Morgan, L. W., Boddy, L., Clark, S. J. & Wilding, N. (1995). Influence of temperature on germination of primary and secondary conidia of Erynia (Accepted 18 December 1998 )

1269 neoaphidis (Zygomycetes ; Entomophthorales). Journal of Invertebrate Pathology 65, 132–138. Nadeau, M. P., Dunphy, G. B. & Boisvert, J. L. (1996). Development of Erynia conica (Zygomycetes ; Entomophthorales) on the cuticle of the adult black flies Simulium rostratum and Simulium decorum (Diptera ; Simuliidae). Journal of Invertebrate Pathology 68, 50–58. Newman, G. G. & Carner, G. R. (1975). Environmental factors affecting conidial sporulation and germination of Entomophthora gammae. Environmental Entomology 4, 615–618. Oduor, G. I., de Moraes, G. J., van der Geest, L. P. S. & Yaninek, J. S. (1996 a). Production and germination of primary conidia of Neozygites floridana (Zygomycetes ; Entomophthorales) under constant temperature, humidities and photoperiods. Journal of Invertebrate Pathology 68, 213–222. Oduor, G. I., Yaninek, J. S., van der Geest, L. P. S. & de Moraes, G. J. (1996 b). Germination and viability of capilliconidia of Neozygites floridana (Zygomycetes ; Entomophthorales) under constant temperature, humidity and light conditions. Journal of Invertebate Pathology 67, 267–278. Page, R. M. & Humber, R. A. (1973). Phototropism in Conidiobolus coronatus. Mycologia 65, 335–354. Papierok, B. (1986). Some noteworthy biological problems within the genus Conidiobolus. In Fundamental and Applied Aspects of Invertebrate Pathology (ed. R. A. Samson, J. M. Vlak & D. Peters), pp. 197–200. Society for Invertebrate Pathology : Wageningen, Netherlands. Prasertphon, S. (1963). Conidial formation in Entomophthora coronata. Journal of Insect Pathology 5, 318–335. Sampedro, L., Uziel, A. & Latge! , J.-P. (1984). Aggressivite! de C. obscurus visa' -vis du puceron du pois. Mycopathologia 86, 3–19. Smith, M. F. & Callaghan, A. A. (1987). Quantitative survey of Conidiobolus and Basidiobolus in soils and litter. Transactons of the British Mycological Society 89, 179–185. St Leger, R. J. (1991). Integument as a Barrier to Microbial Infection. In The Physiology of Insect Epidermis (ed. A. Retnakaran & K. Binnington), pp. 286–308. CSIRO : Australia. van Roermund, H. J. W., Perry, D. F. & Tyrrell, D. (1984). Influence of temperature, light, nutrients and pH in determination of the mode of conidial germination in Zoophthora radicans. Transactions of the British Mycological Society 82, 31–38. Wardlaw, A. C. (1985). Practical Statistics for Experimental Biologists. Wiley : Chichester, U.K. 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. Yendol, G. W. (1968). Factors affecting germination of Entomophthora conidia. Journal of Invertebrate Pathology 10, 116–121. Zahari, P. & Shipton, W. A. (1988). Growth and sporulation responses of Basidiobolus ranarum to changes in environmental parameters. Transactions of the British Mycological Society 91, 141–148.