Survival of Conidiobolus spp. and Basidiobolus ranarum in relation to relative humidity and temperature

fungal ecology 3 (2010) 148–159

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Survival of Conidiobolus spp. and Basidiobolus ranarum in relation to relative humidity and temperature Ian J. HOPKINS, Arthur A. CALLAGHAN* Institute for Environment, Sustainability and Regeneration, Staffordshire University, Mellor Building, College Road, Stoke-on-Trent, ST4 2DE, UK

article info

abstract

Article history:

Survival of saprotrophs and opportunistic pathogens of arthropods was studied in 8

Received 20 January 2009

Conidiobolus spp. and in Basidiobolus ranarum. Response to brief adverse relative humidity

Revision received 18 June 2009

(RH < 98 %) during conidium discharge ranged from large immediate loss of conidium

Accepted 2 July 2009

viability (C. adiaeretus) to little effect (B. ranarum). Primary conidia stored under different

Available online 17 November 2009

combinations of RH (90–98.5 %) and temperature (2–35  C) varied in median survival time

Corresponding editor: Fernando Vega

(ST50 and ST10) from <1 d to >90 d. Conidia of C. osmodes and B. ranarum survived best at 2  C; for other species 10  C was optimal. For samples buried in a Larix plantation, primary

Keywords:

conidia survived 28 d for C. adiaeretus (>81 d for its secondary conidia and microconidia)

Abiotic factors

and 338 d for (B. ranarum). Retrieved mycelium (including resting spores) for all species,

Entomopathogens

renewed growth after at least 81 d. Implications for niche differentiation and trophic

Entomophthorales

modes are discussed.

Niche

ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.

Saprotrophs Survival

Introduction Conidiobolus (subphylum Entomophthoromycotina, order Entomophthorales; James et al. 2006; Hibbett et al. 2007; Humber 2007) and Basidiobolus ranarum, a fungus whose phylogenetic position remains uncertain (Hibbett et al. 2007) have been isolated from a very wide range of soils and litters (King 1976; Humber 1989; Evans 1988; Smith & Callaghan 1987; Coremans-Pelseneer 1974; Nelson et al. 1998). Arthropod pathogens pre-dominate in Entomophthorales. In Conidiobolus some species are obligate pathogens of insects, e.g., C. obscurus, C. gustafssonii, C. destruens; or mites C. chlapowskii (Latge´ et al. 1982; Ba1azy 1993) or they are opportunistic pathogens, e.g., C. coronatus, C. thromboides and C. osmodes (Humber 1989; Ba1azy 1993). B. ranarum usually termed a ‘saprotroph’

sometimes associated with amphibians (Coremans-Pelseneer 1974), is occasionally reported as a pathogen (Hywell-Jones & Gillespie 1986). The Conidiobolus species mentioned above usually seem to be associated with ‘above-ground’ arthropod targets and sometimes cause epizootics. In contrast, more than 20 Conidiobolus species, not implicated as arthropod pathogens, live in sub-surface situations, soil and litter (King 1976; Smith & Callaghan 1987; Humber 1989). The ecology of Conidiobolus species has been relatively little studied and their precise trophic relationships with invertebrates remain unclear. Our previous attempts to describe the niche differentiation of some of these ‘sub-surface’ species emphasised life cycle morphological stages (Waters & Callaghan 1989, 1999; Callaghan et al. 2000), interactions with invertebrate cadavers (Manning et al. 2007) and possible pathogenicity

* Corresponding author. E-mail address: [email protected] (A.A. Callaghan). 1754-5048/$ – see front matter ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2009.06.005

Survival of Conidiobolus spp. and Basidiobolus ranarum

(Manning & Callaghan 2008). These studies used fresh isolates obtained (see Methods) from an ongoing long-term (>20 yr) quantitative sampling of a group of adjacent managed habitats at Keele, Staffordshire, UK, (Nat. Grid. Ref. SJ 821441; Smith, & Callaghan 1987). In the current study, test species were those most frequently disclosed from litter of a larch (Larix) plantation (C. adiaeretus, C. iuxtagenitus, C. lamprauges); from an arable field (C. firmipilleus, C. heterosporus) and those frequently occurring in several habitats (C. coronatus, C. osmodes, C. thromboides and B. ranarum). The larch plantation has, in our surveys, the lowest species diversity for Conidiobolus (consistently 6 species) and is our model target habitat. This small guild of species using arthropods as substrata may give a realistic chance of explaining the coexistence of the Conidiobolus spp. (and B. ranarum), and their interaction with cooccurring arthropods and with other fungi (Mac Nally 1983). The present study concerns the tolerance of test isolates to the abiotic factors, relative humidity (RH) and temperature. A total of 20 isolates comprising the above 8 Conidiobolus species and 3 isolates of B. ranarum were used. We report their differing sensitivities to very brief periods of adverse RH during discharge of conidia and the variation in long-term survival of conidia stored at different combinations of RH and temperature. We also present survival data for whole mycelium (including resting spores) and conidia buried in habitat litter. The primary globose conidia of the fungi tested can, in the absence of nutrients, form a repetitional second generation of similar secondary conidia (and further generations of repetitional conidia not studied here). Additionally, several species can form alternative conidia. Conidiobolus coronatus and C. firmipilleus form microconidia. Basidiobolus ranarum and C. heterosporus form capilliconidia, and C. iuxtagenitus develops actively discharged elongate conidia (ADEC). Conidiobolus adiaeretus has distinctive life history features (Callaghan et al. 2000). It can form clusters of dischargeable microconidia on primary and, more readily, on secondary conidia. These microconidia are distinct from those of other Conidiobolus species and, on each, a capilliconidium can form. Responses of some of these spore variants were recorded. Earlier work (Waters & Callaghan 1999) showed the influence of water potential on the formation of the different conidial types and this informed the choice of RH for habitat burial experiments.

Materials and methods Sources of fungi Test isolates were obtained by canopy methods (Callaghan 2004). For some, the canopy was a filter paper (dia 12.5 cm, pore size 3 mm), bearing a filtered thin wet layer of homogenised litter, adhering to the lid of a Petri dish (dia 14 cm) above malt extract agar (MEA; malt extract, 2 % w/v; Agar No. 3, 1.2 % w/v, Oxoid, Ltd, Basingstoke, UK). Other isolates were obtained by canopying filter papers impregnated with a supernatant, pre-filtered through coarse nylon mesh, from shaken litter suspensions (Callaghan 2004). Each sampling yielded counts of colonies from spore-discharging units (SDU) in the canopies. Each SDU was able to discharge one or more conidia within 3 d before other fungi intruded. Thus, the data

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reflect the relative frequency of fungus loci immediately able to germinate but not the unknown proportion of propagules which may have specific requirements for breaking their dormancy. Isolates from selected colonies were stored at 0–5  C, and maintained by twice-yearly subculture on MEA.

Choice of conidial catch surface Trials in which conidia of all test isolates were showered at 20  C directly onto 2 % MEA through a near-saturated atmosphere, used Petri dish source cultures inverted and sealed rim-to-rim over dish bases of substrates. More than 95 % germinated within 2–4 h by forming a germ tube, at least twice as long as its diameter. Conidia caught on water agar, (WA; Agar No. 3, 1.2 % w/v, Oxoid Ltd, Basingstoke, UK) and transferred immediately by direct touching onto MEA, gave the same rapid, full germination. With glass cover slips (cleaned with 70 % ethanol and thoroughly washed and dried) as catch surface supported on or above WA, germination levels of showered conidia immediately transferred to MEA were usually very low (0–20 %) with an occasional high value (80–100 %). This erratic germination, only partly explained by the presence of condensed water droplets, precluded the use of cover slips as chosen catch surface. In contrast conidia caught on nylon mesh in the humid atmosphere and then transferred to MEA, germinated as normally as those caught directly on MEA. Thus, discs of monofilament nylon mesh, pore size 15 or 20 mm, dia 2.5 cm and thickness ca. 0.2 mm (Saatifil, Sericol Ltd, Broadstairs, Kent,UK) were used in all main experiments on conidial germination.

Effect of initial transient adverse RH Effect on germination of conidia Preliminary experiments indicated that even a brief exposure of freshly discharging primary conidia to air of reduced RH lowered the level of subsequent germination. As an extreme example, conidia showered for 1 min onto nylon mesh, through an atmosphere of RH 10 % (achieved with anhydrous CaCl2), failed to germinate at 20  C even 24 hr after their immediate transfer to MEA. To investigate this early-stage effect separately from that of long-term storage at low humidities, the experimental requirements must include the release of discharging conidia from undisturbed source cultures into a well-conditioned atmosphere for a precise short period of discharge. Immediately after this period, collected conidia need to be rapidly transferred to a suitable growth medium in a saturated atmosphere to test short-term ability to germinate. The procedure adopted used purposedesigned spore shower regulating devices (SRD). Two versions were used. One (SRD1, Fig 1A) was used with individual test isolates. The other (SRD2, Fig 1B) allowed near-simultaneous handling of spore deposits from up to 10 test isolates. For initial experiments source cultures were pre-grown on MEA for 3 d in Petri dishes (9 cm diam for SRD1 and 3 cm diam for SRD2). Each dish was inoculated at several points and inverted, with lid removed, over the aperture (6 cm diam, SRD1 and 2.5 cm, SRD2) in the plastic cover of the humidity chamber (Fig 1). Experiments were conducted at 20  1  C, and underlighting was by fluorescent white lamps. Initially the slider closed the hole; rubber gaskets, silicone grease and

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drops of a 20 % v/v solution of methanal (formaldehyde). Subsequently conidial deposits were scored for % germination. In experiments in which up to 9 different species were manipulated in parallel (SRD2) the plastic support for the showered discs had a central handle allowing simultaneous removal of all nylon discs. At 2, 4, 6 or 8 hr the replicate sets of MEA deposits were killed and subsequently scored for germination.

Effect on viability of conidia

Fig 1 – Side views of spore regulating devices (SRDs) which allow controlled short periods of catch as primary conidia discharge into an atmosphere of conditioned RH. (A) collects conidia from a single Petri dish source culture. (B) collects conidia simultaneously from up to 10 source cultures of different species.

In some short-term experiments nylon discs were included alongside the discs eventually used to estimate % germination. Immediately after showering, these extra discs were temporarily stored in pre-equilibrated Petri dishes lined with wet filter paper (near-saturated atmosphere) and placed in a refrigerator to slow down spore activity. From one disc at a time, conidia were gently removed aseptically (using a fine paint brush) into a drop of mixed stains, fluorescein diacetate (FDA) and propidium iodide (PI). These were prepared and used according to Firstencel et al. (1990). Stained deposits were viewed on a Nikon Microphot FX photomicroscope with epifluorescence attachment and excitation filter 330–380 nm and barrier filter 420 nm. Trials confirmed that conidia freshly caught from sources, when treated as above, fluoresced green. However, those given brief final contact with methanal vapour, fluoresced red. Thus with the deposits from the extra nylon discs, conidia were quickly scored as ‘living’ (yellow/ green fluorescence) or ‘dead’ (red fluorescence) making it possible to compare the proportion of conidia viable immediately after discharge with the proportion eventually showing the ability to germinate after several hours of incubation.

Effect of long-term storage of conidia in different combinations of temperature and RH a mixture of petroleum jelly and candle wax (vaspar) ensured well sealed containers. In most experiments using SRD1, WA containing appropriate proportions of glycerol and water conditioned the RH in the chamber (Dallyn & Fox 1980; Scott 1957); in all other experiments, particularly using SRD2, a liquid mixture of glycerol and water was used. The nylon conidium-catching discs were supported just above the conditioning agar or solution by a metal washer or a plastic frame (Fig 1). Separate detailed monitoring of changes of humidity with time (Electronic Temperature Instruments Ltd, Worthing, W. Sussex, UK) showed that the growth period of 3 d was ample time to allow equilibration of RH (ERH) in the chambers. Conidium formation and discharge in the source dishes were optimal on day 3. To give discharging conidia a brief exposure to atmospheres of different ERH, the slider was opened for chosen periods (1–15 min). This allowed a shower of primary conidia to accumulate on each disc. Three replicates of the apparatus were used for each ERH value and discharge period. After each period of spore catch, each nylon disc was immediately and aseptically placed spore-side downwards onto MEA in a Petri dish, then removed, leaving conidia on the culture medium. Subsequent incubation (at RH ca. 100 %) was in the same conditions as for growth of source cultures. In experiments on single isolates, conidia and germlings were killed after 8–10 h by vapour from

Experimental set up To separate the early effects of adverse RH during discharge and collection of conidia at 20  C from effects due to subsequent storage in various combinations of RH and temperature, the initial deposits of conidia on nylon discs were obtained in a saturated atmosphere provided by deionised water in the base dish of multiple copies of SRD2. Pre-inoculated Petri dish source cultures were incubated on top of each container (9 species simultaneously) as indicated earlier. On day 3 the nylon discs were showered with primary conidia for 30 min. Immediately, each set of 9 discs on the removable support was quickly transferred to a similar container which had a preconditioned atmosphere of the required ERH. The containers were sealed and placed at the required temperature, in the dark, for storage until retrieval time. Overall, temperatures of 2, 10, 20 and 35  C (all 1  C) were used in combination with ERH of 90, 94, 96, 98 and 98.5 %. For each combination, 15 replicate containers were stored. Retrieved apparatus used in early samplings was reconstituted for subsequent longer-term storage. This allowed, as a typical pattern with slight variation, the removal of 2 replicates after 1 d, 3 after 3 d, 5 after 7 d, 3 after 28 d and 1 after 90 d. These replicate numbers were partly dictated by availability of adequately controlled storage space. No attempt was made to monitor RH. It is recognised that at the

Survival of Conidiobolus spp. and Basidiobolus ranarum

different temperatures actual values of RH would differ from the nominal stated values (Johnson 1940). Glycerol/water mixtures were chosen for being minimally affected by temperature variation (Grover & Nicol 1940). At each time of retrieval of a set of nylon discs (9 species handled simultaneously) individual discs were processed as described above; an 8 h incubation period on MEA was allowed before germination was assessed. Number of conidia scored per replicate was usually between 200 and 800.

Determination of survival coefficients For each combination of storage temperature and RH a time course of germination was plotted using the mean % germination from each sampling. This gave a preliminary indication of the storage time leading to 50 % and 10 % germination. It also acted as a check on the feasibility of predicting these values from the data. Next, probit-transformed % germinations of all replicates were plotted against log10 days. In most cases this linearised the germination-time relationship (with a significant regression, usually p < 0.01), and allowed the interpolation of storage time corresponding to 50 % germination (median survival time, ST50) and 10 % (ST10). For each estimate of ST50 and ST10, confidence limits (CL) were determined by a modification of Wardlaw (1987). Thus, standard error of the mean (SEM) of log ST50 ¼ 1/bO5 nw; where, for the 5 sampling times, b ¼ slope of probit/logdays line, n ¼ mean number of conidia scored per replicate and w ¼ mean weighting coefficient associated with the fitted probits (Wardlaw 1987). Approximate 95 % CL were then estimated as ST50  1.96 SEM with eventual return to ‘days’ via antilogs. For clarity they are omitted from Fig 6 but were used to assess the significance of generalisations derived from the bar charts. Where an ST estimate was >90 d, and thus extrapolated beyond the regression line, the bar is stopped at nominal value ‘90 d’.

Burial of conidial deposits and entire mycelia in habitat litter Deposits of primary conidia, secondary conidia (sometimes with small numbers of alternative conidia) and entire mycelium (including resting spores and conidia) were buried in 25-well repli dishes sealed with aluminised tape (at a depth of ca. 5 cm) in the litter of a larch plantation. Eight Conidiobolus species and 2 isolates of B. ranarum were treated together in the same repli dishes. The atmosphere in the containers was pre-conditioned (at 20  C) at either 97.5 or 96 % RH as nominal initial values, though these would probably have fluctuated to slightly higher values (probably up to ca. 97 and 98.5 %) (Grover & Nicol 1940). Values below 98 % RH were used to preclude additional formation of alternative conidial types in the sealed containers (see the Introduction) and to allow for the likely slight rise of RH at the initially low winter temperatures. The temperature in the litter was monitored using Tinytalk data loggers (Orion Components, Chichester, UK; see Supplementary Fig).

Preparation of burial dishes For each of 7 planned retrievals, a set (block) of 5 differently prepared 25-well repli dishes was used. In each set one dish, eventually to be buried as the ‘whole mycelium’ sample,

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contained MEA. Ten pairs of its wells were each inoculated with one of the 8 Conidiobolus species and 2 isolates of B. ranarum. Incubation for 3–4 d at 20  C, inverted over lights prepared this dish as the source for both primary and secondary conidia for all 10 test isolates simultaneously. The other repli dishes, eventually to enclose conidium deposits on washer-supported nylon discs, contained glycerol/water agars. Two contained 12.1 % w/w glycerol and were pre-equilibrated at 20  C to 97.5 % RH. The other two contained 15.5 % w/w glycerol and were pre-equilibrated at 20  C to 96 % RH.

Preparation and insertion of conidial deposits For deposits of primary conidia, the 3 d old repli dish source culture was inverted over a repli dish containing nylon discs resting on WA. At 20  C and with underlighting, conidia were allowed to shower for 1.5 h. Discs were then immediately aseptically transferred onto supporting washers in the wells of one of the pre-conditioned 97.5 % RH repli dishes. The conidia-showering procedure was repeated to place nylon discs bearing primary conidia in one of the pre-conditioned 96 % RH dishes. To set up deposits of secondary conidia on nylon discs an extra step was included in the showering process. The now 4 d old repli dish source culture was inverted over a repli dish containing only WA. Primary conidia were showered for 2.5 h. The showered dish was then inverted over a matching repli dish containing nylon discs on water agar and canopied for 6 h. Thus, deposits of secondary repetitional conidia accumulated. The nylon discs were quickly transferred to supporting washers in the second pre-conditioned 97.5 % RH dish. To put disc deposits of secondary conidia in the remaining pre-conditioned 96 % RH repli dish, the whole 2-stage procedure was repeated using the repli dish source again after several hours of ‘recovery’ time to allow a further crop of primary conidia to form. As mentioned above, the 5th dish of each block was this original source, ready for burial on day 5. As they were being prepared, all dishes were thoroughly sealed with aluminised adhesive tape and together stored overnight in a refrigerator until all 7 blocks were ready for burial. Six blocks were buried close together with individual dishes at a depth of ca. 5 cm. The remaining block was used to determine germination and outgrowth responses at the start (0 d). One block each was retrieved after 2, 6, 28, 43, 81 and 338 d. For dishes containing conidial deposits on nylon discs, the latter were removed and their conidia were printed onto MEA. After 8 h incubation at 20  C (and follow up checks) counts of germinating spores were made. From the ‘entire mycelium’ dish 10 small cubes were taken from the two wells of each test isolate and used to inoculate MEA. They were observed for hyphal outgrowth and evidence of freshly discharged conidia nearby after several days of incubation at 20  C. In parallel with the main experiments, mycelium of test isolates was grown on nylon mesh discs in contact with MEA. The discs were stored as above on support washers in repli dishes of pre-conditioned RH buried in the habitat. On retrieval they gave additional evidence of the ability to form fresh conidia and to grow hyphae from the mycelium. They were retrieved after burial for 22, 37, 50 and 75 d.

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Results 100

C. adiaeretus

Initial effects of adverse RH during conidial discharge 80 60 40 20

0

0 1 (100)

1 (90)

5 (90)

10 (90)

20 (100)

Fig 3 – Responses of primary conidia of C. adiaeretus and B. ranarum to lowered RH during discharge and catch. Germination on MEA, mean (%) ± 1 SE, following different periods of exposure to 90 % RH then incubation for 8 h at 20 8C and 100 % RH.

a delayed rise (to 67 % germination) at 8 h. The control, showered in a saturated atmosphere, rose to 100 % germination after only 4 h. For B. ranarum (Fig 4B) conidial germination was delayed rather than suppressed by the initial catch

A 100

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Fig 2 – Germination of primary conidia of 8 different Conidiobolus species and B. ranarum simultaneously discharged and caught for 15 min in atmospheres of 6 different RH levels, then incubated for 10 h at 20 8C and 100 % RH. Key to abbreviations of named test fungi: (adi) C. adiaeretus; (cor) C. coronatus; (het) C. heterosporus; (iux) C. iuxtagenitus; (fir) C. firmipilleus; (lam) C. lamprauges; (osm) C. osmodes; (thr) C. thromboides; (Br) B. ranarum. Species are displayed in order of increasing tolerance.

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In simultaneous testing (Fig 2), germination after 10 h, following 15 min discharge through different pre-conditioned atmospheres, varied with test RH and species. When showered through saturated air, all species showed >98 % germination. For germination after 94–98 % RH treatments, five species, C. firmipilleus, C. lamprauges, C. osmodes, C. thromboides and B. ranarum, retained high germination (78–100 %). Species most affected by 94–98 % RH were C. adiaeretus, C. coronatus, and C. heterosporus. C. iuxtagenitus also gave low germination after 94 % RH but was little affected by 96–98 % RH. Discharge through air of 90 and 92 % RH affected subsequent germination the most and showed the greatest differences between species. Most affected were C. adiaeretus, C. coronatus, C. heterosporus and C. iuxtagenitus. Increasing tolerance was shown by the other fungi from C. firmipilleus through to C. thromboides. Only B. ranarum was little affected (85 and 83 % germination) after discharge at 90 and 92 % RH. The effect of different periods of lowered RH during discharge was investigated for several species individually (Fig 3). The most sensitive, C. adiaeretus, had low germination (0–12 %) after discharge at 90 % RH for periods of 1, 5, 10 and 20 min; controls at 100 % RH for 1 and 20 min gave 72 and 77 %. In contrast, for B. ranarum (Fig 3), conidial germination was less depressed (18–55 %); controls for 1 and 20 min gave 96 and 97 % germination. For both fungi, levels for the different adverse exposure periods were not significantly different ( p > 0.05). For these two fungi of contrasting extremes, timecourses of germination (Fig 4A, B) were very different. For C. adiaeretus (Fig 4A) initially exposed to 90, 92, 94, or 96 % RH, germination remained at low levels (0–16 %); curves showed no tendency to rise. Initial conidial catch at 98 % RH showed

Germination (%)

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Fig 4 – Time-courses of primary conidia germination on MEA at 20 8C and 100 % RH after initial 15 min discharge and catch in atmospheres of different adverse RH. (A) C. adiaeretus; (B) B. ranarum.

Survival of Conidiobolus spp. and Basidiobolus ranarum

conditions. After 4 h incubation, conidia caught at 94, 96, 98 and 100 % RH germinated at similar high levels (95–100 %). After initial catch at 90 or 92 % RH, germination reached slightly lower levels at 4 h (73, 81 %) with a slight rise at 8 h (86, 87 %). Conidia of all species, as expected, retained their viability (92–100 %) revealed by fluorescent staining, when discharged through saturated air. With test RH of 80, 90 or 96 %, viability of C. adiaeretus conidia dropped markedly (22.7, 33.9 and 8.8 % respectively). Values for B. ranarum remained much higher (63.5, 86.7 and 93 %). The other examples (Fig 5), C. iuxtagenitus and C. thromboides were intermediate in their loss of conidial viability at 90 or 96 % RH. When RH was 80 %, three species showed great loss of viability (down to 22.7–29.3 %). Again, B. ranarum was least affected; discharge at 80 % RH reduced its conidial viability to 63.5 %.

Effect of storage at different combinations of temperature and humidity Estimates for the 50 % and 10 % survival times of primary conidia stored at 2  C and at 5 different levels of RH are presented as 3-dimensional bar charts (Fig 6A, ST50; 6B, ST10); with corresponding plots for 10  C (Fig 6C, D) and for 20  C (Fig 6E, F). Confidence limits are omitted for clarity. Survival times at 35  C were particularly low (data not shown). At this temperature, for all humidities (90–98.5% RH), nearly all ST50 and ST10 were: 1 d. The ST10 exceptions were, at 98 % RH, C. firmipilleus and C. lamprauges 2 d, C. thromboides 3.4 d; at 98.5 % RH, C. firmipilleus 3 d, C. lamprauges 5 d, C. thromboides 7 d and B. ranarum 2 d. At each of the other temperatures the pattern of ST50 values (Fig 6A, C, E) was, predictably, reflected in the similar pattern of the larger values of ST10 (Fig 6B, D, F). However, at highest RH, 98 and 98.5 %, ST10 were so large at 2 or 10  C (Fig 6B, D) that the bar charts arbitrarily stop at 90 d (the last day of sampling) to avoid ambiguities of extrapolation outside

C. adiaeretus

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C. iuxtagenitus

Viability (%)

B. ranarum

80

C. thromboides

60 40 20 0 80

90 96 100 RH % during 5 min conidial catch

Fig 5 – Viability of primary conidia of four test fungi, immediately after 5 min discharge and catch in atmospheres of different RH. Viability (%) was determined by differential fluorescent staining of conidia by propidium iodide and fluorescein diacetate (see text). For each sampling a single determination was done on deposits of 200–300 conidia.

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the regression used for estimates. For 20  C (Fig 6E, F) conidial survival was markedly poorer than at 2 or 10  C. An exception to this may be C. thromboides with an ST10 of >90 d at all temperatures (Fig 6B, D, F). A different, more specific, response to temperature was shown by C. osmodes and B. ranarum (Fig 7): at 2  C at 98 % RH (and at 98.5 % RH), values of ST50 and ST10, were significantly higher ( p < 0.05) than values at 10 or 20  C. Although at 98 % RH conidia of C. thromboides and C. coronatus showed similar trends for their ST50 values (Fig 7), at 98.5 % RH, the ST50 was higher at 10  C than at 2  C (C. thromboides) and, for C. coronatus, estimates were >90 d. Also, for both these fungi, all ST10 values were estimates >90 d. These features prevent any firm conclusions about temperature preferences for these species. Conidia survived briefly when stored at 90 % RH; all ST50 and ST10 were 1 d (Fig 6). Again, at 94 % RH, ST50 were 1 d (except C. thromboides 2 d) and ST10 were all 2 d (except C. firmipilleus and C. thromboides, 6 d at 2 and 10  C). The conidia of most species tolerated storage at an RH of 96 % (Table 1) slightly better than at 90 or 94 %. The most affected were C. adiaeretus, C. heterosporus and C. coronatus. Comparison of conidial survival times at 96 % RH with those at 98 or 98.5 % RH (Fig 6A, C, E) clearly separated the relatively sensitive species from the more tolerant ones. For 4 species, C. adiaeretus, C. firmipilleus, C. iuxtagenitus and C. heterosporus, although almost all their ST50 values for 98 or 98.5 % RH at 2 and 10  C were significantly larger ( p < 0.05) than corresponding values for 96 % RH (Fig 6A, C), conidia were much shorter-lived compared with those of other species at 98 or 98.5 % RH (Fig 6A, C). The ST10 values of C. adiaeretus and C. firmipilleus (Fig 6B, D) remained the smallest; ST10 values for C. iuxtagenitus and C. heterosporus become comparable with those of the others.

Effect of burial in larch plantation In containers with the initial RH of 96 %, primary conidia of test Conidiobolus species remained capable of germinating for ca. 6 d (4 species) or >28 d (3 species) (Table 2). Conidia of B. ranarum (2 isolates) were much longer-lived, >81 or >338 d. At the rather higher initial humidity, RH 97.5 %, primary conidia survived longer; at least 28 d for C. adiaeretus and C. osmodes; >43 d for C. iuxtagenitus and >81 d for 4 species. Again B. ranarum conidia remained viable the longest, >81 and >338 d. Conidiobolus coronatus primary conidia were conspicuously short-lived, 2 d at both humidities. Secondary conidia of nearly all test species at initial RH of 96 %, were distinctly shorter-lived than primary spores; for C. lamprauges survival was equally brief, >6 d. At initial 97.5 % RH, secondary conidia had equal or shorter longevity than primary conidia. Only for C. adiaeretus were secondary conidia and microconidia (which were present in the initial deposits at the start of the experiment) distinctly more tolerant than primary conidia (>81 d compared with >28 d). At initial RH of 97.5 % after burial for 81 d some secondary conidia and microconidia of C. adiaeretus were able to germinate (Table 3). The present study shows survival times >90 d for C. coronatus conidia during the storage experiment but, ambiguously, very limited survival in the buried, sealed containers (Table 2). The pronounced loss of viability after brief adverse RH (Fig 2) may show the initial vulnerability of the conidia before they change into more

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98.5 98 96 RH % 94 90

adi fir

cor osm thr Br iux het lam

100 ST 80 10 60 (days) 40 20 0

Fig 6 – Estimates of survival times (ST) for conidia of 8 Conidiobolus spp. and B. ranarum. Conidia were stored at 2 8C (A,B), 10 8C (C,D) or 20 8C (E,F) in atmospheres of 5 different RH; A,C,E are ST50 and B,D,F are ST10 values; 95 % confidence limits are omitted. Bars of 90 d indicate an arbitrary minimum avoiding extrapolation beyond regression lines (see text). Key to abbreviations of named test fungi; (adi) C. adiaeretus; (fir) C. firmipilleus; (iux) C. iuxtagenitus; (het) C. heterosporus; (lam) C. lamprauges; (osm) C. osmodes; (thr) C. thromboides; (Br) B. ranarum; (cor) C. coronatus.

resistant villose (lorico-) conidia. Conceivably, in the initial preparation of buried dishes, the conidia reacted adversely to the initial low pre-conditioned atmosphere during overnight storage in the refrigerator. On day 0 (burial of dishes) germination of C. coronatus primary conidia was 24 % (96 % RH) and 10.7 % (97.5 % RH); these values are out of line with the other species. Intact mycelium/resting spores showed renewed growth following burial (Table 2). At 96 % RH this ceased after ca. 6 d for C. adiaeretus, after 28 d (3 species) or 43 d (C. thromboides) and after 81 d for 5 species including C. iuxtagenitus. At 97.5 % RH all test species, including C. adiaeretus, still showed fresh growth and produced fresh conidia after 81 d.

Discussion The context of the current study is the examination of those features of Conidiobolus spp. and B. ranarum which facilitate their coexistence (niche differentiation), their trophic relationships with arthropods, and their survival in habitat. The recognition that conidial survival was affected by brief adverse RH during discharge poses particular difficulties of interpretation and, for the most affected fungi, a positive

contribution to their fitness is difficult to discern. The subsequent effect (loss of viability or, at least, inability to germinate) was independent of the period of exposure, suggesting that the reduced ambient RH acts just before the moment of release of the discharging conidia. We note only one other report (Griggs et al. 1999) of a rapid effect of adverse humidity during discharge; the viability of primary conidia of Zoophthora radicans dropped to ca. 5 % after 5 min discharge into air of RH 60 %. However, in contrast with the present study, incubation after discharge was in the same adverse RH as for discharge. Our findings suggest that rapid surface changes are involved in loss of viability. Some reports discuss enzymes associated with discharge (Phadatere et al. 1989) and ultrastructural changes (Eilenberg et al. 1995) but none discuss rapid surface change. The larch litter species differed considerably in the extent of their response to initial adverse RH and to more prolonged exposure to lowered RH (storage), following normal discharge through saturated air. C. adiaeretus primary conidia were the most sensitive and had the shortest survival times. The chlamydospores of this fungus, enhanced in numbers by low temperatures (Drechsler 1953), presumably provide longerterm survival. In the present study secondary repetitional conidia and microconidia of C. adiaeretus survived much longer

Survival of Conidiobolus spp. and Basidiobolus ranarum

ST50 (days) + 95% CL

100

+

o

Temperature C

80

2

10

20

60 40 20 0 C.osmodes

B.ranarum

C.thromboides C.coronatus Fungi stored at RH 98%

Fig 7 – Survival times (ST50) for primary conidia of C. osmodes, B. ranarum, C. thromboides and C. coronatus when stored at 2, 10 and 20 in atmospheres of 98 % RH. The ‘D’ above the bar of value 90 d denotes an arbitrary minimum avoiding extrapolation beyond a regression line (see text). Survival at 35 8C was <1d.

than primary conidia when buried in sealed containers in the larch litter. This links with survey findings (I J Hopkins, unpubl.) that C. adiaeretus is the Conidiobolus most frequently detected from larch litter at all times of year except midsummer. The greater tolerance of conidia of C. iuxtagenitus and, particularly of C. lamprauges, to reduced RH, e.g. at 96 % RH (Table 1), contrasts with C. adiaeretus. Conidiobolus iuxtagenitus conidia with optimal survival at 10  C, develops ADECs and has zygospores as resting spores (Waters & Callaghan 1989). Conidia of C. lamprauges were long-lived at all test temperatures except 35  C. This species has no reported alternative conidia but has zygospores. For these three species there seems little commonality of life history features or responses to RH and temperature and little to indicate why they consistently cooccur and predominate in larch litter. The two test species most frequently isolated from soil of an arable field (winter wheat) adjacent to the larch plantation, C. firmipilleus and C. heterosporus, had conidia with survival times intermediate

155

between those of C. adiaeretus and the longer-lived C. lamprauges (and the other four longer-lived species below). Resting spores for these species are chlamydospores for C. firmipilleus and zygospores for C. heterosporus. Again, reported features give little insight into their co-occurrence in the particular field habitat. Conidia of the remaining four test species (C. thromboides, C. coronatus, C. osmodes and B. ranarum), the widely occurring opportunistic arthropod pathogens, all show the longest survival times. C. osmodes and B. ranarum conidia have optimal survival at 2  C, tying in with their widespread occurrence in the cool temperate zone; tropical strains are presumably more likely to have higher temperature optima. The ability of mycelia to persist for >81 d at the temperatures prevailing in the larch habitat at RH < 100 % is matched by the ability of conidia of the more tolerant species (those mentioned above and C. lamprauges) to survive for >90 d in humid air. Resting spores are usually the agents of long-term survival in a particular habitat (Hajek 1997), so the long-lived conidia may allow aerial transfer to other habitats, although their intolerance to RH < 98 % counters this. Conidia are more likely to provide a reservoir from which immediate germination can occur at any time of the year when potential arthropod vectors/substrata are available. The interaction with arthropod vectors, some of which may be eventual cadaver substrata or potential targets for pathogen attack, also suggests the importance of even a short survival period for primary conidia in litter, particularly at RH < 98 %. Thus primary conidia, even of the sensitive C. adiaeretus, are probably essential for eventual contact and adherence to arthropod external surfaces. They could withstand low humidities for hours or days before the animal died, or until they form secondary repetitional or alternative conidia. For species not arthropod pathogens, this may well be important. However, even when air humidity is low, there could often be quite high humidity in cavities within the litter (e.g. Coleman et al. 2004). Earlier reports on C. coronatus and C. thromboides (Yendol 1968) showed that an RH of 95–100 % was needed for germination of primary conidia. Stimmon (1968) showed that the infectivity of C. coronatus primary conidia for symphylans was retained for at least 30 d when stored on plaster/charcoal.

Table 1 – Estimates (days) of survival times, ST50 and ST10, for primary conidia of 9 test species listed in approximate order of increasing tolerance stored at 96 % RH and at 2, 10 or 20 8C. SEM given in parentheses except for small survival values Storage temperature (oC) 2 Test fungi C. thromboides C. lamprauges C. firmipilleus C. iuxtagenitus B. ranarum C. osmodes C. coronatus C. heterosporus C. adiaeretus

10

20

ST50

ST10

ST50

ST10

8 (0.16) 2 5.6 (0.2) 1.8 1.7 2 <1 1 <1

26 (0.52) 11 (0.22) 14.5 (0.45) 6.7 (0.3) 6 (0.17) 7 (0.14) 1 3 2

5 (0.1) 4 (0.1) 5.6 (0.2) 1.7 ()b 3 <1a 1 <1

17 (0.3) 18 15.8 (0.45) 6.6 (0.3) (30)a 5 (0.1) 2a 4 (0.08) 3

a From time course; regression not significant. b Erratic time course; regression not significant.

ST50 3 1 1.4 2.5 <1 2 3 1 <1

ST10 6 (0.1) 6 (0.12) 5.6 (0.25) 6.4 (0.25) 7 (0.14) 3 9 (0.18) 2 2

For all test fungi renewed growth still occurred after 81 days 6.1 0.7 61.1 2.8 1.2 13.2 4.8 10.4 4.6 41.3 81 43 6 28 81 28 6 28 338 81

Mycelium 2 y conidia

Retrieval day and germination (%)

14.0 6.4 0.9 11.7 3.5 5.7 7.0 7.4 16.5 8.3 28 43 81 81 81 28 2 81 338 338 a See Supplementary Fig to locate retrieval day in overall burial sequence. b Isolate from larch litter. c Isolate from grazed pasture.

6 81 28 28 81 28 81 43 81 81 6 6 28 28 6 6 <2 28 338 81 C. adiaeretus C . iuxtagenitus C. heterosporus C. firmipilleus C. lamprauges C. osmodes C. coronatus C. thromboides B. ranarum (04)b B. ranarum (01)c

1 y conidia Test fungi

21.9 77.8 2.6 6.3 41.5 40.7 0 6.9 <1.0 3.2

6 6 6 6 6 6 2 2 43 6

2.0 11.1 4.0 15.6 42.7 7.8 15.0 57.1 3.1 52.0

Mycelium 2 y conidia

Last day to regrowth Retrieval day and germination (%)

a

96 %

1 y conidia

a

97.5 %

Last day to regrowth

I.J. Hopkins, A.A. Callaghan

Nominal initial conditioned RH

Table 2 – Survival of primary (1 y), secondary (2 y) conidia and mycelium of 9 test species buried in litter of a larch (Larix) plantation. Values are, for conidia, number of days to the last demonstration of the ability to germinate and % germination; and, for mycelium, the number of days to the last demonstration of regrowth

156

Conidia of C. coronatus in the present study showed the same RH threshold and were long-lived (>90 d) although they rapidly lost viability in the burial experiment. Conidia of C. obscurus, an obligate entomopathogen had a higher infectivity for aphids when stored on soil at 5  C (mortality 75 % after 60 d) than when stored at 10 or 20  C (Latteur 1980). The responses to RH and temperature by the arthropod pathogens comprising most of the Entomophthorales (sensu lato) have been reviewed, often in the context of potential use of the fungi as biological control agents (Keller & Zimmerman 1989; Hajek & St. Leger 1994; Hajek 1997; Pell et al. 2001; Steinkraus 2006). The diversity of habitat and geographical area are reflected in the diversity of findings. The emphasis is on aboveground, foliar pathogens in contrast to the predominantly soil/ litter habitats of most Conidiobolus species and B. ranarum. Nevertheless, several comparisons are interesting. Many of the arthropod pathogens in the Entomophthorales have, like all the current test fungi, a threshold of relative humidity, often ca. 90– 95 %, below which their primary conidia fail to germinate. Examples include Entomophthora (Batkoa) apiculatus (Yendol 1968), Entomophthora (Pandora) gammae (Newman & Carner 1975), Entomophthora (Pandora) delphasis (Shimazu 1977) and many species of Zoophthora and Neozygites in which primary conidia germinate to form capilliconidia. These include: Zoophthora phalloides with a 98 % RH threshold (Glare et al. 1986); N. fresenii, ca. 85 % RH threshold at 25  C (Steinkraus & Slaymaker, 1994); and N. floridana >95 % RH threshold (Oduor et al. 1996a). The latter contrasts with the ability of the capilliconidia to germinate at lower humidities (Oduor et al. 1996b). In the above examples, primary conidia were maintained at test humidities and examined directly for germination over a short period, usually hours, occasionally days. In other work, cited below, closer to the approach in the current study, conidia were stored at test RH then removed at intervals and placed in conditions conducive to germination or host infection. These reports of the retention of ability of primary conidia to germinate (persistence), relate the duration of survival to various factors such as temperature, relative humidity, soil moisture, the nature of the surface and whether the storage was laboratory controlled or in habitat. These various influences are illustrated by the behaviour of primary conidia of Pandora neoaphidis. These conidia retained some infectivity for aphids up to14 d in the field (Brobyn et al. 1985). In controlled storage on leaf or glass surfaces survival was better at lower RH; even at 50 % RH some infectivity persisted (Brobyn et al. 1987). Storage of primary conidia on moist soil for up to 96 d showed the longest survival at 5  C with conidia still infective after 63 d and still forming replicate conidia after 96 d. (Neilsen et al. 2003). A detailed laboratory and field study (Baverstock et al. 2007) confirmed that primary conidia stored on soil still had some infectivity after 80 d. For a contrasting species, Z. radicans, Uziel & Kenneth (1991) emphasised the shorter persistance of primary conidia at low humidity compared with the capilliconidia. Thus, after storage for 1 week on cellophane surfaces at 50 % RH, germination of primary conidia had dropped to zero whereas, even after 5 weeks, capilliconidia still showed 19 % germination. If the above timings do relate to the situation in habitat they presumably reflect the adaptation of the pathogen to the timing of particular target arthropods.

Survival of Conidiobolus spp. and Basidiobolus ranarum

157

Table 3 – Survival following burial of C. adiaeretus microconidia and secondary conidia (2 y) compared with primary conidia (1 y). The microconidia were part of the initial deposit of secondary conidia prepared for burial (see Methods). The test % germination are given for each retrieval day Retrieval day 0 (Initial conditioned RH 96 %) 76.3 Microconidiab 1 y conidia 96.0 2 y conidia 57.4

a

2

6

28

43

81

338

186 249 343

65.8 0 26.5

38 196 49

6.0 21.9 2.0

32 224 295

0 0 0

73 190 131

0 0 0

77 110 148

0 0 0

28 200 65

0 0 0

0 0 0

(Initial conditioned RH 97.5 %) 51.2 168 Microconidiab 1 y conidia 89.6 384 2 y conidia 42.7 227

65.2 30.1 57.0

138 226 151

92.9 8.9 41.2

168 304 161

21.4 14.0 13.1

294 236 237

12.3 0 3.5

163 109 311

3.0 0 6.1

132 122 114

0 0 0

0 0 0

a Day zero control; no burial. b Callaghan et al. 2000.

Survival times of primary conidia of our test species show some important differences. Five test species have conidia which have quite long survival times (>90 d) when stored at RH 98 %. A tentative consideration of extrapolated values beyond the strict limits of the regressions used suggests possible survival times of 150–250 d, and for B. ranarum direct evidence gives >300 d (Table 2). These are longer than times for some of the arthropod pathogens (Hajek 1997), for example Z. radicans (Furlong & Pell 1997), Entomophthora schizophorae (Kalsbeek et al. 2001) and P. neoaphidis (Neilsen et al. 2003) but they do depend on relatively high humidities (>96 %). Retention of the ability to renew growth after at least 81 d buried in sealed containers in larch litter coupled with the general temperature preference for 10  C (2  C for B. ranarum and C. osmodes) reflects their occurrence in soil or litter of a cool temperate area. The different patterns of survival for the test Conidiobolus spp. and B. ranarum may reflect trophic links with different types of arthropod targets. The five species with long conidial survival times (C. lamprauges, C. osmodes, C. coronatus, C. thromboides and B. ranarum) may be generalist saprotrophs of arthropod cadavers. The primary conidia would potentially be available much of the year to germinate on or near a wide range of vectors/potential substrata. Their life cycle would not be tied to the season of any one target arthropod. If some of the co-occurring arthropods are stressed, this could favour an opportunist attack. A previous finding showing that C. coronatus (and possibly C. thromboides and C. osmodes) could kill some co-occurring arthropods may support this (Manning & Callaghan 2008). Conidiobolus thromboides, C. osmodes and C. lamprauges have no alternative conidia. The microconidia of C. coronatus, scattered by discharge, could give extra chance of contacting potential target arthropods especially if they aggregate, and, in this apparently asexual fungus, the portioning out of nuclei during formation of microconidia may be a mechanism for genetic variation. Basidiobolus ranarum has large capilliconidia, able to be widely deployed on plant fragments (Manning et al. 2007) as contact points for mobile vectors. Of the remaining 4 test species, C. adiaeretus is likely to be a quite specialised saprotroph adapted to a particular arthropod (or nematode) vector/substratum, possibly one

which aggregates and is present much of the year. Its primary conidia are intolerant of low RH and have relatively short survival times even at RH 98 % but its secondary replicative conidia and microconidia survived longer in habitat burial. It will be of interest to know the tolerance to low RH of the capilliconidia which form on the microconidia. Conidiobolus heterosporus and Conidiobolus firmipilleus, with their short-lived primary conidia, could be linked with specific seasonal arthropods in the field soil; the former, exploiting capilliconidia to contact mobile vectors, and the latter using its microconidia as in C. coronatus (its resting spores are asexual chlamydospores). Finally, C. iuxtagenitus may be a relatively specialised saprotroph for particular types of arthropod occurring near to the litter surface. Its actively discharged elongate conidia, also seen on larch litter fragments (Manning et al. 2007), are triggered and directed by light (Waters & Callaghan 1989) and could contact mobile vectors. Ongoing research is testing the above suggestions by: correlating arthropod profiles with those of Conidiobolus and Basidiobolus; by analysis of arthropod visitors to fungal baits; and by direct disclosure of fungi carried on mobile arthropods.

Acknowledgements We thank Mrs. Audra Jones and the other biology laboratory staff (Faculty of Sciences) for their continued support. We also thank referees for useful comments.

Supplementary material Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.funeco.2009.06.005

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