The growth response of some Chytridiomycota to temperatures commonly observed in the soil

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1 Mycol. Res. 109 (6): (June 2005). f The British Mycological Society 717 doi: /s Printed in the United Kingdom. The growth response of some Chytridiomycota to temperatures commonly observed in the soil Frank H. GLEASON 1 *, Peter M. LETCHER 2, Zoe COMMANDEUR 1, Cho Eun JEONG 1 and Peter A. MCGEE 1 1 School of Biological Sciences A12, University of Sydney, 2006 Australia. 2 Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA. frankjanet@oz .com.au Received 17 February 2004; accepted 1 December Chytridiomycota were isolated into pure culture from cool temperate and warm semi-arid soils of eastern Australia. In pure culture these fungi responded variably to the range of temperatures commonly recorded in their environment. All members of the Blastocladiales, Spizellomycetales and Chytridiales grew in culture at temperatures up to 30 xc. Some isolates from the Blastocladiales and Spizellomycetales continued to grow at or above 37 x. Some isolates of the Chytridiales grew up to but not beyond 35 x. All isolates in the Chytridiales were able to resume growth at 20 x after brief exposure to temperatures higher than the maximum growth temperature, but were killed by exposure to higher temperatures for 7 d. Because in the natural soil habitat temperature may exceed the maximum for growth it may be a limiting factor that determines the distribution of chytrids in the soil. INTRODUCTION The majority of fungi grow between 5 and 35 xc (Dix & Webster 1995). Some fungi tolerate higher temperatures (Emerson 1968, Gleason 1971, Tansey & Jack 1976, Dix & Webster 1995), and thermophilic fungi may continue to grow at up to 62 x (Brock 1978). Autoecological studies have resulted in lists of thermophilic and thermotolerant fungi found in compost (Miller 1993) and sun heated soils (Tansey & Jack 1976). Though the fungi listed in such studies are from many taxonomic groups, members of the Chytridiomycota (chytrids) are absent. While the absence of chytrids may be due to difficulties in isolating and identifying chytrids (Bills et al. 2004), chytrids may be more intolerant of higher temperatures than other groups of fungi. Cultures of the aerobic members of the Chytridiomycota are commonly grown on solid agar media at approximately 15 to 25 x in the laboratory. Growth in culture ceases above 25 x in the psychrophile, Triparticalcar arcticum (Barr 1970b) and above 42 x for isolates of some species of Allomyces (Nielsen 1982). The obligately anaerobic chytrids of the order Neocallimastigales are found in the digestive tracts of a * Corresponding author. large group of herbivores, where temperatures remain around 37 x (Theodorou et al. 1994, Trinci et al. 1994). The anaerobic rumen fungi may also continue to grow up to 42 x (Lowe et al. 1987, Theodorou et al. 1994). Except for Allomyces and the rumen chytrids, growth of chytrids has not been reported above 35 x (Ajello 1948, Haskins & Weston 1950, Rothwell 1956, Goldstein 1960, Barr 1969, 1970a, b, Nolan 1970, Hasija & Miller 1971, Piotrowski et al. 2004). Many aerobic chytrids are commonly found in soil (Sparrow 1960). Dry soils exposed to sunlight over the summer in Australia may experience temperatures well above 35 x (McGee 1989). Fungi in soil used for cropping may be adapted to higher temperatures than those in heavily vegetated habitats, or they may have alternative mechanisms to enable survival through summer. The fungi may either become quiescent as temperatures rise above the maximum temperature for growth or form heat resistant structures. Some chytrids can resume growth after drying for 7 d at 20 x and after drying for 5 d at 20 x followed by heating at 80 x for two days (Gleason et al. 2004). Thus formation of structures that resist heat is one likely mechanism enabling survival. Alternatively, the fungi may, in some way, become conditioned by rising temperature enabling growth at higher temperatures. Up-regulation of proteins that

2 Growth response of some Chytridiomycota to temperature 718 Table 1. Source, form and maximum temperatures for growth of chytrids. Fungus and order a Location of collection b and collector c Bait d and form of colony e Maximum temperature for growth (xc) Maximum temperature of recovery f (xc) Allomyces anomalus AUS 1 1 SB, 1 C, H 40 nt A. anomalus C/C2 1 NC, 3 S, H 40 nt Spizellomyces sp. M Ad 2 2 NR, 3 P, M 40 nt Catenaria sp. Poly Ad 2 1 NR, 3 S, H 37 nt Catenophlyctis sp. CC 4 1 NC, 3 P, M 37 nt Spizellomyces sp. M C/C2 2 NC, 3 P, M 37 nt Spizellomyces sp. D C/C4 2 NC, 3 P, M 37 nt Rhizophydium sp. AUS 2 3 SB, 1 P, M 35* 6 40 x Rhizophydium sp. AUS 6 3 SB, 1 O, M 35* 6 40 x Rhizophydium sp. AUS 7 3 SC, 1 P, M x Rhizophydium sp. AUS 8 3 SB, 1 P, M x Rhizophydium elyense AUS 9 3 SB, 2 S, M 35* 6 40 x Rhizophlyctis rosea AUS 13 2 SB, 1 O, M 35 nt Cladochytridium sp. AUS 11 3 T, 1 O, P x Rhizophydium sp. AUS 3 3 SB, 1 P, M x Rhizophydium sp. AUS 10 3 CC, 1 P, M x Rhizophydium sp. AUS 12 3 CC, 1 P, M x Chytriomyces hyalinus AUS 5 3 SC, 1 C, M x Chytriomyces hyalinus AUS 14 3 CC, 1 C, M x Chytriomyces sp. M Ad 14 3 NR, 3 P, M 30** 6 37 x Rhizophydium sp. AUS 15 3 SB, 1 P, M x Rhizophlyctis sp. AUS 16 2 SB, 1 P, M 30 nt Powellomyces sp. AUS 17 2 CC, 1 C, M 30 nt a 1 Blastocladiales; 2 Spizellomycetales; and 3 Chytridiales. b SB, Sydney Basin; SC, South Coast; CC, Central Coast; NC, Narrabri, crop; NR, Narrabri, roadside and T, Tasmania, alpine. c Collector: 1, P. Letcher; 2, C. Briggs; and 3, Z. Commandeur. d Bait used for isolation: C, chitin; O, onion skin; P, pollen; and S, snake skin. e Form of colony: h, hyphal; m, monocentric; and p, polycentric. f nt, Not tested because heat resistant structures are formed (Gleason et al. 2004, Gleason, unpubl.). * Inconsistent growth at 37 x. ** Inconsistent growth at 33 x. increase tolerance of stress, especially heat, has been demonstrated for a wide diversity of fungi (Plesofsky- Vig & Brambl 1993). Thus, protective proteins or some other physiological mechanism may be up-regulated by increasing temperature, enabling fungi adapted to hot climates to continue growing as soil temperatures increase from winter to summer. Conditioning may be more likely in fungi found in soils subject to episodic high temperatures, such as cropping and arid soils in hot climates, thereby enabling adapted fungi to survive local conditions. The objective of this study was to compare the growth, and to identify responses to increasing temperature, of chytrids in the Blastocladiales, Spizellomycetales and Chytridiales. Chytrids were isolated from soils representing two distinct environments in Australia: cooler, native vegetation of temperate forests, mostly in the Sydney Basin, and warmer, semiarid soils in northern New South Wales. MATERIALS AND METHODS Maximum temperature of growth We selected 23 chytrids (Table 1) from the Sydney Basin, coastal central and southern New South Wales, the alps of Tasmania (Letcher, McGee & Powell 2004a) or Narrabri, NSW (Commandeur, Letcher & McGee, unpubl.) for study. These fungi were isolated from soil samples by using pollen, chitin (purified shrimp exoskeleton), keratin (purified snake skin) or cellulose (onion epidermis) as baits (Sparrow 1960). Stock cultures were grown on half strength YpSs in 2% agar or PYG in 2% agar (Fuller & Jaworski 1987) in 9 cm Petri dishes wrapped with parafilm and maintained at 20 x (Table 1). Fungi were transferred to fresh growth media at least once every three months. In growth experiments cultures grown for 7 d or less at 20 x were used for inoculum. The temperatures chosen for testing fungal growth were selected to represent different soil temperature regimes. In the summer months the soil temperature 10 cm below the surface of cropping soils may reach 40 x at Narrabri ( au/tools/weather/wx.htm). Further, the air temperatures at Narrabri are much higher than the Sydney Basin or the Tasmanian Alps (Bureau of Meteorology, Commonwealth of Australia, au./climate). Subcultures of all isolates were incubated at 25, 30, 33, 35, 37, 40 or 42 x in temperature-controlled incubators for 7 d. Growth on solid media was determined as an increase in volume, diameter or number of

3 F. H. Gleason and others 719 Table 2. Mean ( S.D.) dry weight (mg) of Spizellomyces sp. (M Ad 2) grown at 20 or 37 x. Time of harvest after inoculation (h) Temperature xc ND a a ND, not determined. colonies by visual observation. All growth experiments were repeated at least three times, and the maximum temperature for growth of each isolate was recorded (Table 1). Recovery of fungi following incubation at temperatures above the maximum temperature for growth was tested. Cultures of fungi in the Chytridiales were placed at either 37 x or 40 x for 3 or 6 h, and then returned to 20 x for another 7 d to determine whether fungi could resume growth after exposure to temperatures at which growth ceased. All recovery experiments were repeated at least three times. Voucher cultures are permanently preserved in the collection at the Department of Biological Sciences, University of Alabama. Growth of Spizellomyces Determining growth visually on solid media can sometimes be difficult. Zoospores of Spizellomyces sp. Mar Ad 2, a species tolerant of high temperatures, were obtained by flooding cultures with five ml of de-ionized water. After 2 h, 0.2 ml of zoospore suspension was added to 100 ml Schott bottles containing 25 ml PYG liquid medium. The bottles were then placed on a rotary shaker and incubated at either 20 or 37 x. After 38, 50, 61, 73, 85, 109 or 133 h, five replicate bottles from the rotary shaker were placed in ice. The thalli were then filtered from the liquid under vacuum onto pre-weighed Whatman filter paper. The thalli were dried in an oven at 80 x, and the dry weight was determined to 0.1 mg. Growth data were analysed by ANOVA and the two tailed t-test (Microsoft Excel). Conditioning of growth Selected fungi were tested for the capacity to upregulate or condition tolerance of higher temperatures. Rhizophydium sp. AUS 2, Rhizophydium sp. AUS 6, Rhizophydium elyensis AUS 9, Chytriomyces sp. M Ad 14, and Spizellomyces sp. M Ad 2 were grown from zoospores on half strength YpSs in 1% agar for 1 d at either 20 or 30 x. Three replicate plates were then transferred either to 30 x (from 20 x only), 33, 37 or 40 x and grown for a further week. The contents of each Petri dish was then passed through a 25 mm screen. The thalli remaining on the screen were removed and placed in clean Petri dishes, then they were dried at 80 x, and the dry weights were determined. Growth of Rhizophydium elyensis AUS 9 under controlled conditions was investigated further. Three Petri dishes of stock culture held at 31 x for 10 d were flooded with 5 ml sterile deionised water for 2 h. A 0.1 ml aliquot of the zoospore suspension was then placed in 20 ml of sterile half strength YpSs liquid media in Schott bottles. The bottles were capped and gently shaken to distribute the zoospores. Bottles were then placed in incubators at either 31, 35 or 37 x each for either 1, 2 or 3 d, and then either left at that temperature or moved to 37 x for the remainder of the growth period. Fourteen days after inoculation, the contents of five replicate bottles of each treatment were filtered through a 25 mm screen, the thalli were dried on pre-weighed filter paper at 80 x, and the dry weights were determined. Growth data were analysed by ANOVA (Microsoft Excel). RESULTS Maximum temperatures for growth All fungi grew on solid media at 20 x and at 30 x, and none at 42 x. The maximum temperature for the growth of these fungi varied by isolate (Table 1). Seven members of the Blastocladiales and Spizellomycetales grew at or above 37 x. Five isolates of Rhizophydium grew at 35 x. The growth of three isolates of Rhizophydium (AUS 2, AUS 6 and AUS 9) was inconsistent at 37 x. The diameter of the colonies did not appear to increase in all replicates. With the exception of those chytrids that form heat resistant structures (Table 1), no cultures resumed growth when returned to room temperature after exposure to 7 d incubation at temperatures above the maximum for growth. After exposure to a temperature higher than the maximum for growth for 3 6 h, 13 fungi resumed active growth at 20 x (Table 1). Growth of Spizellomyces sp. M Ad 2 In liquid culture growth of Spizellomyces sp. M Ad 2 was first apparent after 38 h at 37 x and after 61 h at 20 x. Though the dry weight of this fungus had significantly increased by 109 h at both temperatures, at 20 x the dry weight was significantly greater than at 37 x (t=7.5, P=0.0017: Table 2). At 20 x growth continued throughout the duration of the experiment. At 37 x

4 Growth response of some Chytridiomycota to temperature 720 Table 3. Mean ( S.D.) dry weight (mg) of chytrids after 14 d growth on YpSs in 1% agar. Temperature (xc) Conditioning Growth Fungus Rhizophydium sp. AUS 2 Rhizophydium sp. AUS 6 Rhizophydium elyense AUS 9 Spizellomyces sp. M Ad 2 Chytriomyces sp. M Ad Table 4. Comparison of the effect of conditioning on dry weight at 40 xc ofspizellomyces sp. M Ad 2 after 14 d growth in liquid culture. n.s., not significantly different; S.D., statistically significant difference at the 0.05 level of confidence. Temperature of conditioning Temperature of growth (xc) P=0.059 F=5.4, n.s. P=0.001 F=32, S.D. P=0.024 F=9, S.D. 30 P=0.017 F=10.7, S.D. P=0.2 F=2.1, n.s. 33 P=0.39 F=9, n.s. Table 5. Mean ( S.D.) dry weight (mg) of Rhizophydium elyensis AUS 9 after 14 d at 31, 35 or 37 xc without conditioning, or after 14 d at 37 x after conditioning for one, two or three d at a lower temperature. Temperature of growth (xc) Conditioning d (temperature) Mean d wt S.D. (mg) (31) (31) (31) (35) (35) (35) there was no sign of growth after 50 h (F=1.65, P=0.2). 35 x were significantly greater than at 37 x (F=190, P<0.01; Table 5). Conditioning of growth After 14 d in liquid culture the dry weights of five fungi tested were greatest at 30 x. Rhizophydium sp. AUS 2, Rhizophydium sp. AUS 6, Rhizophydium elyensis AUS 9 and Chytriomyces sp. M Ad 14 formed detectable dry weights after growth at 33 x, and Spizellomyces sp. M Ad 2 up to 40 x (Table 3). Except for Spizellomyces sp. Mar Ad 2, the dry weights decreased as the growth temperature increased, regardless of the temperatures at which cultures had been conditioned. The dry weight of Spizellomyces sp. Mar Ad 2 was significantly greater at 40 x when conditioned at 33 x compared to 20 x or 30 x (F=7.7, P=0.004), and it was also greater when conditioned at 37 x compared to 20 x (Table 4). R. elyensis AUS 9 grew in liquid culture when incubated for the whole time at 31 and 35 x, but not at 37 x. When the fungus was first incubated at 31 x or 35 x for one day and then placed at 37 x, the fungus formed detectable dry weight, but the temperature of conditioning had no significant effect (F=1.27, P>0.31). The dry weights of fungi incubated entirely at 31 x or DISCUSSION Soil fungi may survive high temperatures by various mechanisms. Some species become quiescent, thermotolerant fungi have the pre-existing capacity to grow up to a maximum temperature, and others may be conditioned to grow by slowly increasing temperatures. All three mechanisms appear to apply to some chytrids, albeit to a limited extent. On solid media growth was determined as an increase in volume, diameter or number of colonies. We chose solid media partly because growth can be seen visually and partly because conditions on the surface of solid media may be closer to conditions in the soil than total immersion in liquid media. In the soil chytrids are found growing on solid substrates at the interfaces with water and air. According to these criteria cultures of two isolates of Allomyces anomalus and one isolate of Spizellomyces sp. continued to grow at 40 x but not 42 x. Some isolates of Allomyces spp. have been reported to grow at 42 x (Nielsen 1982). The maximum temperature for growth

5 F. H. Gleason and others 721 of the other members of the Blastocladiales and Spizellomycetales ranged from 30 to 37 x. All of the fungi in the Chytridiales, including eight isolates of Rhizophydium, ceased growth above 35 x on solid media. In a molecular phylogenetic study of 29 isolates of Rhizophydium and other genera of chytrids Letcher et al. (2004b) identified groupings that contained both American and Australian isolates of Rhizophydium. The isolates used in the present study represent four of these groupings: (a) AUS 3; (b) AUS 2, 6 and 9; (c) AUS 7 and 8; and (d) AUS 12. These groupings appear to include species with similar temperature maxima for growth. The maximum temperature for isolates in (a) and (d) was 30 x, and the maximum temperature for isolates in (b) and (c) was 35 x. This apparent pattern of phylogenetic relationships reflecting similarities in maximum temperature for growth between species of Rhizophydium needs to be examined further with a larger sample of isolates from each grouping. The results of our research do support the generalisation that most chytrids do not grow above 35 x (Ajello 1948, Haskins & Weston 1950, Rothwell 1956, Goldstein 1960, Barr 1969, 1970a, b, Nolan 1970, Hasija & Miller 1971, Piotrowski et al. 2004). However, despite the capacity of a few chytrids to form heatresistant structures (Gleason et al. 2004) and the fact that a few chytrids can grow at 37 x and 40 x (in the present study), the overall response of chytrids indicates sensitivity to the higher temperatures found in soils of Australia during summer. A comprehensive explanation for their mechanisms of survival remains to be elucidated. Most Chytridiales tested recovered growth after short exposure to 37 x and 40 x. The time of incubation was approximately equivalent to the average length of time that soil temperature is inclement during a hot summer day ( Weather/wx.htm). Therefore, the ability to endure brief durations of high temperatures may be one further mechanism enabling chytrids to tolerate transient periods of high temperature in soil in the summer in warmer climates. Evidence for conditioning in the response to high temperatures was restricted and highly qualified. Of those fungi tested in liquid culture, there was some evidence for conditioning in only one fungus. The dry weight of Spizellomyces sp. was greater at 40 x if the fungus had been exposed to 30 x or 33 x during the conditioning period. Three isolates of Rhizophydium and one isolate of Chytriomyces (all Chytridiales) showed no evidence of conditioning. Two different patterns were found in an experiment comparing the growth of Spizellomyces sp. (M Ad 2) at 20 x and 37 x (Table 2). The pattern of growth at 20 x showed a continuous increase in dry weight from 50 to 133 h which is consistent with the hypothesis that formation of sporangia is followed by release of zoospores and subsequently by formation of fresh sporangia. The pattern of growth at 37 x appeared to indicate that sporangia are initiated and mature rapidly up until 50 h, but then growth slowed. The reason for the different growth patterns is unclear. The importance of the various survival mechanisms to chytrids in soil in the field remains to be explored. One chytrid from the Sydney basin and several from Narrabri grew at or above 37 x, and all were in either the Blastocladiales or the Spizellomycetales. These fungi may also form survival structures upon drying enabling tolerance of temperatures higher than experienced in the field (Gleason et al. 2004). Members of the Chytridiales survive only for short periods above the maximum temperatures for their growth and they cannot survive drying (Gleason et al. 2004). Thus we would expect that members of the Blastocladiales and Spizellomycetales would be more likely to be found at Narribri than in the Sydney basin. Similar numbers of all three orders were obtained from enrichment cultures with soil from Narrabri although only a small number of species in total were observed (Commandeur, Letcher & McGee, unpubl.). Further testing and a larger sample size are needed to clarify whether these differences in response to temperature are important determinants of the distribution of chytrids in their native environment. In some experiments different parts of the life cycle of chytrids appeared to react differently to temperature. Sporangia appeared to develop at higher temperatures than zoospores could be released. The impact of temperature on formation and release of zoospores remains unclear. Some chytrids are unable to survive drying in the laboratory (Gleason et al. 2004) and possibly in the soil. In addition the amount of moisture in the soil is important for release and swimming ability of zoospores. In arid regions of Australia the soil often dries during periods of high temperatures. Temperature and moisture are factors which may influence the distribution of chytrids in the soil profile and across the landscape. Consideration of the interaction between temperature and available moisture may be necessary in further studies of chytrid growth and survival. ACKNOWLEDGEMENTS This research was supported in part by NSF-PEET Grant no. DEB We wish to thank Martha Powell for her continuing encouragement. REFERENCES Ajello, L. (1948) A cytological and nutritional study of Polychytrium aggregatum. Part II. Nutrition. American Journal of Botany 35: Barr, D. J. S. (1969) Studies on Rhizophydium and Phlyctochytrium (Chytridiales). II. Comparative physiology. 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