Mycol. Res. 108 (5): 583–589 (May 2004). f The British Mycological Society 583 DOI: 10.1017/S0953756204009736 Printed in the United Kingdom.

Some in soil recover from drying and high temperatures

1 2 1 Frank H. GLEASON *, Peter M. LETCHER and Peter A. MCGEE 1 School of Biological Sciences A12, University of Sydney, 2006, Australia. 2 Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA. E-mail : [email protected]

Received 21 November 2003; accepted 28 January 2004.

Rhizophlyctis rosea was found in 44% of 59 soil samples from national parks, urban reserves and gardens, and agricultural lands of eastern New South Wales, Australia. As some of the soils are periodically dry and hot, we examined possible mechanisms that enable survival in stressful environments such as agricultural lands. Air-dried thalli of R. rosea in soil and pure cultures of R. rosea, two isolates of Allomyces anomalus, one isolate of Catenaria sp., one of Catenophlyctis sp. and one of Spizellomyces sp. recovered following incubation at 90 xC for two days. Powellomyces sp. recovered following incubation at 80 x. Sporangia of all seven fungi shrank during air-drying, and immediately returned to turgidity when rehydrated. Some sporangia of R. rosea released immediately upon rehydration. These data indicate that some Chytridiomycota have resistant structures that enable survival through periodic drying and high summer temperatures typical of soils used for cropping. Eleven Chytridiomycota isolated from soil did not survive either drying or heat. Neither habitat of the nor morphological type correlated with the capacity to tolerate drying and heat.

INTRODUCTION and Blastocladiella (Cantino 1969) are reported to survive as resistant sporangia, and Allomyces is well Temperature directly influences the rate of physiologi- known to survive air-drying (Youatt 1991b). Thick- cal processes of biota, and it affects physicochemical walled sporangia may function as survival structures in processes in soil. Soils may experience large fluctuations Olpidium (Campbell 1985, Weber & Webster 2000), and in temperature. Soils used for cropping have a tem- Synchytrium is known to form resting spores (Laidlaw perature range that exceeds that of soils with a heavy 1985, Hampson et al. 1994). The survival structures are vegetative cover. The temperature of a soil is closely unknown for Rhizophlyctis (Dogma 1974, Willoughby related to its water status (Russell 1973). Thus fungi in 2001) and rumen chytrids (Davies et al. 1993a, b, cropping soils may be better adapted to rapid change Theodorou et al. 1994) though resting spores of R. rosea in moisture and temperature than fungi in heavily were illustrated by Dogma (1974). vegetated soils. A few chytrids are thought to be prevalent in soil. Fungi must have mechanisms that enable survival Rhizophlyctis rosea and several species of Allomyces through conditions unfavorable for growth. For in- have been reported from around the world (Sparrow stance, the presence of a wall that is impermeable 1960, Karling 1977), including Australia (Harder & to water (Sussman 1968) might ensure that resistant Gallwitzer-Uebelmesser 1959, Jeffrey & Willoughby structures survive through dry periods. Very little is 1964, Willoughby 1965, Karling 1988). These fungi known about the mechanisms of survival of fungi in were particularly prevalent in soils used for agriculture the Chytridiomycota (herein called chytrids) through and horticulture (Weber & Webster 2000). Most of periods of environmental stress (Powell 1993). Chytrids the surveys were limited in extent and results must be may survive fluctuations in temperature and moisture viewed as indicative. Recently, Allomyces sp. was found content of the soil as resistant structures (Sparrow once and R. rosea only five times in an intensive 1960). For instance, species of Allomyces (Machlis & sampling from native vegetation of the Sydney Basin, Ossia 1953, Skucas 1968, Olson 1984, Youatt 1991a) Australia (Letcher et al. 2004). The fungi may have been uncommon, or inappropriate techniques or timing * Corresponding author. used for isolation of chytrids. Survival of chytrids 584

In many studies of distribution of chytrids, soil paper and one small piece of Whatman lens paper samples were collected and then shipped long distances (Haskins 1939, Stanier 1942, Griffiths & Jones 1963, before analysis. The steps taken to prepare fungi for Dogma 1974, Weber & Webster 2000, Willoughby shipping, and the effect of storage and shipping on the 2001) placed on the surface of the solution. fungi in these soil samples are unknown. A considerable The dishes were incubated at 20 x for 7–10 d. The period of time between collection and analysis may baits were then removed from the dishes and examined result in loss of propagules of sensitive species from microscopically. The presence of R. rosea was recorded the soil sample. Thus these studies may have under- after 7 d. Diagnostic features were red sporangia with estimated diversity of chytrids. characteristic rhizoids and discharge tubes (Sparrow This paper reports the first part of an investigation 1960, Dogma 1974, Karling 1977). into the ecology of chytrids in soil. We focused on R. rosea because it is apparently widespread in agri- Survival of Rhizophlyctis rosea cultural soils and is easy to discern microscopically. The main objective of the research was to clarify A soil sample collected from the experimental garden in whether chytrids tolerate high temperatures especially February was air-dried in a Petri dish in the laboratory in dry soil. at 20 x. After 16 wk, the soil was wetted, and then baited. Baits were examined after 7 d for the presence of Rhizophlyctis rosea. Soil samples from eight sites containing abundant MATERIALS AND METHODS R. rosea were air-dried at room temperature. The sites We first sought to determine whether Rhizophlyctis represented undisturbed and highly disturbed soils. rosea is found in soils that might experience high Ten g of each air-dried soil was incubated separately in temperatures. We then tested whether R. rosea could a temperature-controlled oven (Thermoline, Australia) tolerate high temperatures. Finally, we compared tol- at 70, 80, 84, 90, or 95 x for 48 h. Incubation at 80 x was erance of R. rosea with a range of other chytrids in repeated, making six separate experiments. Below 90 x, culture to determine whether tolerance of heat was samples were incubated in plastic Petri dishes. At 90 x widespread among the chytrids. or above, dry soil was first transferred to aluminum foil dishes prior to heating. After cooling the soil was baited as above, and baits were examined microscopically Distribution of Rhizophlyctis rosea after 7 d. Only one dish was used for each site in each To clarify the distribution of Rhizophlyctis rosea, experiment. We chose 70 x as the baseline temperature samples of approx. 150 g soil were collected from 59 because soil maxima at this level have been observed sites across eastern New South Wales, mostly close to (McGee 1989). Sydney. Eight sites experience high temperatures, two Soil water solution consisting of approx. 100 g soil from under open heath vegetation, and six from fields from the experimental garden shaken in one L of used to grow cotton. Permission to collect on private de-ionized water was passed through a Whatman No. 1 lands was granted by the owners. Authorization to con- filter paper, bottled and autoclaved. Cultures were duct research and to collect soil samples on land owned established in sterile soil water by transferring pieces of by the New South Wales State Government was filter paper colonized by R. rosea and associated granted under scientific investigation license no. A3375 microbes from the experimental garden in Petri dishes. (National Parks and Wildlife Service). Soil was taken Fresh sterile baits were added. After 7–14 d, the newly from the surface after scraping away any litter, and colonized pieces of filter paper were transferred again placed in 17 by 18 cm plastic snap lock bags. Samples to fresh soil water with fresh sterile baits or removed were transported to the laboratory within 24 h of from the water and dried at room temperature in sterile collecting, and then either processed immediately or Petri dishes. Dried pieces of colonized filter paper were stored at 6 x. Only one soil sample was collected at each stored at room temperature until further use. A small site, except that soil from an experimental garden near amount of thallus from a pure culture of R. rosea the laboratory was sampled over a six month period. (Table 1: Fungus D; Letcher et al. 2004) growing on When bags were sub-sampled more than once, the bags PYG agar (Fuller & Jaworski 1987) was placed in the were returned to the cool room immediately after sterile soil water culture with fresh sterile baits and removing the subsample. treated in the same way as the isolates from soil. Air dried pieces of filter paper with thalli of R. rosea from the experimental garden and from the pure Baiting for Rhizophlyctis rosea culture of R. rosea were incubated at 70, 80 and 90 x for Approx. 10 g of soil from each sample was placed in a 24 h, and then returned to a fresh sterile soil water 9 cm sterile plastic Petri dish. Approx. 20 ml of sterile culture. Small amounts of thallus from cultures on agar de-ionized water was added and the soil and water were were transferred to pieces of sterile filter paper, which mixed. Each dish was baited with cellulose substrates were then placed in sterile Petri dishes and air-dried. consisting of seven small pieces of Whatman filter These air-dried thalli on filter paper from the pure F. H. Gleason, P. M. Letcher and P. A. McGee 585

Table 1. Cultures of chytrids obtained from New South Wales used in the study.

Substrate used No. Fungusa Collection site Vegetation type Collectorb Form of colony for isolation

A Allomyces anomalous A1 Garrigal National Park Casuarina P. Letcher Hyphal chitin B A. anomalous C/C2 Narrabri Cotton Z. Commandeur Hyphal keratin C Powellomyces sp. A17 Ourimbah Rainforest P. Letcher Monocentric chitin D Rhizophlyctis rosea A13 University of Sydney Garden P. Letcher Monocentric cellulose E Catenaria sp. Dec Ad 2-0 Narrabri Mixed riparian Z. Commandeur Polycentric keratin F Catenophlyctis sp. C/C 4-10 Narrabri Cotton Z. Commandeur Monocentric pollen G Spizellomyces sp. Mar Ad 2-0 Narrabri Roadside Z. Commandeur Monocentric pollen H Rhizophydium sp. A2 Dharug NP Wet sclerophyll P. Letcher Monocentric pollen J Rhizophydium sp. A3 Lane Cove NP Wet sclerophyll P. Letcher Monocentric pollen K Chytriomyces sp. A4 Wheeler Creek NP Wet sclerophyll P. Letcher Monocentric chitin L Chytriomyces hyalinus A5 Morton NP Open heath P. Letcher Monocentric chitin M Rhizophydium sp. A6 Dharug NP Wet sclerophyll P. Letcher Monocentric onion skin N Rhizophydium sp. A7 Morton NP Open heath P. Letcher Monocentric pollen P Rhizophydium elyensis A9 Yarramundi Mud from pond C. Briggs Monocentric keratin Q Rhizophydium sp. A10 Ourimbah Rainforest P. Letcher Monocentric pollen R Rhizophydium sp. A12 Ourimbah Rainforest P. Letcher Monocentric pollen S Chytriomyces hyalinus A14 Ourimbah Rainforest P. Letcher Monocentric chitin T Rhizophydium sp. A15 Ku-ring-gai NP Dry sclerophyll forest P. Letcher Monocentric pollen

a Identifications putative. b Voucher cultures are permanently preserved in the collection at the University of Alabama, Department of Biological Sciences. culture of R. rosea were incubated at 80 and 90 x for experimental garden), Allomyces anomalus (Fungi A 48 h. After cooling these were transferred to fresh and B), Powellomyces sp. (Fungus C), Catenaria PYG agar. Sporangia of thalli were examined micro- sp. (Fungus E), Catenophlyctis sp. (Fungus F) and scopically. Spizellomyces sp. (Fungus G) were placed on slides Controls consisting of uncolonized filter paper under a coverslip without water, and examined micro- indicated the absence of chytrids on the fresh filter scopically. To determine whether the sporangia or paper baits or in sterile soil water. rhizoids were capable of recovering, de-ionized water was added to the edge of the cover slip, and rehydration was examined microscopically. To determine whether Survival of other chytrids the reaction was possibly due to differences in water Pure cultures of fungi isolated from soil in temperate potential, 1 M sucrose was added to rehydrated thalli of rain forest, wet sclerophyll forest, dry sclerophyll forest, R. rosea, and the reaction followed. The sugar solution open heath, or agricultural sites, fungi that were mono- was then replaced by de-ionized water. centric or polycentric, and fungi originally isolated using various baits, were grown on YPSS/2 agar or PYG agar (Fuller & Jaworski 1987) in Petri dishes at RESULTS 20 x and 30 x (Table 1). As data were identical, only Distribution of Rhizophlyctis rosea those from 20 x are reported. Small amounts of thallus from the cultures with or without agar were transferred Rhizophlyctis rosea was observed on baits from 26 of to four small pieces of sterile filter paper, which were 59 sites, including 15 of 24 samples from agricultural or placed in sterile Petri dishes. The fungi were then dried horticultural sites, and 11 of 35 soils from undisturbed on the laboratory bench for 5–7 d. Air-dried thalli on sites with natural vegetation from private lands, filter paper were placed back on nutrient agar to deter- national parks and reserves. R. rosea was isolated from mine survival of air-drying. The experiment was re- one site that experiences high temperature. The density peated. Further dried fragments of all fungi were of sporangia varied on the baits. Sporangia of R. rosea incubated at 80 x for 48 h and then cooled. The pieces were more easily seen on lens paper than on filter paper of filter paper with the fungi attached were transferred because soil particles that stuck to the filter paper to fresh solid growth media in Petri dishes and incu- obscured sporangia, and because sporangia were deeply bated on the laboratory bench at 20 x for up to 14 d. embedded in the filter paper. Further, filter paper had Freshly dried samples of the fungi that survived incu- sunk to the bottom of the solution, while the lens paper bation at 80 x were then tested for survival after heating remained on the surface. However, partially decom- for two days at 90 x. posed filter paper was easier to manipulate than par- tially decomposed lens paper. The presence of R. rosea was recorded on samples usually within 7 d, because Rehydration of dried thalli baits were rapidly digested, and bacteria colonized Air-dried thalli of Rhizophylctis rosea (from pure thalli. Bacteria and protists were commonly present culture, Fungus D; and from dried soil from the with sporangia. Because of the variable level of Survival of chytrids 586 contamination and difficulty in observing sporangia, a sporangia were not quantified. ( ) During a drought in Feb. 2003, R. rosea was abun- dant in soil samples obtained from the experimental garden, and these samples were used in the subsequent experiments. The fungus was absent on baits for 2 wk following storms in March, but was present when the garden was sampled again in June, when the soil was dry.

Survival of Rhizophlyctis rosea Baits placed in the rehydrated soil samples from the experimental garden that had been stored air-dried for 16 wk became colonized by Rhizophlyctis rosea. R. rosea was recovered from soil samples from eight different sites after those soils were heated for 48 h at 70, 80, 84 and 90 x.At95x, R. rosea was recovered from soil of five of the eight sites. Air dried thalli of R. rosea growing in filter paper from a soil water culture survived incubation at 70, (b) 80 and 90 x for 48 h in three separate experiments. The zoosporangia discharged, and the zoospores colonized fresh filter paper. Thalli of the pure culture of R. rosea placed on pieces of filter paper survived incubation at 70 and 80 x but not 90 x.

Survival of other chytrids Of the 18 fungi, only two isolates of Allomyces ano- malus, and one of Catenaria sp., Powellomyces sp., Rhizophlyctis rosea, Catenophlyctis sp. and Spizello- myces sp. survived drying at room temperature. After drying at room temperature, isolates of A. anomalus, Catenaria sp., Rhizophlyctis rosea, Catenophlyctis sp. and Spizellomyces sp. also survived incubation at 80 and 90 x for 48 h. Powellomyces sp. survived incubation at 80 x for 48 h (Table 1).

Rehydration of dried thalli Fig. 1. Thalli of Rhizophlyctis rosea:(a) air dried from soil water culture; and (b) another thallus after rehydration. Sporangia of Rhizophlyctis rosea from soil appeared Walls of sporangia arrowed. shriveled and collapsed after air-drying (Fig. 1a). Sporangia rapidly returned to their normal shape after addition of de-ionized water (Fig. 1b). Hydrated spor- soils of nearly half of the sites sampled, and was slightly angia were slightly plasmolysed by the addition of 1 M more common in disturbed soils such as those used for sucrose. Subsequent washing with de-ionized water re- horticulture and agriculture. While it was found in only versed plasmolysis. Addition of water to dry sporangia one soil that experiences high temperatures, subsequent also resulted in immediate discharge of zoospores in extensive sampling at Narrabri found R. rosea at low some cases. densities in soils of all the sites on the cotton farm (Zoe Pure cultures of two isolates of Allomyces anomalus, Commandeur, unpubl.). Thus, while high temperature Catenaria sp., R. rosea, Powellomyces sp., Cateno- and disturbance may influence isolation from soil, they phlyctis sp. and Spizellomyces sp. shriveled on drying, appear to be unimportant in the survival of R. rosea. and rehydrated rapidly on addition of water. In a few soils, R. rosea appeared to be abundant. Rehydrated fungi resembled untreated fungi in the The factors determining distribution and abundance light microscope. The survival structures were not are unclear. Repeated sampling over 5 m from an elucidated. experimental garden near the laboratory indicated that abundance of the fungus may be affected by incidence and intensity of rainfall. The fungus was not found on DISCUSSION baits placed in soil samples collected immediately after Rhizophlyctis rosea was widespread in eastern New heavy rainfall (data not shown), yet was abundant South Wales, Australia. The fungus was baited from during an earlier drought period and a later dry period F. H. Gleason, P. M. Letcher and P. A. McGee 587

(data not shown). Thus, while presence on baits con- 2001). In our investigation, resistant structures in soil firms presence of the fungus in a sample, absence does survived temperatures up to 90 x. Capacity to survive not necessarily indicate absence from the soil. air-drying and high temperatures indicates one mech- R. rosea is possibly more prevalent than is indicated anism to explain the survival of R. rosea in habitats in this investigation. We did not attempt to collect used for cropping that are subject to periodic and fre- air-dry soil samples, nor did we air-dry the samples quent summer drought. The air-dried sporangia are following collection. Apparent absence of R. rosea also possible dispersal units. They could be carried by from some soils may have been due to the fungus being wind with soil particles from site to site. These propa- in a stage of its life cycle where colonization of baits gules may partly explain the widespread distribution of was unlikely. The relationship between quantity of R. rosea in disturbed habitats. sporangia on bait and abundance of the fungus in soil In contrast to Willoughby (2001), sporangia of must be treated cautiously. For instance, addition of R. rosea became shriveled during drying, and rehy- baits to soil stored damp in cool conditions for a long drated to their normal morphology within a few period may result in lower apparent abundance, only seconds of exposure to water (Fig. 1). Because the because some propagules have exhausted their internal sporangial wall appears readily permeable to water, we energy supplies. The apparent disparity between propose that the cytoplasm itself is resistant to drying abundance and sporangia on baits has implications for and heating when the fungus is in a quiescent state. The ecological studies. If other chytrids have a similar mechanism for coping with rapid and cyclic changes in response to soil moisture, then air-dry samples, kept hydration, are unclear. High concentrations of salts or dry are likely to realize a greater abundance and osmoticum may influence metabolic processes within diversity of fungi. The mechanisms regulating changes zoospores and the sporangium. Cytoplasmic mechan- in abundance require more careful examination, and in isms enabling survival warrant further investigation. part rely on understanding the life-cycle of each chytrid Many other fungi tolerate high temperatures in the soil. (Warcup & Baker 1963, Sussman 1968, Bayne & Thalli of R. rosea recovered from drying. The walls Mitchener 1979). Indeed, dormancy is broken by heat of the sporangia were clearly permeable, for desiccated in some (Warcup & Baker 1963). Little is sporangia imbibed immediately on exposure to water. known of heat tolerance in chytrids (Remy 1948, Imbibition of water by dried sporangia was immediately Teakle & Thomas 1985). In our study, air-dried followed by release of zoospores in some cases. Thus it Allomyces anomalus (Fungi A and B), Catenaria sp., might be argued that during periods of dry weather, R. rosea, Powellomyces sp., Catenophlyctis sp. and the fungus survives as dried sporangia in soil, and that Spizellomyces sp. survived at 80 x for 48 h, though zoospores are released soon after adequate rainfall. If not other chytrids from various habitats. Survival is so, the fungus will survive if zoospores establish on apparently unrelated to morphology of the fungus, substrates to form sporangia before the soil dries. Soils habitat from which the fungi were isolated, and the were baited with cellulose in these experiments, and bait on which the fungi were isolated. However, it is colonization was apparent on baits. We do not know interesting to note that R. rosea survived sometimes in what substrate the fungus colonizes naturally in soil. soil held at 95 x, indicating that the thermal death R. rosea was baited successfully on cellulose only, point of the fungus is in the range of 90–95 x. indicating that cellulose might be a good substrate for Many chytrids are subcultured regularly, with con- maintenance and growth of the fungus (Haskins 1939, sequent potential alteration of the genetic character- Stanier 1942, Griffiths & Jones 1963, Dogma 1974, istics of these strains in the laboratory. Many of these Weber & Webster 2000, Willoughby 2001). It is curious fungi are difficult to maintain in culture and are lost then, that R. rosea was not more common in soils for no apparent reason. Indeed, different parts of one where reasonable levels of cellulosic carbon are found. culture may be at different physiological stages. Several Although not quantified, all of the 59 soils collected chytrids recovered from air-drying indicating the in this investigation contained at least some decaying potential of using air-drying for the long-term storage plant material, providing a source of cellulose, and of these chytrids. Issues that need to be further in- possible nutrients for the fungus. R. rosea was observed vestigated to clarify the potential of drying for storage to co-occur with other microbes on baits; either the include whether the tolerance to drying is induced or fungus was present with many different microbes, or staged in soil, the stage of the life-cycle where drying the fungus was absent and other microbes were enables quiescence, and the role of nutrients and water infrequent. It was unclear whether the diversity of during storage and recovery. microbes was present because of R. rosea or because The process used to test the survival of extreme the absence of R. rosea indicated some general anti- temperature by these fungi was direct and presumably microbial factor. The interaction between R. rosea harsh. The thalli were placed on filter paper, dried and other microbes during colonization of cellulosic rapidly, and then placed in incubators at very high substrates in soil warrants further study. temperatures. It is unlikely that any protective function Survival of air-dried R. rosea in soil on the lab bench could have been induced in the short time allowed. for 16 wk has been observed previously (Willoughby Further, lack of survival at 80 x need not indicate the Survival of chytrids 588 capacity of these fungi to tolerate more realistic rates of Dogma, I. J. jr (1974) Developmental and taxonomic studies on drying or soil temperatures, or even to grow when rhizophlyctoid fungi, Chytridiales. II. The Karlingia (Rhizophlyctis) edaphic conditions are amenable. We have clarified one rosea-complex. Nova Hedwigia 25: 1–49. Fuller, M. S. & Jaworski, A. (1987) Zoosporic Fungi in Teaching and mechanism enabling survival of several chytrids in soil. Research. Southeastern Publishing, Athens, GA. Other chytrids are found in these soils, and they must Griffiths, E. & Jones, D. (1963) Colonization of cellulose by soil have other adaptive mechanisms that enable them to micro-organisms. Transactions of the British Mycological Society tolerate the soil drying, and exposure to high tempera- 46: 285–294. tures. These mechanisms remain to be explored. Hampson, M. C., Yang, A. F. & Bal, A. K. (1994) Ultrastructure of Synchytrium endobioticum resting spores and enhancement of Rhizophlyctis rosea can dry in air, and subsequently germination using snails. 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