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The effect of metals on growth, reproduction and attachment of zoosporic true fungi

By Linda Henderson

School of Life and Environmental Sciences The University of Sydney

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of The University of Sydney 28 February 2018 Declaration

The work described in this thesis is exclusively my own investigation and has not previously been submitted for the award of a degree at this or any other institution.

Linda Henderson

February 2018

i Authorship attribution statement

Chapter 1 of this thesis, excluding Section 1.1, is published as; Henderson, L, Lilje, E. Robinson, K, Gleason FH, Lilje O, 2017. ‘Effects of toxic metals on chytrids, fungal-like organisms and higher fungi’ In: The fungal community: its organisation and role in the ecosystem 4th Ed. J. Dighton and F. White (eds.) CRC Press.

The various contributions to this chapter are published within the Contributions section (p. 448) of Henderson et al. 2017. Only those sections of the chapter which I have written (30.1; 30.2.1; 30.3; 30.4; 30.7 in Henderson et al. 2017) have been included in this thesis.

Chapter 2 of this thesis is published as; Henderson, L., Ly, M., Robinson, K., Gleason, F., Lilje, O. 2018. Maximum temperature for growth and reproduction is similar in two soil isolates of Gaertneriomyces semiglobifer (Spizellomycetales, Chytridiomycetes) from different soil environments in north eastern New South Wales, Australia. Nova Hedwigia: Journal of Cryptogamic Science 106: 485-497.

I designed the study, extracted the data and wrote the draft of the manuscript.

Chapter 3 of this thesis is published as Henderson L, Pilgaard B, Gleason FH, Lilje O, 2015. Copper (II) lead (II), and zinc (II) reduce growth and zoospore release in four zoosporic true fungi from soils of NSW, Australia. Fungal Biology 119: 648–55.

I co-designed the study, extracted the data and wrote the drafts of the manuscript.

Chapter 4 of this thesis has been submitted to the journal Nova Hedwigia for publication as; Henderson, L, Marano, AV, Truszewski E, Gleason FH and Lilje, O, 2018. Copper (ll), Lead (ll) and Zinc (ll) reduce the rate of attachment in three zoosporic true fungi from soils of NSW, Australia.

I designed the study, extracted the data and wrote the drafts of the manuscript.

ii

In addition to the statements above, in cases where I am not the corresponding author of a published item, permission to include the published item has been granted by the corresponding author.

Linda Henderson 20th February 2018

As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.

Dr Osu Lilje 20th February 2018

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Acknowledgements

Thanks to both my supervisors, to whom I owe a great deal. Dr Osu Lilje for teaching me essential laboratory techniques, also for her consistent encouragement and support throughout my research. Dr Frank H Gleason has inspiring my passion for research in the field of zoosporic fungi and for his constant encouragement and advice. I would like to thank all the members of the Lilje microbiology lab and the Dr Peter McGee fungal lab for help and support when I needed it most. Especially, thanks to Dr Katie Robinson and Dr Mai-An Ly for help with molecular techniques. Thanks to Bo Pilgaard for help in designing the growth experimental protocol. Dr Agostina Marano and Dr José Ivanildo de Souza provided invaluable advice on chytrid attachment protocol. The Australian Microscopy & Microanalysis Research Facility node (Sydney Microscopy & Microanalysis) at the University of Sydney is gratefully acknowledged for training and providing access to microscopy facilities. Most of all, I would like to acknowledge Gavin, Ellen, Lauren and James, for their continuous love and support, without which this work would not have been possible. Gratias tibi ago, Domine.

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Table of contents

Declaration...... i

Authorship attribution statement ...... ii

Acknowledgements ...... iv

Abstract ...... 1

Chapter 1: General introduction ...... 2

1.1 The lifecycle of zoosporic true fungi (chytrids) and their role in soil ...... 2

1.2 Effects of toxic metals on chytrids, fungal-like organisms and higher fungi ...... 6

1.2.1 Introduction ...... 6

1.2.2 Fungal Communities in metal-polluted and metal-rich soils ...... 7

1.2.3 Mechanisms of fungal resistance and tolerance to metals ...... 12

1.2.4 Physiological responses of fungi to toxic metal stress ...... 13

1.2.4.1 Toxicity ...... 13

1.2.4.2 Sporulation and germination ...... 15

1.2.4.3 Enzyme and metabolite activity ...... 17

1.2.4.4 Non-enzymatic responses ...... 18

1.2.5 Concluding remarks ...... 20

Chapter 2: A comparison of temperature effects on the same isolate

(Gaertneriomyces semiglobifer) from different locations in NSW ...... 22

2.1 Abstract ...... 23

2.2 Introduction ...... 24

2.3 Materials and methods ...... 26

2.3.1 Isolate collection and maintenance ...... 26

v

2.3.2 Preparation of inoculum ...... 26

2.3.3 Identification of isolate ...... 27

2.3.4 Temperature effects on colony growth size (growth on solid PYG) ...... 28

2.3.5 Temperature effects on biomass over time (growth yield in liquid PYG) ...... 28

2.3.6 Temperature effects on the number of zoospores released ...... 29

2.3.7 Maximum temperature for growth recovery ...... 29

2.3.8 Statistical Method ...... 29

2.4 Results ...... 30

2.4.1 Identification of fungal isolate ...... 30

2.4.2 Temperature effects on colony size (growth on solid media) ...... 30

2.4.3 Temperature effects on biomass over time (growth yield in liquid PYG) ...... 30

2.4.4 Temperature effects on the number of zoospores released ...... 31

2.4.5 Maximum temperature for growth recovery ...... 31

2.5 Discussion ...... 34

Chapter 3: The effect of three metals on growth and reproduction of four isolates of zoosporic fungi ...... 38

3.1 Abstract ...... 39

3.2 Introduction ...... 40

3.3 Materials and methods ...... 42

3.3.1 Selection and maintenance of isolates ...... 42

3.3.2 Preparation of inoculum ...... 43

3.3.3 Growth in liquid media...... 43

3.3.4 Zoospore release ...... 43

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3.3.5 Survival ...... 44

3.3.6 Statistical Analysis ...... 44

3.4 Results ...... 45

3.4.1 Biomass production (growth)...... 45

3.4.2 Zoospore production ...... 48

3.4.3 Survival (recovery of growth after incubation with toxic levels

of Cu II, Pb II or Zn II) ...... 48

3.5 Discussion ...... 50

Chapter 4: The effect of Cu, Pb and Zn on the attachment of four zoosporic fungi to organic substrates ...... 54

4.1 Abstract ...... 54

4.2 Introduction ...... 55

4.3 Materials and Methods ...... 57

4.3.1 Selection and Maintenance of isolates ...... 57

4.3.2 Preparation of inoculum ...... 57

4.3.3 Measurement of attachment to substrates ...... 57

4.3.4 Measurement of rhizoid length ...... 58

4.3.5 Statistical analysis ...... 59

4.3.5 Preparation of fungi for SEM imaging ...... 59

4.4 Results ...... 59

4.4.1 Attachment to substrates in the presence of metals ...... 59

4.4.2 Measurement of number and length of R.rosea rhizoids after incubation with

Pb (ll) ...... 62

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4.4.3 Imagery of attached sporangia incubated with different concentrations of

Pb (ll) ...... 63

4.5 Discussion ...... 65

Chapter 5: Metals, metal uptake and morphological change in fungi: A review ...... 68

Chapter 6: General discussion ...... 75

Reference ...... 78

Appendix ...... 99

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Abstract

Many metals are required for cellular metabolism, however in excess they are toxic. Metals have complex effects on soil fungi, both at the level of the individual fungus and for the fungal community. Fungi have developed adaptive responses to enable them to complete their lifecycles within the soil, while in the presence of metals. Different stages of the fungal lifecycle are affected differently by metals. Soil zoosporic fungi (chytrids) are common in soils and attach to surfaces by means of a system of rhizoids. They contribute to the soil carbon cycle by converting complex carbohydrates to more accessible forms for uptake by other microbes. Chytrids are also important in biogeochemical cycling of elements such as nitrogen and sulphur. It is also likely that chytrids have developed efficient transport and sequestration pathways for scavenging often low levels of essential metals in the soil environment. However, the roles of chytrids in many soil processes, and their responses to environmental stress, are not well understood. In order to explore the response of chytrids to temperature stress, here we initially examine the effect of increased temperature on chytrid isolates from different geographic and climatic regions. Isolates of Gaertneriomyces semiglobifer from different regions grow at similar rates and have similar patterns of zoospore production at different temperatures. This data allows prediction of the distribution, growth and abundance of the fungus and potential changes due to the effects of climate change. We then examine the effect of Cu (ll), Pb (ll) and Zn (ll) on growth, zoospore production and attachment of chytrids to common organic substrates. The four isolates, representing four orders within the phylum Chytridiomycota, showed greatest sensitivity in growth, attachment and zoospore production in response to Cu (ll) and least sensitivity to Pb (ll). All the fungi responded similarly in sensitivity to growth in response to different metals as they did in rates of attachment to organic substrates. Interestingly, some metals also caused increases in growth, zoospore production and attachment below the toxic threshold concentrations. In particular, Rhizophlyctis rosea increased the number and length of rhizoids when incubated with Pb (ll). Chytrids are known to be widespread and common throughout soils world-wide. Our work allows us to predict that the levels of Cu (ll), Pb (ll) and Zn (ll), found here to be toxic, will be detrimental to soil chytrid populations and reduce colonisation of organic substrates. Toxic effects of metals on the lifecycle of chytrids are expected to reduce the rate of mineralisation of soil organic matter, thereby reducing nutrient availability for the soil microbial loop, to the detriment of ecological function in soils. 1

Chapter 1: Introduction

1.1 The lifecycle of zoosporic true fungi (chytrids) and their role in soil. Zoosporic true fungi (chytrids) have been frequently observed growing in many habitats in soil (Shearer et al. 2007). Soil chytrids in the phyla Chytridiomycota and Blastocladiomycota are either saprotrophic, mutualistic or parasitic (Sparrow 1960; Powell 1994). These eukaryotic microorganisms produce motile spores with single posteriorly directed whiplash flagella (Barr 2001). They are widely distributed in soil, most commonly in moist temperate climates (Sparrow 1960; Gleason et al. 2006). They are expected to have keystone roles in both soil and aquatic ecosystems (Gleason et al. 2008; Gleason et al. 2012). In Chapter one, initially we describe the lifecycle of chytrids and their roles in soil. As very little information is available in the literature on exposure of zoosporic fungi to metals, we examine next the effects of toxic metals on chytrids, fungal-like organisms and higher fungi. Chapter two of this thesis examines the ability of two isolates of the chytrid species Gaertneriomyces semiglobifer, isolated from two different climatic regions and soil types in NSW, to grow and reproduce at increased temperatures. Chapter two is important because currently little is known of the distribution of the various species of soil chytrid and how environmental change affects their abundance and growth. Temperature may be an important factor in limiting the geographical distribution of chytrids (Gleason and McGee 2008) and chytrid response to temperature may also allow predictions of the response of the fungi to climate change. Chapter three then examines the growth and zoospore production responses of four soil chytrids, isolated from soils in different regions in NSW, to various concentrations of toxic metals. The ability of chytrids to colonise organic substrates in the presence of the same toxic metals is explored in Chapter four. Finally, in Chapter five we review potential pathways for metal uptake and resultant morphological change in fungi.

Saprotrophic fungi, including chytrids, are required to attach to nutritive substrates because this close contact is necessary for heterotrophic fungi to obtain essential nutrients in oligotrophic environments. Attachment also prevents displacement of the fungus by water and wind (Epstein and Nicholson 1997). For attachment to occur the zoospore must often first overcome a hydrophobic surface or an electrostatic repulsion

2 barrier (Jones 1994). Attachment of fungal spores to the surface of a host involves an initial passive, reversible attachment followed by active, irreversible attachment process (Jones 1994). Passive attachment includes random contact with the surface, entanglement (Lilje and Lilje 2008), chemotaxis and spore appendage mediated attachment. Active attachment involves excretion of extracellular mucilaginous matrix (ECM) (Epstein and Nicholson 2016), spore differentiation and germination (Jones1994), possibly followed by penetration of the substratum/host. The zoospores of some chytrids have ECM cell coats which are produced during zoosporogenesis (Powell 1994) glycoproteins have been identified as a component of zoospore cell coats (Dalley and Sonneborn 1982). Upon encystment the cell coat composition changes to become sticky, either due to synthesis of new material or due to secretion from material stored in vesicles (Powell 1994). However, the chemical compositions of ECM and other fungal glues are yet to be characterised (Epstein and Nicholson 2016). Fungal ECMs are known to be comprised of proteins, carbohydrates, lipids, and nucleic acid; however there is still considerable complexity and diversity between the fungi (Zarnowski et al. 2014). Although most microorganisms live within self-produced ECM biofilm in the environment, the composition and functions of these complexes are poorly understood (Zarnowski et al. 2014).

In heterotrophic fungi attachment to substrates is often necessary in order to initiate germination (Jones 1994). The ascomycete, Phyllosticta ampelicida, requires conidial attachment to hydrophobic surfaces in order for germination to proceed (Shaw et al. 2006). However, when grown in high nutrient media in vitro chytrids do not require attachment to substrates in order to encyst and germinate. The swimming zoospores may attach either to vessel surfaces or to each other, followed by germination and growth of sporangia (Richardson et al. 1998; Lilje and Lilje 2008). Similarly, the anaerobic rumen fungus Neocallimastix frontalis may germinate and form mature thalli in vitro followed by attachment to filter paper (Richardson et al. 1998). In the soil and aquatic environments from which many chytrids are isolated nutrients are limited and numerous other heterotrophic microorganisms are competitors for nutritive substrates. In these environments chytrids require close contact with the organic substrate in order to absorb nutrients through the cell wall (Jones 1994).

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Fungal attachment to organic substrates is a necessary step in the carbon cycle and within the soil food web. The degradation of organic substrates occurs by the attachment and sometimes penetration of hyphae and rhizoids and also through the activity of excreted enzymes. Degradation of organic substrates provides organic and inorganic nutrients which may also be taken up by other microorganisms. Certain chytrid species are known to digest crystalline cellulose or carboxymethyl cellulose (Gleason et al. 2011), thereby converting particulate carbon to more accessible forms for uptake by other microbes .Over time as chytrid fungi degrades organic compounds (Gleason et al. 2012) in the soil they will reduce the concentration of inorganic nutrients, such as nitrate and sulphate (Digby et al. 2010,) and also dissolve relatively insoluble phosphate (Midgley et al. 2006). Fungal attachment, in the form of aggregation of individual thalli to form colonies, provides habitat for other organisms which assemble around the outside of fungal colonies where ECM is a source of nutrition for bacteria (De Boer et al. 2006). Internally, colonies may provide protection of organic material from degradation. This persistent organic material is hypothesized to include microbial debris and hyphal fragments (Tisdale and Oades, 1982). Increased microaggregation of chytrids may protect organic matter, including dead chytrid thalli, from chemical and physical decay, by controlling pH and oxygen levels and excluding or reducing enzymatic activity of other microbes, thereby increasing soil carbon storage.

Zoospores are known to attach to substrates of plant and animal origin, such as pollen, keratin, cellulose and chitin (Haskins 1939; Joblin 1989; Mitchell and Deacon 1986; Richardson et al. 1998; Sparrow 1960) which are sources of nutrition for the developing sporangia. The zoospores of some chytrid species preferentially colonize certain substrates over others (Mitchell & Deacon 1986; Powell 1993; Gleason et al. 2011), indicating surface recognition by zoospores (Gleason and Lilje 2009). The rumen chytrid Neocallimastix frontalis attaches to cellulosic material with rhizoids (Richardson et al. 1998), which are then associated with the physical disruption of the material (Gleason et al. 2011; Joblin 1989). Rumen chytrids are also known to attach to organic particles through an adhesion pad, possibly with the aid of ECM (Powell 1994). The triggers for rhizoidal growth and ECM production are not well known (Powell 1994). Chytrids also attach to each other, forming colonies, both in liquid and on solid media (Lilje and Lilje 2008). Colony formation is hypothesized to promote survival and growth of Boothiomyces sp. (AUS6) by facilitating the formation of a microclimate (Lilje and Lilje 2008), which may

4 be essential for survival in soil. Adherence of zoospores may be due to ECM which is produced at different stages in the chytrid life cycle, including during encystment (Powell 1994). In AUS6, ECM has been described on both the rhizoids and the sporangial cell wall (Lilje and Lilje 2008). The benefits of colony formation are likely to include reduced predation, increased water availability, increased nutrient availability and reduced exposure to toxic levels of metals.

The toxic effect of metals in soil is determined by the bioavailability of the individual metal (Nolan et al. 2003). In aquatic environments the free cupric ion (Cu2+) is a better indicator of toxicity than the cuprous ion (Cu ll) (Jonas 1989). However many additional chemical and physical environmental variables affect the lability and therefore toxicity of metal ion activities in soils. Lability is determined by a number of soil physicochemical characteristics including electrical conductivity, redox potential, pH, ionic strength, organic carbon and metal hydroxides in combination with soil-metal contact time and the intrinsic lability of each metal (Mao et al. 2017). While the role of different anions in metal salt

-, 2- toxicity to fungi has not been fully explored, it is expected that anions such as NO3 PO4

2+ and SO4 which are essential nutrients will be toxic at higher concentrations than others such as Cl- and Fl- . For example, development of the soil arthropod Poecilus

(Pterostichus) cupreus was found to be more susceptible to CuCl2 than CuSO4 (Schrader et al. 1997). However, the zoosporic lifecycle stage of P. cinnamomi was much more sensitive to cations than anions, with most cations causing encystment and Ca2+ also

- - - inducing subsequent germination, while none of the anions, including Cl , NO3 , F ,

2- 2- - H2PO4 , SO4 , CH3COO caused encystment (Byrt et al. 1982).

Chytrids grow saprotrophically and are therefore directly exposed to metals in soils, both in the soil solution and bound to organic substrates. Metals which are essential micronutrients are regulated at the cellular level in order to maximise growth and reproduction, thus increasing numbers of attached chytrid sporangia and entire colony biomass. For example, ferritin-regulating genes are modulators of cellular iron levels and occur in zygomycetes and chytrids (Canessa and Larrondo 2013). It is also expected that all metals will prove toxic to chytrids at increased concentrations as has been established for oomycetes (Leano and Pang 2010, Slade and Pegg 1993). The toxic effect of metals on eukaryotic microorganisms in soil environments is complex and likely to vary on the microscale. Fungi interact with metals in the soil through important biogeochemical

5 processes (Gadd and Raven 2010). Fungi cause redistribution and transformation of metals in soils through a balance between mobilization processes, such as; solubilisation, redox mobilization and methylation, and immobilization processes including; biosorption, intracellular accumulation, biomineral formation, redox immobilization and sorption to biominerals (Gadd 2010). The role of fungi in metal transformation depends on physicochemical factors which determine growth and activity, including the availability of nutrient sources, such as metals (Gadd and Raven 2010). We predict that the presence of micro molar quantities of essential metals will cause increased growth and attachment until toxicity thresholds are reached. We also predict that when toxicity thresholds are reached all metals are expected to reduce the ability of chytrids to grow, reproduce, complete their lifecycle, and to colonise organic materials. This work will contribute significantly to our limited understanding of the responses of chytrids, at different stages in the lifecycle, to soluble metals, thus improving our knowledge of the roles of chytrids in the soil ecosystem.

1.2 Effects of toxic metals on chytrids, fungal-like organisms and higher fungi

The section has been published within: Henderson LE, K Robinson, FH Gleason and O. Lilje 2017: Effects of toxic metals on chytrids, fungal-like organisms and higher fungi. – In: DIGHTON J. & J.F. WHITE (Eds): The Fungal Community: its Organisation and Role in the Ecosystem 4th Edition, pp. 433 – 458. – CRC Taylor and Francis.

1.2.1 Introduction

Some metals, particularly the toxic metals, may accumulate in soils and in aquatic food webs, negatively affecting microorganisms and ecosystem functions, including nutrient cycling. The effect of metals in soils is a product of inherent toxicity due to chemical properties as well as the availability of the metals in solution. Many aspects of fungal growth, metabolism and cell differentiation are affected by metals (Gadd 1993). Toxic metals require immobilization in non-bioavailable forms to become less toxic. Toxic effects are apparent in fungi at the individual species level (gene expression and physiology, activity of enzymes), population levels (ecotypes) and community levels. The

6 challenge is to understand how toxicity effects at each of these levels impact on structural and functional diversity in soil and aquatic environments.

The study of community fungal composition and dynamics has become an emerging field of research (Colpaert et al. 2011). The use of biomarkers, such as Phospholipid Fatty-acid Analysis (PLFA), to study changes in community structure has largely been replaced by DNA and rRNA analysis used for phylogenetic resolution. These new techniques in molecular biology include; multiplex-terminal restriction fragment length polymorphism (M-TRFLP), denaturing gradient gel electrophoresis (DGGE), ribosomal intergenic spacer analysis (RISA), and randomly amplified polymorphic DNA (RAPD). An increased understanding of the impact of toxic metals on fungal communities will provide strategies for remediation of metal-polluted landscapes and waterways. Our ability to predict and adapt to climate change also requires understanding ecosystem functions mediated by fungi. This chapter considers the effect of metals on soil and aquatic fungal populations and their physiological and morphological strategies for surviving toxic metals.

1.2.2 Fungal Communities in metal-polluted and metal-rich environments

Soil conditions, such as moisture content, organic matter content, pH and nutrient availability have long been known to influence the effect of toxic metals on soil microbial diversity (Lofts et al. 2004; Stefanowicz 2012; Azarbad et al. 2013; Op-De Beeck et al. 2015). The availability and speciation of metal ions in the soil environment also determines toxic effect, for example, the resistance of soil microbiota to Cr, Cd and Pb is influenced by bioavailability (Caliz et al. 2013). The combination of soil conditions in the presence of metals affects the growth and metabolism of microorganisms, causing changes in community composition (Frey et al. 2006, Akerblom et al. 2007). In a study on metal exposure effects on soil microorganisms, Khan et al. (2010) found that bacteria were more sensitive to Cd and Pb than fungi and actinomycetes. Further, in a unique 11 year study on sewage sludge addition to agricultural soils, the use of M-TRFLP found a dose- dependent shift in fungal, bacterial and archaeal species composition for Zn and Cu- affected soils, presumably toward metal-tolerant species (MacDonald et al. 2011).

Other soil organisms may mediate the toxic effects of metals on microorganisms. Plant species diversity is positively correlated with soil microbial biomass, respiration,

7 activity and functional richness in the presence of toxic metals (Stephanowicz 2012) while Sumi et al. (2015) found that root exudates in a Zn and Cd contaminated soil have a protective effect on fungal metabolic activity within the rhizosphere. The effects of toxic metals on populations of fungivorous soil invertebrates must also be considered. For example, mycophagous isopods are known to increase fungal diversity and function as keystone species within a fungal community (Crowther et al. 2013). Reproduction rate of the mycophagous Folsomia candida declined when fed mycelium grown on media with high levels of Cu (Pfeffer et al. 2010).

Predicting the effect of toxic metals on soil fungal communities under climate change is complex. It is certain that climate change is having an impact on soil fungi (Kauserud et al. 2010). In a study on soil macrofungi from 1960 – 2007 in the United Kingdom and Norway, Kauserud et al. (2010) found a significant shift of fruiting to earlier in the spring. It is expected that changes in plant physiology (especially root structure) in response to climate change will impact on the diversity and activity of rhizosphere microbes, potentially changing the rates of metal uptake. Also, changes in soil chemical (CEC, OC and pH), physical and biological characteristics may affect metal bioavailability. In studying arbuscular mycorrhizal fungi (AMF) under climate change, community composition in soils, but not in roots, was significantly affected by global warming (Yang et al. 2013), with warming alone increasing the abundance of Glomeraceae.

Certain fungi have a number of adaptive physiological responses which allow them to avoid exposure to metals. For example, increased resistance to toxic metals has been found in arbuscular mycorrhizal (AMF) fungi isolates from metal polluted soils (Gonzalez- Chavez et al. 2002). Glomus intraradices significantly increased presymbiotic hyphal length in the presence of Cd and Pb, even at metal concentrations that partially inhibited spore germination (Pawlowska and Charvat (2004), presumably to allow penetration into less toxic microsites. Isolates from metal-affected soil may also reduce metal uptake rates to symbiotic partners. Langer et al. (2012) showed that the inherent mycorrhizal community from a metal-polluted soil acted as a barrier to uptake of zinc by Populus tremula at high Zn levels in the soil.

Stress caused by the presence of toxic metals may affect the structure of fungal communities in the soil. Metal-resistant fungi were isolated from agricultural soil which had received long-term applications of municipal and industrial wastewater, predominantly;

8

Aspergillus, Penicillium, Alternaria, Geotrichum, Fusarium, Rhizopus, Monilia and Trichoderma (Zafar et al. 2007). A shift towards increased frequency of tolerant fungi at the expense of non-tolerant may change fungal community composition. Increased frequency of tolerant species or ecotypes and decline of non-tolerant may change soil functional diversity or cause a loss of soil function. With the availability of molecular tools for analysis of the soil fungal community it is now possible to explore structural fungal diversity shifts in response to metals in the soil environment.

Certain toxic metals appear to have an obvious impact on some fungal communities. A reduction in the number of ectomycorrhizal (ECM) species and a shift in species composition was observed near a copper smelting site, using PCR amplification and morphotyping (Rudawska et al. 2011). The shift in species composition was predominantly from Phialophora finlandia to Atheliaceae species. With the use of community-level fingerprinting via DGGE Chu et al. (2010) observed a significant effect of increasing Cu on Fusarium and Trichoderma community composition. In a post-mining pine-ectomycorrhizal community, increasing Mn correlated with the increased presence of Atheliaceae and decrease of Russulaceae (Huang et al. 2014). Although Huang et al. (2012) found no effect of Pb, Zn or Cd on ECM community structure, Rhizopogon buenoi, Inocybe curvipes, Tomentella ellisii, and Suillus granulatus occurred in soils which were significantly higher in Cu. The addition of Hg changed ECM composition and also species richness in an in vitro study on a community isolated from non-polluted pine soils (Crane et al. 2012). In the presence of 88 μg g-1 Hg there was a significant decline in species richness, with the resultant ECM community dominated by one or two presumably Hg tolerant morphotypes.

The ability of ECM communities to function in soils contaminated with toxic levels of heavy metals may explain why they are often encountered in pioneer situations, for example, in metal-rich mine soils (Huang et al. 2012). Ecotypes of Suillus luteus are often found in pioneer conditions on Zn- and Cd-polluted sites in the Campine region in Belgium (Colpaert et al. 2000, 2004) There is considerable evidence that these communities are necessary for the restoration of plant communities by alleviating metal toxicity (Meharg and Cairney 2000, Adriaensen et al. 2006, Colpaert et al. 2011). However, the evidence is conflicting on the effects of metals on ECM communities; in some field studies species diversity declines and in others there is simply a change in abundance of fungi within an on-going highly diverse ECM community. Reduced diversity of ectomycorrhizal fungal

9 communities in toxic metal-contaminated areas (Staudenrausch et al. 2005, Ruotsalainen et al. 2009) contrast with studies showing highly diverse ECM fungal communities in metal-rich sites, with no selection for toxic metal-tolerant species (Blaudez et al. 2000b, Cripps 2003, Krpata et al. 2008, Colpaert et al. 2011, Hui et al. 2011). In soils of reafforested Hunan province minelands Huang et al. (2012) found ECM colonization rate, abundance, diversity and richness on Masson pine root tips was not significantly affected by the occurrence of elevated Pb, Zn and Cd. In contrast, although Op De Beeck et al. (2015) found little effect on fungal diversity, they found a significant difference in abundance of ECM species correlated with Zn and Cd levels. Metal polluted soils contained more abundant Suillus luteus, Roussel sp., Sagenomella humicola, Cadophora finlandica, Wilcoxina mikolae and Inocybe lacera while metal un-affected soils contained more abundant Rhizopogon luteolus, Rhizoscyphus ericae and Vonarxia vagans.

Evidence of adaptive tolerance of some fungi can be found from soil types which are naturally high in certain toxic metals. Soils developed on serpentine geological substrates, containing high Ni levels, have a broad taxonomic range of ECM and co-associated fungi (Urban et al. 2008, Moser et al. 2009). However, Cenococcum geophilum isolates from serpentine soils were found to have significantly higher tolerance to Ni than isolates from non-serpentine soils (Goncalves et al. 2009) and are therefore tolerant ecotypes. The majority of ECM isolates from high selenium soils in the Eastern Rocky Mountain ranges were also found to have significantly higher tolerance to Se than those from low Se control soil (Wangeline et al. 2011). Kohout et al. (2015), however, found AMF root colonisation rates declined as the serpentine character and Ni concentration of the soil increased. Doubkova (2012) also found lower root colonisation for Glomus overall in serpentine soils, however, there was increased colonisation by tolerant over non-tolerant Glomus; also AMF isolates from serpentine soils significantly enhanced above ground plant growth in these soils whereas those from non-serpentine soils did not. There was no evidence of a unique microbial community within serpentine soils and no increase in the proportion of AMF, although these soils have a distinct chemistry (increased B, pH, Cd, Ni, Cr) (Fitzsimons et al. 2010). Richness diversity and evenness are not different from non- serpentine soils. Therefore, plant growth on these soils does not rely on a unique community of AMF soil mutualists. However, other studies have shown a distinct difference in AMF community structure (Schechter and Bruns 2008). It is also clear that

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Ni-tolerant AMF may differ in their abilities to protect plants from symptoms of toxicity, presumably by increasing P and decreasing Ni uptake (Amir et al. 2013).

Hassan (2011) explored the diversity of AMF in urban soils heavily polluted with trace metals (As, Cd, Cu, Sn, Pb and Zn). AMF diversity index in uncontaminated soils was significantly higher than those in contaminated soils and species richness was significantly lower in contaminated soils than in uncontaminated soils. Community structure was modified, resulting in a small number of Glomus sp., including Glomus mosseae, dominant in polluted soil. In the vicinity of a copper smelter the diversity and evenness of the AMF and the entire soil fungal community declined with increasing metal in the soil litter layer, however the ECM community composition did not change significantly (Mikryukov et al. 2015). In a severely metal polluted Cu and Fe mine site in Guangdong, China (Cu 532 – 968 mg kg-1, Zn 1554 – 3125 mg kg-1, Pb 3628 – 7116 mg kg-1 and Cd 2.4 -12.7 mg kg-1), Long et al. (2010) using DDGE analysis, found 60% of AMF genotypes in plant roots and rhizosphere soil were related to Glomus. Also, in a similarly high Cu, Pb and Zn ash dump, Glomus, including Glomus intraradices/fasciculutum were the most abundant genotype (Bedini et al. 2010).

AMF sequence types were found to correspond to four categories of heavy metal contamination (Zarei et al. 2010) in a Pb and Zn mine, indicating tolerant and non-tolerant isolates. Variation in the Glomus community was also affected by Pb, Zn, plant-available P and calcium carbonate levels in soils. He et al. (2014), using meta-analysis of AMF data from 1970 – 2012, found the beneficial effect of Glomeraceae on plant growth increased and that of non-Glomeraceae decreased under high levels of toxic metal. This relationship was reversed when toxic metals were not present, indicating constitutive tolerance of Glomeraceae to toxic metals. This tolerance allows the fungi to function in toxic metal environments and employ specific metal resistance mechanisms.

Most of the work on toxic metal effects in fungal communities has been focused on ECM and AMF, due to the importance of these fungal groups in forest ecosystems and mine rehabilitation. Little research is available on toxic metal effects on many other commonly occurring soil fungi. For example, the ascomycete Trichoderma is a commonly- occurring plant symbiont. One available study found the in vitro growth of thirteen Trichoderma isolates was completely inhibited by 20 µmolL-1 Cu (Petrovic et al. 2014). Toxic metal effects on another group of soil fungi, melanin-rich Dark Septate Endophytes

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(DSE) have recently been explored. Likar and Regver (2009) looked at AMF and DSE isolated from Salix caprea (goat willow) roots in the vicinity of a Pb smelter. The frequency of DSE colonisation of Salix caprea roots was positively correlated with soil Pb, Cd and plant-available P with Phialophora, Phialocephala and Leptodontidium increasing in abundance in metal-rich locations. A 250-year old Pb and Zn slag heap yielded DSE dominated by Exophiala and Phialophora, 82% of isolated root-associated fungi were DSE related (Zhang, Li and Zhao 2013).

Although little has been published on metal-mediated community changes in fungal- like soil organisms, plant pathogens such as oomycetes have received some attention. The effect of metals on aspects of the lifecycle of oomycetes Phytophthora and Pythium (Slade and Pegg 1993) found metals, in general, are toxic at lower concentrations for zoospores than at any other stage of the life cycle, with toxicity in the order Ag>>Cu>Ni>Co>Zn. Ag was toxic at 50 ppb. Increased Cd, Ni and Zn weakened the antagonistic interaction of Trichoderma against Pythium irregular (Naar 2006). However, Kredics et al. (2001) found metal resistant strains of Trichoderma had a strengthened antagonistic response to Pythium.

1.2.3 Mechanisms of fungal resistance and tolerance to metals

Many fungi grow well in the presence of toxic metals (Gadd 1993, Baldrian and Gabriel 2002). Fungi may even accumulate some of the more toxic metals such as Hg, Pb and Cd (Das et al. 1997). Metal resistance in fungi has been defined as the ability of an organism to survive toxic levels of metal by active mechanisms employed in direct response to metal species (Zafar et al. 2007). Extracellular mechanisms include precipitation, complexation and crystallisation of metals via the production of organic acids, polysaccharides, melanins and proteins (Gadd, 1993; Baldrian 2003). Intracellular mechanisms include decreased influx, increased efflux, compartmentation (in vacuoles) and sequestration of metals by metallothionein (proteins) and glutathione peptides (phytochelatins) (Gadd 2007, Das and Guha 2009). Fungal resistance is metal and species specific and depends on environmental conditions, such as pH and nutrient source.

The fungal cellular responses involve a range of tolerance and resistance mechanisms which together determine the response of the fungi and its survival in the presence of toxic metal. This explains why, for example, there was no direct relationship

12 found between tolerance of Aspergillus and Rhizopus isolates from metal contaminated agricultural soils and biosorption ability (Zafar et al. 2007). Likewise, the tolerance of four DSE of Salix variegate to Cd was not significantly related to Cd uptake by the fungi (An et al. 2015). If fungi can accumulate toxic metals in their cytoplasm during growth, they may provide a method which can be used to remove toxic metals from contaminated soils.

1.2.4 Physiological responses of fungi to toxic metal stress

Metal homeostasis in fungi requires the uptake, storage and secretion of metals to prevent cell damage. A large number of gene-encoded, highly specific low and high- affinity metal transporters act in organelles and the plasma membrane to maintain metal ion homeostasis (Nevo and Nelson 2006). At the cellular level, metals exert toxic effects by interacting with proteins and nucleic acids, or by the induction of reactive oxygen species (ROS). Toxic metals change enzyme expression, damaging DNA and proteins. Increased ROS and resultant oxidative stress leads to redox imbalance causing cell membrane damage and lipid peroxidation. In order to counteract oxidative stress, fungi utilize enzymatic and non-enzymatic detoxification systems. Enzymatic responses include catalases (CAT), peroxidases and superoxide dismutases (SOD), while non-enzymatic responses include phenolic compounds, glutathione, thiols, cysteine, polyamines, ascorbate and carotenoids (Valco et al. 2006).

1.2.4.1 Toxicity

Fungi generate ROS as a metabolic reaction to various environmental stressors including; light, ionizing radiation, temperature changes, mechanical damage (Gessler et al. 2007). ROS are also important in cellulose and lignin decomposition in wood decomposing fungi and possibly as protection against competitive microflora. ROS, including hydrogen peroxide (H2O2), may act as messengers in the extracellular environment and also within the cells of eukaryotes (Rhee 2006) including for cell signalling in differentiation and proliferation. The induction of reactive oxygen species leads to oxidative stress and eventually overwhelms the cellular defences as metal concentration increases. Cd inhibits the mitochondrial electron chain and causes the formation of superoxide radicals, which in turn cause lipid peroxidation (Chen et al. 2014). Pb changes the redox levels in the cells of Phanerochaete chrysosporium (white rot fungi) (Wan et al. 2015) in a concentration and time-dependent manner. Cd accumulation in P.

13 chrysosporium and subsequent increased H2O2 triggers the production of SOD and CAT enzymes (Xu et al. 2015b).

Toxicity affects the cell membranes and cellular transport processes. There is a difference in cellular uptake depending on the metal species, for example, Cuny et al. (2004) found a metal resistant lichen Diploschistes muscorum, took up soluble and residual Cd intracellularly, while Zn was predominantly taken up extracellularly in residual form. In gene deletion studies of Saccharomyces cerevisiae, Pb has been found to affect many cellular processes, with cellular transport processes particularly sensitive to Pb and Cd ions (Du et al. 2015). A combination of Cd, Pb and Zn reduced membrane permeability and cell proliferation of the yeast Pichia kudriavzevii over time (Mesquita et al. 2015). The loss of cell permeability was attributed predominantly to Pb, which also accumulated at a faster rate within the cells. Cd causes significant cell damage in P. chryosporium including; rigidification of the plasma membrane, reduction in the H+-ATPase activity in the plasma membrane, increase in mitochondrial membrane permeability, mitochondria membrane- potential breakdown and eventual cell death (Chen et al. 2014).

The role of ATPases in the resistance of fungi to toxic metals is an emerging field of study. ATPases are membrane-bound ion channels with distinct metal-binding domains which facilitate ion movement, coupled with the synthesis or hydrolysis of ATP. The highly conserved protein V-ATPase is a proton pump of cellular organelles, including vacuoles, which regulate metal homeostasis. V-ATPase regulates intracellular pH, receptor- mediated endocytosis, coupled transport of small molecules and ions, and oxidative stress response. Vacuolar storage of toxic metal is an effective resistance mechanism. Loss of V-ATPase activity leads to sensitivity to metals (Ramsay and Gadd 1997), ROS accumulation, endogenous oxidative stress ( Milgrom et al. 2007) and impaired growth, for example in Candida albicans (Jia et al. 2014). Reduction of Ca ATPase function in golgi of the filamentous entomopathogenic fungi Beauveria bassiana facilitated sensitivity to Zn2+,

2+ 3+ Cu and Fe (Wang et al. 2013). In S. cerevisiae a P1B-type ATPase upregulates the export of intracellular Cd (Adle et al. 2007), while inactivation of the gene which regulates Ca ATPase slows Cd uptake (Mielniczki-Pereira et al. 2011). This indicates a potentially critical role for plasma membrane ATPases in both intracellular signalling and resistance to toxic metals.

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1.2.4.2 Sporulation and germination

Ni had a dose dependent effect on Glomus, reducing both rates of colonization and sporulation for two isolates from serpentine (high Ni) soils in New Caledonia (Amir et al. 2013). In contrast, Pawlowska and Charvat (2004) found spore germination to be less sensitive than all other stages of the lifecycle for two Glomus isolates exposed to Cd, Pb or Zn. The two Glomus isolates also responded differently to Cd, Pb and Zn. Symbiotic sporulation declined in G. intraradices as hyphal density declined and metal level increased, however spore germination rates between G. etunicatum and G. intraradices differed. Spores of G intraradices germinated following incubation in 1 mM of Cd or 10 mM of Zn, while G. etunicatum spores did not germinate (Parlowska and Charvat 2004). G intraradices spores also recovered from incubation with metal. Cu stimulated the early production of sclerotia initiation and maturation in Penicillium thomii (Zhao et al. 2015).

Aquatic hyphomycetes are essential in aquatic energy and nutrient cycling as they perform the first step in the degradation of aquatic plant detritus, enhancing nutritional availability for invertebrates (Pascoal et al. 2005). Both essential (Ca, Zn, Cu) and non- essential (Cd) metals reduced sporulation in a hyphomycete population from a Canadian stream; sporulation declined at 500 µg L-1 for Ca, 250 µg L-1 for Cu, 62.5 µg L-1 for Zn and 2 µg L-1 for Cd. Sporulation ability of Anguillospora filiformis, A. longissima, and Clavatospora tentacula in the presence of all metals indicated higher tolerance than Tricladium angulatum or Varicosporium elodeae. A sporulation peak occurred between 0.1 and 5 µg L-1 for aquatic hyphomycetes exposed to the above metals. This is interpreted as a hormetic, or compensatory, response to toxic metal (Sridhar and Baerlocher 2011). There are implications for the effect on life cycles of aquatic hyphomycetes given widespread low levels of metal contaminants and the effects of increased bioavailability of metals due to acidification from industrial pollution.

Zoosporic fungi and fungal-like organisms are impacted by numerous metals differently at different stages in the lifecycle. Many soluble cations affect oomycete zoospores, causing encystment and in some cases increased germination, however few studies have confirmed these results in vivo. Cysts are often the most tolerant to high levels of cations and motile zoospores the least tolerant. In a study of the response of four soil zoosporic true fungi to Cu, Pb and Zn, Cu (60 ppm) was found to be most toxic to growth and reproduction and Pb (100 ppm) least toxic (Henderson et al., 2015). Four

15 mangrove community oomycetes Halophytophthora sp. produced abnormal oogonia when incubated with 10 ppm Cu (Leano and Pang 2010). Ag is highly toxic to zoospores of Phytophthora sp. at 50 ppb (Slade and Pegg 1993). There was a dose-responsive relationship to increasing Cu for Labyrinthulomycota (thraustochytrids) isolated from mangrove communities, with growth and sporulation success declining with increasing Cu. However, exposure to low (2 – 64 mg L-1) levels of Cu had a stimulatory effect on sporulation and growth for some isolates (Pang et al. 2015). Increased growth and zoospore release at low levels of Cu (10 ppm), Pb (60 ppm) and Zn (10 ppm) was also demonstrated for soil zoosporic true fungi (Henderson et al. 2015), however this response varied with fungal isolate and metal species. The aquatic zoosporic fungus Blastcladiella emersonii is responsive to Cs and Rb causing spontaneous germination (Soll and Sonneborn 1972), however Li did not cause germination. An osmotic response was reported for the oomycete Aphanomyces astaci with metals differentially inducing encystment and germination (Sensson and Unestam 1975). Cysts of three Pythium isolates increased in germination rate in response to 35 mM Ca, Mg or Sr but declined in response to La (Donaldson and Deacon 1992). Ca or Sr at 30 mM caused encystment of 90% of Phytophthora cinnamomi zoospores, and Mg caused 50% encystment, however spontaneous germination only occurred in those encysted with Ca (Byrt et al. 1982).

Metal nanoparticles are an emerging risk to terrestrial and aquatic environments. Toxic metals are increasingly common within the environment in particulate form as nanoparticles. Metal nanoparticles are prone to sorption to organic particles and are readily transported in aquatic environments. They have been found to decrease species richness and shift fungal species composition. In a study on fungal leaf litter decomposition in a forested stream, sporulation declined at 100 ppm for both Ag and CuO nanoparticles, with shift in species composition away from Flagellospora sp. and towards Heliscus lugdunensis (Pradhan et al. 2011).

A warming climate is expected to decrease the length of the fungal growth cycle. It is expected that spore production and germination will increase (Kauserud et al. 2010). Also the level and bioavailability of contaminants in soils and waterways will be affected by climate change (Batista et al. 2012). In a study of a freshwater ecosystem, the effect of increased Cd and higher temperature was observed (Batista et al. 2012). The cadmium concentration inhibiting 50% of reproduction was found to be lower at 21°C than 15°C, suggesting that toxicity of Cd is higher as temperature increases. Increased Cd caused a

16 decrease in fungal diversity, assessed as sporulating species (Batista et al. 2012, Moreirinha et al. 2011); however, some fungi were found to increase spore production, including Fusarium, Alatospora pulchella, Tetrachaetum elegans and Triscelosphorus acuminatus. An overall decline in fungal sporulation and microbially mediated leaf decomposition occurred at 1.5 mg L-1 Cd (Batista et al. 2012). After release from Cu and Zn stress, fungal reproduction recovered (Duarte et al. 2008).

1.2.4.3 Enzyme and metabolite activity

Up regulation of enzymes may be the first line of defence for fungi against toxic metals. Toxic metal exposure induces the production of ROS such as hydrogen peroxide, superoxide, and hydroxyl radicals, causing lipid peroxidation, which increases membrane permeability (Xu et al. 2011) and causes DNA damage (Collin-Hansen et al. 2005). Levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and guaicol peroxidase (POD) in P. chrysosporium are responsive to Cd concentration and time of incubation in liquid culture. Low concentrations or short time (2 hr) exposure to Cd increases enzyme production (Chen et al. 2014). Cellulolytic hydrolase and lipid peroxidase (LiP) expression are modulated by Cu, Pb, Zn and Mn in Pleurotus ostreatus (Baldrian et al. 2006). Enzyme production, in particular SOD, coincides with lipid peroxidation levels in P. chryosporium, with SOD significantly correlated to ROS production at lower concentrations of Cd (Zeng et al. 2012). SOD upregulates at lower concentrations of Cd, even before glutathione production. It is likely that changes in the activities of antioxidant enzymes such as SOD, CAT, POD and glutathione reductase (GR) are an adaptive response to toxic metal exposure. These enzymes remove ROS and their products (Bai et al. 2003). SOD catalyzes the dismutation of the superoxide radical to

H2O2 and O2, which is then degraded by POD and CAT to H2O and O2.

Li et al. (2015) found SOD and POD levels in soil were increased by Tricholoma lobayensis in the presence of Pb. This increase was further accentuated by co-inoculation with a Pb-tolerant Acillus thuringiensis. SOD rates were significantly upregulated by Cd in the metal resistant lichen Diploschistes muscorum, which took up soluble and residual Cd intracellularly (Cuny et al. 2004). In Bjerkandera adusta (white rot fungi) low concentration of Se (0.5 mM) doubled manganese peroxidase (MnP) production, whereas higher concentrations of Se (200 mM) inhibited MnP production and increased lipid peroxidation levels (Catal et al. 2008). While production of ligninolytic oxidase (LiP) and MnP by P.

17 chryosporium were inhibited by the addition of Cd (Baldrian 2003, Xu et al. 2015a), low levels of Cu (1.2 µM) and Zn (18 µM) increased LiP and MnP (Baldrian 2003). In Penicillium thomii Cu stress upregulated the enzymes SOD, CAT, APX, and GR at 100 µg ml-1 CuSO4 (Zhao et al. 2015). SOD, CAT and GR were upregulated in Oudemansiella radicata, increasing at low Cu levels then reducing at high Cu (Jiang et al. 2015). This is indicative of upregulation followed by depletion due to reaction with Cu.

Species of metal or metal combinations affect the secretion profile differently. All hydrolases (acid phosphatase, b-glucosidase, b-galactosidase and N-acetyl-b- glucosaminidase) of the saprophyte Trametes versicolor were inhibited while all oxidases (laccase, Mn-peroxidase) increased in the presence of Cu, Cd and combined Zn, Cu, Pb and Cd (Lebrun et al. 2011). Pb was the most inhibitory metal and lignin peroxidase was only produced in the presence of Cu. Apart from Hg, all other metals tested did not inhibit extracellular enzyme production in Trichoderma isolates, even at the same levels at which mycelial growth was inhibited (Kredics et al. 2001) and in some cases β-glucosidase, cellobiohydrolase and β-xylosidase enzymes increased when incubated with Al, Cu or Pb. Ferric reductase activity was significantly enhanced in Pythium when incubated with nanoparticles of ZnO and significantly reduced with nanoparticles of CuO (Zabrieski et al. 2015).

There is little work available on the effect of metals on enzyme expression in aquatic hyphomycetes, however some studies found detrimental effects on sporulation and growth. Some aquatic hyphomycetes increase the activity of antioxidant enzymes, particularly SOD and CAT in the presence of Zn and Cu (Azevedo et al. 2007). Generally toxic effects are greater for ionic rather than nanoparticle forms of metals (Pradhan et al. 2011). Hg had a negative effect on laccase production in the hyphomycete Chalara paradoxa (Robles et al. 2002). It is expected that enzymatic responses of oomycetes to toxic metals are common as marine thrustochytrids contribute significant β–glucosidase, aminopeptidase and phosphatase to marine sediments (Bongiorni et al. 2005).

1.2.4.4 Non-enzymatic responses

The production by fungi of low molecular weight organic acids (LMWOA), such as oxalic acid, is a non-enzymatic response dependent on pH, source of N (NH4 or NO2) and species of metal ion (Sazanova et al. 2015). Oxalic acid is the most common secreted organic acid in filamentous fungi of Ascomycota, Basidiomycota and Zygomycota (Makela

18 et al. 2010). Oxalic acid immobilizes toxic metal by the formation of metal oxalate crystals (Sutter and Jones 1985; Xu et al. 2015a). The white rot fungus P. chrysosporium, well known for its ability to take up metals, produces oxalic acid in the presence of numerous metals and chelates metal in the form of organic acid crystals. Oxalic acid production by P. chrysosporium in the presence of Cd is rapid and dependent on Cd concentration (Xu et al. 2015a), while the addition of oxalic acid alleviates toxicity, increases MnP and LiP activity and also increases Cd uptake. Production of LMWOA varies depending on metal, pH, type of media and nitrogen source for Aspergillus niger and Penicillium citrinum. Only citric and succinic acid are produced in the presence of Cu, while oxalic acid is also produced in the presence of Zn (Sazanova et al. 2015).

The metallothionein-encoding CUP1 gene is implicated in evolved Cu resistance in Sarchomyces cerevisiae as it is upregulated in Cu resistant strains. Metallothionein induction is encoded by two genes in S. cerevisiae, regulated by Cu, through the transcription factor ACE 1, which also induces the transcription of the SOD 1 gene, encoding cytosolic Cu-Zn SOD (Adamo et al. 2012). A Cu-resistant strain of S. cerevisiae accumulated the same amount of intracellular Cu and expressed similar levels of SOD as a non-resistant strain, however non-resistant cells accumulated more oxidative stress as shown by protein carbonylation assays (Adamo et al. 2012), implying the importance of metallothionein induction in Cu resistance. Upregulation of low-molecular weight proteins also occurs in Candida tropicalis when incubated with 100 mg L-1 As, Cd, Cr, Pb and Cu (Ilyas and Rehman 2015), indicating over-expression of proteins such as metallothionein may be a mechanism of resistance to toxic metals.

The tripeptide glutathione is a low-molecular weight sulfur-containing compound abundant in all eukaryotes and is believed to be an important resistance mechanism in the detoxification of intracellular ROS. Reduced glutathione (GSH) is oxidised by reacting with

- oxidative agents such as O and H2O2 resulting in oxidised glutathione (GSSG). Induced oxidative stress involves over production of GSSG, tipping the cellular redox balance towards the oxidative state and toxicity (Ilyas and Rehman 2015). GSH/GSSG ratios varies between metals, with large increases in GSH and cysteine occurred in C. tropicalis when incubated with 100 mgL-1 As, Cd, Cr, Pb and Cu and high levels of GSSG occurred during incubation with As, Cr and Pb (Ilyas and Rehman 2015). Aquatic hyphomycetes produce thiol-containing compounds, including glutathione, to sequester metal ions

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(Guimarães-Soares et al. 2007). However, there is also evidence that Cu tolerance may not be related to GSH activity (Bi et al. 2007, Gharieb & Gadd 2004).

Melanin and carotinoids are also part of the antioxidant defence system of fungi. Melanin is biologically active as an antioxidant, antimicrobial and as a metal chelator (Manivasagan et al. 2013). Melanin protects yeast against degradation by the enzymes produced by other organisms (Garcia-Rivera et al. 2001). In an in vitro study melanisation levels increased in Cryptococcus neoformans as Cu levels increased (Mauch et al. 2013). The black yeast genus Exophiala (order Chaetothyriales), isolated from an arsenic mine, is able to tolerate As at 10 mg L-1 (Seyedmousavi et L. 2011). This tolerance is related to the high concentration of protective melanin in the cell wall. The metal tolerant lichen thalli Pyxine cocoes, when exposed to Cr, declined in Chlorophyll a, b and total chlorophyll content while significantly increasing in carotenoid content (Bajpai et al. 2015). Cu-induced oxidative stress increased the biomass, carotenoid, ascorbate and glutathione content of Penicillium thomii sclerotia (Zhao et al. 2015). Oudemansiella radicata accumulated Cu in the fruiting bodies in a concentration and time-dependant manner, with increased thiols and glutathione (Jiang et al. 2015).

The production of ECM is also an important defence mechanism. The epiphytic lichen Xanthoria parietina has a thick layer of extracellular hydrophobic parietin which functions as UV protection (Kalinowska et al. 2015). This layer is postulated to form a physical hydrophobic barrier to toxic metal ions and metal particulates which are precipitated by secondary metabolites, including norstictic, psoromic and usnic acids. Cysteine, glutathione and phytochelatin contents were lower in X. parietina thalli from which the parietin layer had been removed by washing in acetone. Biofilm growth also allows Candida tropicalis to tolerate levels of toxic Ag which kill planktonic cells (Harrison et al. 2006. Ag was toxic at 25 mM for planktonic cells but biofilm cells were not killed at 150 mM, due to metal precipitation within biofilms and restricted penetration of metals into the biofilm matrix (Harrison et al. 2006).

1.2.5 Effects of toxic metals on chytrids, fungal-like organisms and higher fungi – concluding remarks

Structural changes in fungal diversity occur in response to toxic metals in the soil environment. These changes may or may not be permanent. The shift towards increased

20 frequency of metal-tolerant fungi at the expense of non-tolerant may change soil functional diversity or cause a loss of soil function. Field studies have found a reduction in the number of ECM species and a shift in species composition in long-term metal-polluted soil. Fungal community compositions are also expected to change due to the combined effects of toxic metals and climate change. For example Glomeracae are more tolerant than many other fungi to both toxic metals and increased temperatures. Further study of these shifts in abundance is required.

Fungi are able to both tolerate and resist toxic metals. Extracellular tolerance mechanisms include precipitation, complexation and crystallisation of metals; intracellular resistance mechanisms include decreased influx, increased efflux, compartmentation in vacuoles and sequestration of metals by metallothionein and glutathione. Resistance to toxic metals also includes the upregulation of enzymes such as superoxide dismutase (SOD) and catalase (CAT) and production of low molecular weight organic acids (LMWOA).

Toxic metals cause morphological changes in fungi, including sporulation, germination, hyphal extension and mycelium expansion. Fungi and oomycetes employ different strategies according to the type of metal, its concentration and the environmental conditions. They are highly adaptive; using explorative or exploitative growth strategies to survive. Sporulation of aquatic hyphomycetes and zoospores of true fungi and fungal-like organisms are highly sensitive to toxic metals, which in some cases may stimulate sporulation.

Toxic effects are apparent in fungi at the individual species level (gene expression and physiology, activity of enzymes), population levels (ecotypes) and community levels. Resultant changes in community structure are now determined by DNA and rRNA analysis for phylogenetic resolution. These new techniques in molecular biology include; multiplex- terminal restriction length fragment polymorphism (M-TRFLP), denaturing gradient gel electrophoresis (DGGE), ribosomal intergenic spacer analysis (RISA), and randomly amplified polymorphic DNA (RAPD). These current technologies are also able to identify many individual genes or gene families conferring resistance, such as those encoding metal transport proteins, transcriptional regulators and enzymes involved in GSH metabolism.

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Chapter 2: Maximum temperature for growth and reproduction is similar in two soil isolates of Gaertneriomyces semiglobifer (Spizellomycetales, Chytridiomycetes) from different soil environments in north eastern New South Wales, Australia.

The chapter has been published as: Henderson, L., Ly, M., Robinson, K., Gleason, F.H., Lilje, O. (2018). Maximum temperature for growth and reproduction is similar in two soil isolates of Gaertneriomyces semiglobifer (Spizellomycetales, Chytridiomycetes) from different soil environments in north eastern New South Wales, Australia. Nova Hedwigia: Journal of Cryptogamic Science 106: 485-497.

Chapter one reviewed the lifecycle of chytrids, their potential roles in soil ecosystems and we reviewed the effects of toxic metals on chytrids, fungal-like organisms and higher fungi. In this chapter we examine the ability of two isolates of the same chytrid species, Gaertneriomyces semiglobifer, from two different climatic regions and soil types of NSW, to grow and reproduce at different temperatures. Environmental parameters, such as temperature, are important in determining chytrid growth and abundance. Currently little is known about how the various species of soil chytrid are geographically distributed in Australia. Temperature may be an important factor which limits the geographical distribution of chytrids (Gleason and McGee 2008). If so, increases in soil temperature due to climate warming may affect G. semiglobifer distribution, activity within the soil and the resource utilization capability of the fungus. Changes in the interactions between fungi and toxic metals may also occur. For example, cadmium sorption capacity of the metal- resistant Penicillium janthinillum declined as temperature increased to 40 °C (Cai et al. 2016), and chytrids may be likewise affected. Due to the difficulty of isolating soil chytrids and maintaining the isolates in pure culture, only one isolate from each region has been examined here. Therefore, potential variation in site population response has not been determined.

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2.1 Abstract

Two isolates of the zoosporic fungus, Gaertneriomyces semiglobifer (order Spizellomycetales), one from a semi-arid region and one from a mild temperate region of New South Wales, Australia, had similar patterns of growth and sporulation in response to increased temperatures. Both strains of G. semiglobifer were able to grow in temperatures up to a maximum of 37°C and were able to recover growth after incubation on solid PYG media for 7 days at 40 but not 42°C. Both strains produced viable zoospores when incubated on solid PYG media for 7 days at up to 40 but not 42°C. The two strains grew at similar rates in liquid PYG media. Both strains increased in growth over time at 25°C and initially increased in growth at 37°C to 62 hours, but after that growth declined. Changes in rates of zoospore release for both strains were similar to changes in biomass as temperature increased, except for large increases in zoospore release for both isolates when incubated at 37°C. If temperature causes similar effects in the field it is expected that growth and reproduction of the fungus will be reduced in summer months when daytime air temperatures exceed 37°C. This may have implications for the geographical distribution and abundance of G. semiglobifer in the soils of NSW.

Key words: Zoosporic fungi, Chytrids, Spizellomycetales, Temperature, Fungal Growth Rate

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2.2 Introduction

Although there have been many studies estimating the most likely responses of individual animal and plant species to large temperature increases predicted in the future due to global warming (Hochachka & Somero 2002; Atkin & Tjoelker 2003) there are only a few studies addressing the ability of soil fungi to adapt to similar temperature increases (Romero-Olivares et al. 2015). Additionally, work has mainly focused on soil community- level responses rather than those of individual species (Conant et al. 2011). Soil fungi play an essential role in the carbon cycle. Saprotrophic soil fungi produce extracellular enzymes which degrade soil organic matter into soluble forms for assimilation by living organisms (Falkowski et al. 2008). The growth responses of the different stages of the life cycles of saprotrophic soil fungi to increased temperature are not well understood, although these fungi have important effects on the amount of soil organic matter accumulated in soil, both locally and globally (Crowther & Bradford 2013).

Studies of pathogenic fungi, which are resident in soils, provide some insight into differences in growth response between fungal strains under different temperature regimes. For example, the entomopathogenic species Metarhizium anisopliae is a filamentous fungus that has a broad host range and which shows no growth above 37°C. However, a thermotolerant variant of this species which can grow at 37°C is able to be cultured in the laboratory (de Crecy et al. 2009). Populations of the wheat pathogen Zymoseptoria tritici (syn Mycosphaerella graminicola) (Zhan & McDonald 2011) and barley pathogen Rhynchosporium commune (Stefansson et al. 2013) from warmer parts of the world had higher growth rates at higher temperatures than those from cooler regions. Genetic groups of the insect-pathogenic fungus Beauveria bassiana were found to be associated with habitat types. Those isolates from the warmer agricultural soils grew at higher temperatures than those from temperate forests or the Arctic tundra (Bidochka et al. 2002). Elevated temperatures (30 and 35°C) increased the relative abundance of the potentially pathogenic Aspergillus fumigatus and Scedosporium aurantiacum in natural and anthropogenic soils (Marfenina & Danilogorskaya 2017). The parasitic zoosporic fungi Batrachochytrium dendrobatidis had different growth patterns at the upper thermal limit for three strains from three distinctly different Australian climatic regions (Stevenson et al. 2013). It is clear that local climate conditions play a significant role in the adaptation of certain pathogenic fungal species to growth at higher temperatures.

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In general, there are relatively few studies on the tolerance of biotrophic and saprotrophic soil fungi to increased temperatures. In one laboratory study, the arbuscular mycorrhizal species Glomus mosseae increased in extraradical mycelium length by 75% in heated compared to unheated soil (Heinemeyer et al. 2006). Among the ascomycetes, Neurospora discreta was found to acclimate to warmer temperatures by producing more spores than parental strains (Romero-Olivares 2015). Growth rates for Ascomycetous and Basidiomycetous yeast at high temperatures suggest diverse mechanisms for thermotolerance (Robert et al. 2015). Some thermotolerant strains of the ascomycete Trichoderma asperellum had maximum growth at 37°C compared to 30°C for non- thermotolerant isolates (Poosapati et al. 2014). Five cord-forming basidiomycetes were able to acclimate to higher temperatures, however with reduced growth and respiration rates (Crowther & Bradford 2013). Mycelial growth rate is expected to be an indication of the capacity of saprotrophic fungi to utilize organic substrates under increased temperature regimes (Boddy 2000).

Chytrids (zoosporic true fungi) (Phylum Cytridiomycota) are common soil saprotrophs which are free living in the soil and usually reproduce rapidly asexually by motile zoospores (ruderal ecological strategy) but some species can reproduce sexually (Sparrow 1960). Chytrids have been isolated from numerous soil types and environments in eastern Australia (Letcher et al. 2004a; 2004b). The growth responses of many of these isolates to a wide range of temperature have been determined in the laboratory (Gleason et al. 2005; Gleason & McGee 2008). Most chytrid species are not able to grow on solid PYG media when temperature exceeds 35°C, or resume growth if incubated in liquid media at more than a few degrees above the maximum temperature for growth (Gleason & McGee 2008). While there is some evidence for conditioning of the fungi to allow growth at higher temperatures (Gleason et al. 2005), there have been no studies on the difference in heat tolerance between isolates of the same species of chytrid from geographically and climatically distinct regions.

Environmental conditions affecting the distribution of chytrids in nature have not been thoroughly studied (Commandeur et al. 2005), nonetheless, adaptation to local climate conditions is to be expected. Zoosporic fungi have relatively short generation times (approximately 72 hrs) so might be able to acclimate quickly to warming. It is also possible that chytrids adapt to a warmer environment by becoming more thermotolerant. Gaertneriomyces semiglobifer (Barr 1980, 1981, 1984) is a cosmopolitan species which

25 has previously been isolated from agricultural soil at Narrabri and throughout diverse environments world-wide (Wakefield et al. 2010). Our study aims to determine whether two strains of the same species (G. semiglobifer) of zoosporic true fungi, isolated from distinctly different geographic and climatic zones in Eastern Australia, have different thermal maximum temperatures for growth and reproduction in the laboratory. We predict that G. semiglobifer strain from the semi-arid region will grow and reproduce at a higher temperature than the strain from the mild temperate region. We tested this hypothesis in the present study.

2.3 Materials and methods

2.3.1 Isolate collection and maintenance

Two strains of G. semiglobifer (order Spizellomycetales) isolated from soils in New South Wales (NSW) (Table 1) were selected for this study. Mar C/C2 was previously isolated from agricultural soil of Narrabri in north-western NSW in 2003 as described by Commandeur et al. (2005). They identified this fungus by its morphology in the light microscope and with partial rDNA sequences recorded in Genbank (Gleason et al. 2010; Wakefield et al. 2010). LEH 1 was isolated from soil samples collected in September 2014 from a dry sclerophyll forest near Wingen in the Upper Hunter, NSW (0 – 5 cm depth, ) and was identified using the same methods as Mar C/C2. Both cultures were maintained on solid PYG agar containing peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 3 gL-1 and agar 20 g L-1 at 20°C. Fungi were transferred to fresh growth media at least once every 7 days.

2.3.2 Preparation of inoculum

After subculturing on solid PYG (peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 5 g L-1) agar in petri dishes and incubated for 7 days at 20°C, zoospore release was induced by flooding the cultures with 8 mL sterile de-ionised water and further incubating for two hr at 20°C. Zoospore numbers were counted using a haemocytometer. Each 0.2 mL aliquot of inoculum contained a minimum density of 2000 zoospores mL-1.

26

Table 1 – Location and climatic characteristics of the wild isolates of G. semiglobifer.

Isolate Location NSW Mean annual Mean Mean winter Land use Genbank name Geographic Climate precipitation summer minimum type id coordinates Zoneb (mm y−1)b maximum temperature temperature (°C)b (°C)b

LEH1 S 31° 52’ 6 831.3 29.7 – 30.7 2.0 – 3.2 Native MF000989 E 150° 53’ bushland Wingen

Mar S 30° 20ʹ 4 661.6 33.0 – 33.8 3.7 – 5.2 cropping FJ827645 C/C2 E 149° 47ʹ FJ827701 Narrabri a FJ827738 aFrom Gleason et al.2010 bData from Australian Bureau of Meteorology, 2017; http://www.bom.gov.au/climate/data.

2.3.3 Identification of isolate

The LEH1 isolate was grown in 50 mL Falcon tubes in liquid PYG media (peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 5 g L-1) at 25° for 3 days. The thalli were centrifuged in a Sorvall Super T21 (Sorvall Thermo) (2000 RPM, 10 mins, 20°C), washed with 25 mL de-ionised water and centrifuged again.

Tissue was homogenised in a TissueLyser (Qiagen) using 0.1 g zirconia/silica beads (Daintree Scientific) and 200 µL lysis buffer (10 mM Tris-HCl (pH = 7.5), 2mM EDTA, 1% (w/v) SDS). Samples were then treated with Proteinase K (100 ng/µl final concentration; 30 mins, 56°C). DNA was purified using EconoSpin Mini Spin Columns (Epoch Life Sciences) using a standard bind-wash-elute procedure. DNA concentration and quality was determined using a Nanodrop spectrometer. PCR was performed using primers that target a region of the nuclear 18S rRNA gene – 18S_F 5′- AACCTGGTTGATCCTGCCAGT-3′ and 18S_R 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ (Medlin et al. 1988). PCR reactions had a final volume of 50 µL and contained 1x PCR buffer, 200 µM each dNTP, 0.2 µM of each primer, 2 µl [LEH1] template DNA and 1.25U

27

Taq polymerase (Qiagen). Cycle parameters were as follows: 1 cycle of 94°C for 3 mins, 35 cycles of 94°C for 30 sec; 50°C for 30 sec; 72°C for 2 mins, 1 cycle of 72°C for 10 mins.

Sequencing of PCR products was performed by Macrogen Inc. (Seoul, South Korea). DNA sequence analysis was conducted with Geneious Pro 4.8.5 (http://www.geneious.com, Kearse et al. 2012). Homologous sequences were identified with web-based BLAST searches (blastn algorithm with default parameters; Altschul et al. 1997). DNA sequences confirmed by this study are accessible on GenBank under the ID MF000989.

2.3.4 Temperature effects on colony size (growth on solid PYG)

The inoculum for both isolates was a circular plug of 5 mm diameter taken from a PYG plate inoculated with zoospores and incubated for 7 days at 20°C and subsequently transferred to a fresh PYG plate. Subcultures of both isolates were incubated in the dark at 25, 37, 40 and 42°C in temperature-controlled incubators, without shaking, for 7 days. The temperatures selected for testing fungal growth and reproduction were based on the maximum temperature for growth of order Spizellomycetales fungi identified by Gleason et al. (2004). Growth on solid media was quantified by measuring colony diameters at 7 days. All growth experiments were repeated.

2.3.5 Temperature effects on biomass over time (growth yield in liquid PYG)

The temperatures selected for this experiment were 25°C (control) and 37°C (maximum temperature for growth) based on results obtained from temperature effects on colony size (Section 2.4). For each temperature, five replicates of each isolate were inoculated with 0.2 mL of zoospores inoculum into plastic 50 mL centrifuge tubes containing 25 mL of liquid PYG media (peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 5 g L-1 and MgSO4.7H2O 0.12 g L-1). Tubes were incubated on a shaker at 200 rpm in the dark at either 25 or 37 °C. After 38, 50, 61, 73, 85, 98 or 109 h, five replicate tubes from the shaker were placed on ice. The thalli were centrifuged in a Sorvall Super T21 (Sorvall Thermo) (2000 RPM, 10 mins, 20°C), washed with 25 mL de-ionised water and centrifuged again. The thalli were washed into aluminium dishes; dried at 65°C for 24 hours and weighed to determine the dry weight. The dry weight was determined to ± 0.1 mg. All experiments were repeated once.

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2.3.6 Temperature effects on the number of zoospores released

Three petri dishes containing solid PYG agar were inoculated with 1.6 mL of zoospores in suspension from each isolate. As for biomass experiments, the temperatures used in this experiment were based on colony size experiments (Section 2.4). The cultures were incubated without shaking at 25, 37 or 42°C for 7 days, and then flooded with 8 mL of de-ionised water. After 2 hours the number of zoospores released from the cultures was counted using a haemocytometer. To calculate zoospore numbers, the haemocytometer surface was divided into 9 large squares. The volume above each square was 0.1 mm3. Three estimates of the number of zoospores in four of the large squares were recorded at each concentration for each isolate. All experiments were repeated once.

2.3.7 Maximum temperature for growth recovery

The survival of each isolate incubated at 25, 37, 40 and 42°C was tested following zoospore release. Zoospores harvested from the fungi incubated in solid media at each temperature were transferred to fresh solid PYG agar and incubated for 7 days at 25°C. The recovery of growth was microscopically determined. Those isolates that recovered growth within 7 days were determined to have survived the treatment. Experiments were repeated once.

2.3.8 Statistical Method

Results were evaluated statistically via two-way analysis of variance followed by Tukey HSD for multiple comparisons within an isolate and t-test for single comparisons between isolates using IBM SPSS for Windows (Ver 22). Where variance was not homogeneous, data were first Log10 transformed (multiple comparisons) or unequal variances were assumed (single comparisons). The results were considered significant at the level of P < 0.05. The dependent variable is biomass and independent variables fungal isolate and time.

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2.4 Results

2.4.1 Identification of fungal isolate

A blastn search of the ~1.6 kb partial 18S rRNA sequence obtained for LEH1 returned a highest scoring alignment with a G. semiglobifer (Mar CC2) 18S rRNA sequence (99% identity; GenBank accession: AF164247.2) (Wakefield et al. 2010). Further genomic information, such as multilocus sequence typing (MLST), is needed to determine if the isolate may be differentiated as a unique strain of G. semiglobifer.

2.4.2 Temperature effects on colony size (growth on solid PYG)

Both fungi grew well on solid PYG media incubated for 7 days at 25 and at 37 but not at 40 or 42°C (Table 2). Both fungi resumed growth when returned to room temperature for 7 days after incubation at 40°C but not after incubation at 42°C.

2.4.3 Temperature effects on biomass over time (growth yield in liquid PYG)

Both isolates showed similar patterns of growth in liquid media over time at 25°C (Figure 1) and 37°C (Figure 2). At 25°C time was the only significant effect on biomass (P < 0.001). There was no significant effect of isolate on biomass or significant interaction between fungal isolate and time. However, at 37°C there was a significant interaction between fungal isolate and time (P = 0.002), indicating that at the higher temperature the growth response of the two isolates differed significantly over time. When incubated at 25°C both isolates increased in biomass over time, achieving higher growth than the initial 38 hr from 50 hr and for all subsequent time periods (P <0.001). At 25°C higher growth than the initial 38hr was observed for LEH1 for all time periods from 50 hr (P = 0.032) and for Mar CC2 for all time periods from 62 hr (P = 0.026). When incubated at 37°C both isolates achieved lower growth at 109 hr compared to 38 hr. At 37°C lower growth at 109 hr than 38 hr was observed for Mar CC2 (P = 0.044) and for LEH1(P = 0.004), however biomass decline was not significant for LEH1 when the experiment was repeated. In comparing differences in growth between 25°C and 37°C at each time period, both isolates declined significantly in biomass when incubated at 37°C compared to 25°C; Mar CC2 declined from 74 – 109 hr (P = 0.029), and LEH1 from 86 – 109 hr (P = 0.016). However, at the initial 38 hr period LEH1 achieved a biomass 60 – 73% of the control temperature when incubated at 37°C while Mar CC2 achieved a biomass of 103 – 197%.

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Biomass for Mar CC2 was significantly higher at both 38 hr and 50 hr at 37°C compared to 25°C (P = 0.046), although this increase was only significant at 50 hr (P = 0.001) for the repeated experiment.

2.4.4 Temperature effects on the number of zoospores released

Patterns of zoospore production of the two isolates after incubation at 25, 37 and 42°C (Figure 3) were the same, although rates of production were four time higher for LEH1 compared to Mar C/C2 at both 25 and 37°C. The zoospore production of each isolate increased for Mar CC2 (226 – 253%) and LEH1 (270 – 558%) after incubation at 37°C compared to incubation at 25°C. Zoospore production declined for Mar CC2 (42 – 64%) and LEH1 (69 – 87%) after incubation at 42°C compared to 25°C.

2.4.5 Maximum temperature for growth recovery

Both isolates grew on solid PYG from zoospores released after incubation at 25, 37 and 40, but not 42°C.

Table 2 – Mean increase in colony diameter (mm± SD) of two isolates of G. semiglobifer after 7 d growth on solid PYG media.

Isolate Temperature of Incubation

25°C 37°C 40°C 42°C

Mar C/C2 6.6 ± 0.6 6.1 ± 1.3 0.5 ± 0.2 0.2 ± 0.1*

LEH1 8.4 ± 1.6 5.3 ± 1.1 0.3 ± 0.2 0.4 ± 0.2*

*Indicates the isolate did not recover growth after incubation at the prescribed temperature

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20

18

16

14

12

10

8 biomass (mg dry biomass dry (mg weight) 6 Mar CC2 experiment 1 4 LEH1 experiment 1 Mar CC2 experiment 2 2 LEH1 experiment 2

0 26 38 50 62 74 86 98 110

time of incubation (hr)

Fig. 1. Mean growth over time for two isolates of G. semiglobifer after incubation in liquid media at 25°C. Dry weight (mg: mean ± SE).

20 Mar CC2 experiment 1 18 LEH1 experiment 1

16 Mar CC2 experiment 2

14 LEH1 experiment 2

12

10

8 biomass (mg dry biomass dry (mg weight) 6

4

2

0 26 38 50 62 74 86 98 110 time of incubation (hr)

Fig. 2. Mean growth over time for two isolates of G. semiglobifer after incubation in liquid media at 37°C. Dry weight (mg: mean ± SE).

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Fig. 3. Mean release of zoospores from two isolates of G. semiglobifer after incubation on solid media at different temperatures. Number of zoospores released (x103: mean ± SE).

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2.5 Discussion

The maximum temperature for growth and reproduction for two strains of G. semiglobifer from two geographically and climatically distinct regions (Table 1) were the same. Both strains grew at similar rates in liquid PYG media when incubated at the control temperature (25°C) and at the upper thermal limit for growth (37°C). There is a high correlation between maximum temperature for growth and maximum temperature for survival for soil chytrid isolates (Gleason & McGee 2008), with most chytrids unable to grow above 35°C. G. semiglobifer however, grew at up to 37°C in liquid PYG and survived on solid media for 7 days at 40°C. When 37°C is exceeded it is expected that growth of G. semiglobifer in moist soils will be suspended. It is thought that soil chytrids become quiescent or rely on heat resistant structures in order to survive when soil temperatures exceed the upper thermal maximum for growth (Gleason et al. 2004). Soil temperatures in eastern Australia commonly exceed 35°C and have been recorded at up to 70°C (McGee 1989).

Our work indicates that G. semiglobifer is sensitive to temperatures 37°C and above. Beyond this temperature the fungus did not increase in biomass and rates of zoospore production decline. Similarly, Spizellomyces sp. (Mar Ad 2-0) also in the order Spizellomycetales, grew significantly less in liquid media when incubated at 37°C compared to 20°C after 109 hr (Gleason et al. 2005). Zoosporic fungi from the order Spizellomycetales were found to be among the most heat tolerant of those which were tested by Gleason and McGee (2008), however we expect growth and reproduction of G. semiglobifer is likely to be reduced during daytime summer conditions which exceed 37°C at the soil surface. The average number of days when surface temperature exceeds 37°C in a year is greater for Narrabri (17.9 days) than Murrurundi (2.9 days) (www.bom.gov.au/climate/data), therefore we expect that high temperature will limit the growth of G. semiglobifer more frequently in the semi-arid climatic region of Narrabri than the mild temperate region of the upper Hunter.

G. semiglobifer is a globally-distributed saprotrophic zoosporic fungus. Although little is known generally about how environmental conditions affect the abundance and distribution of soil chytrids, the examination of functional traits may allow prediction of these patterns. Temperature has been hypothesized as the major determinant of fungal occurrence and distribution (Pirozynski 1968) and increased temperature causes

34 structural changes in soil fungal communities (Koske 1987; Allison & Treseder 2008; Xiong et al. 2014; Semenova et al. 2015; Marfeninaa & Danilogorskayaa 2017). Stress tolerance is predicted to influence the entire fungal community composition (Crowther et al. 2014) by the interaction of individual-level trait expression and niche processes. In this way, upper thermal tolerance of G. semiglobifer may broadly affect both its distribution and abundance within the soil community across different climate regions.

The reason for substantially higher zoospore production (4x) for LEH1 compared to Mar CC2 at both 25 and 37°C is not clear but may indicate a difference in reproductive fitness. A similar difference in zoospore production was found in Batrachochytrium dendrobatidis (Bd) where isolates from NSW and Tasmania produced significantly more zoospores than an isolate from Queensland over the same temperature range (Stevenson et al. 2013). However, in work undertaken on two lineages of Bd which were thermally adapted to 4°C or 23°C (Voyles et al. 2012) zoospore production rate and minimum time to zoospore production were significantly lower in the cold-adapted lineage. The cold- adapted zoospores were also active for longer when subsequently incubated at either high or low temperature. These temperature-mediated lifecycle shifts may allow zoosporic fungi to adapt to living at the limit of thermal extremes by trading off rapid population growth for longer term survival, albeit at lower population densities.

The two-fold increase in zoospore production for both G. semiglobifer strains, after incubation at 37°C, requires further investigation, although it is possibly a stress response of the fungus. An alternative theory involves the possibility of the sporangia surviving passage through the gastrointestinal tract of herbivorous mammals. After passing through the mammal and being deposited in dung, an increase in zoospore production would be advantageous for growth and survival. Mammals may also provide a vector for dispersal of the fungus across the landscape. In a study of diversity within the order Spizellomycetales, dung-inhabiting chytrids from horse and cow manure where found to cluster within the genera Gaertneriomyces and Triparticalcar and a further unidentified clade (Wakefield et al. 2010). Also, a recently described species within the same order, Triparticalar equi, (Davis et al. 2016) was isolated from horse dung. Although G. semiglobifer is dung- associated, the fungus is also known to be free-living in soil. Both Mar CC2 and LEH1 isolates used here were obtained from baiting soil with pollen and corn husk respectively, however beef cattle grazing was observed in the vicinity of LEH1 soil sampling site. Future

35 work on isolation of G. semiglobifer may further elucidate whether dung-associated fungi have increased zoospore production compared to soil-associated fungi.

G. semiglobifer may survive higher temperatures than those tested here during dry conditions at the soil surface. Both Mar CC2 and LEH1 survived air drying on the laboratory bench for 7 days but did not survive incubation at 80° for 48 hr (data not shown). However, Mar Ad 2 (Spizellomyces sp.), also from cropping soils at Narrabri, survived incubation at 80 and 90°C for 48 hr (Gleason et al. 2004) due to the presence of dormant resistant sporangia (Gleason et al. 2010). These structures are, however, unlikely to form in the moist conditions which may commonly be found in irrigated cotton cropping systems such as the isolation site at Narrabri. Additionally, soil chytrids require at least a thin film of water in order to reproduce by release of zoospores (Gleason et al. 2012). Many factors affect survival in soil ecosystems. At depths below the soil surface or in shaded sites, soil temperatures may be reduced, allowing G. semiglobifer to continue growing. However, it is expected that chytrids predominantly occupy the surface soil layers, utilizing only the leaf litter and topsoil layer of the soil due to availability of suitable organic substrates (Gleason et al. 2012; Sparrow 1960). It is also possible that chytrids may not respond the same way in the soil as they did in the laboratory experiments reported in the present study.

Except for rumen chytrids (Phylum Neocallimastigales), Allomyces (Phylum Blastocladiomycota) and Spizellomyces sp. (Phylum Chytridiomycota) no other species of chytrid has been reported as growing above 35°C. Gleason et al. (2005) found that two zoosporic fungi from of the genus Allomyces and one from Spizellomyces grew up to 40°C. Spizellomyces sp. isolate (Mar Ad 2) also grew significantly more at 40°C when conditioned at 33 and 37°C compared to the control (20°C) (Gleason et al 2005), however the growth rates of the conditioned fungus were still lower than those at the control temperature. Similarly, although the soil ascomycete Neurospora discreta acclimates to warmer temperatures, there appears to be a physiological trade-off in terms of a reduction in mycelial biomass and growth rate (Romero-Olivares 2015). Although it has been predicted that chytrids may adapt to increased temperature, for example, by upregulating physiological mechanisms such as synthesis of specific protein (Gleason et al. 2005), this is not supported by our work here on G. semiglobifer. Our results indicate that Mar CC2 grows initially at a higher rate than LEH1 at 37°C for short periods of time (≤ 62 hr), corresponding to initiation and maturation of sporangia (Gleason et al. (2005) followed by

36 growth decline. However, morphological changes were not observed in either isolate incubated at 37°C or higher. This contrasts with morphological changes observed by Henderson et al (2017) for chytrids incubated with toxic levels of metals.

Two strains of G. semiglobifer isolated from different climatic regions of eastern Australia and incubated at different temperatures in the laboratory grew at the same rate and produced similar patterns of zoospore production; therefore we reject our original hypothesis. Our results indicate that when daytime summer temperatures exceed 37°C it is likely that the growth and reproduction of the fungi will be reduced. Daily summer temperature maximums regularly exceed 37°C in parts of eastern Australia. Hot microclimates, including cropping soils when crop cover is low, are expected to likewise reduce growth and reproduction in comparison to vegetated sites such as those with native bushland. Our work also suggests some zoosporic fungi may not adapt to growing at increased temperatures brought about by climate change or by habitat destruction.

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Chapter 3: Copper (II) Lead (II) and Zinc (II) reduce growth and zoospore release in four zoosporic true fungi from soils of NSW, Australia.

The Chapter has been published as: Henderson L, Pilgaard B, Gleason FH, Lilje O, (2015). Copper (II) lead (II), and zinc (II) reduce growth and zoospore release in four zoosporic true fungi from soils of NSW, Australia. Fungal Biology 119: 648 – 655.

In Chapter two we examined maximum temperature for growth and reproduction for isolates of G. semiglobifer from different climatic regions, establishing that the isolates responded similarly at each temperature. This is important for interpreting how temperature stress affects the chytrid in field situations, which may occur in combination with metal stress. In this Chapter we now examine the effect of three toxic metals on the lifecycle of four species of chytrids. Toxic metal contamination of soils is common and widespread; however the ability of chytrids to grow and reproduce in the presence of these metals is, so far, unknown. The four chytrids chosen here are commonly discovered in soils and are expected to be exposed to toxic metals within soil environments, however the geographic distribution of these species is not well known. The toxic thresholds for growth and reproduction which we determine in vitro here, will be important for establishing the likely effects on chytrids within toxic metal contaminated environments.

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3.1 Abstract

This study examined the responses of a group of four zoosporic true fungi isolated from soils in NSW Australia, to concentrations of toxic metals in the laboratory that may be found in polluted soils. All isolates showed greatest sensitivity to Cu and least sensitivity to Pb. All isolates showed significant reduction in growth at 60 ppm (0.94 mmol m-3) for Cu, while three declined significantly at 60 ppm (0.92 mmol m-3) Zn. The growth of two isolates declined significantly at 100 ppm (0.48 mmol m-3) Pb and one at 200ppm (0.96 mmol m-3) Pb. The rate of production of zoospores for all isolates was reduced when sporangia were grown in solid PYG media with 60 ppm Cu. Three isolates significantly declined in production at 60 ppm Zn and three at 100 ppm Pb. All isolates recovered growth after incubation in solid media with 60 ppm Zn or 100 ppm Pb. Two isolates did not recover growth after incubation in 60 ppm Cu. If these metals cause similar effects in the field, Cu, Pb and Zn contamination of NSW soils is likely to reduce biomass of zoosporic true fungi. Loss of the fungi may reduce the rate of mineralisation of soil organic matter.

Keywords: soluble metal, copper, lead, zinc, zoosporic true fungi, chytrids

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3.2 Introduction

Deposition of toxic metals in soils poses a serious threat to the natural environment and human health. Agricultural, urban and industrial activities may contribute metals to soils (Adriano 2001). Toxic metals may remain in soils and accumulate in produce such as vegetables (Islam et al. 2007; Peralta-Videa et al. 2009). Furthermore, the toxic metals can be transported to aquatic ecosystems in solution or attached to soil particles, affecting these environments as well. Understanding the effect of metals on living biota is crucial for determining appropriate responses necessary to mitigate soil pollution.

Some metals, such as copper and zinc, are necessary for metabolic functions. Others, such as lead, have no known biological function. Excessive quantities of both essential and non-essential bioavailable metals are frequently toxic (Baldrian 2003). Metal pollution may decrease the diversity of species within soil (Fomina et al. 2005) and inhibit the growth, reproduction and physiological processes of groups of soil microorganisms. High levels of metals in leaf litter, including copper, lead and zinc, reduce the biomass of filamentous fungi (Cotrufo et al. 1995) and the rate of decomposition of leaf litter (Johnson and Hale 2004). This is perhaps related to the fact that metals also reduce the production of extracellular enzymes, such as cellulase and laccase, which assist in the decomposition of organic matter (Baldrian 2003).

Toxic metals have a complex and multifaceted effect on soil organisms. Metals in soil originate from many different sources. Concentrations of copper, lead and zinc may be increased in soils proximate to mining and smelting operations, for example, in some coastal soils of New South Wales, Australia (Kachenko and Singh 2006). Application of commercial fungicides may increase the levels of copper in soils. Atmospheric lead from motor vehicle and industrial exhaust locally raises lead levels in soil (Davis and Birch 2011). Polluted soils have a complex set of factors determining the distribution and availability of metals. Metal bioavailability varies spatially and temporally within the soil. Soil parameters (including pH, cation exchange capacity and organic matter content and concentration) determine the availability of metals within the soil solution through ion exchange, solubilisation and assimilation by microorganisms (Adriano 2001).

The response of fungi to metal pollution can be unpredictable since fungi have mechanisms of tolerance and resistance that determine the effect metals have on

40 physiological factors such as growth. Metals can be adsorbed onto fungal cell walls (Dhankar and Hooda 2011), reducing bioavailability. Toxic metals can induce or accelerate melanin production which increases the biosorption capacity of the fungal biomass (Gadd and Griffiths 1980). In the presence of heavy metals fungi may also change patterns of growth and vary biomass distribution, including by temporal or spatial cessation of growth in response to negative chemotropism (Fomina et al. 2005). Fungi also have both extracellular and intracellular mechanisms for reducing the effect of toxic metals. Externally, metals are sequestered by polysaccharides and precipitated by organic acids (Fomina et al. 2005). Internally, metals are sequestered in vacuoles by metallothioneins and phytochelatins (Gadd 1993).

Most studies on the effects of toxic metals on soil fungi involve mycorrhizal species (Blaudez et al. 2000b; Fomina et al. 2005; Hartley et al. 1997; Jones and Muehlchen 1994; Morselt et al. 1986; Pawlowska and Charvat 2004), basidiomycetes (Baldrian 2003; Hoiland 1995) and Saccharomyces sp. (Avery et al. 1996; White and Gadd 1986). The effects of Cu (II), Pb (II) and Zn (II) have been studied in zoosporic lower marine fungi (Leano and Pang 2010). Generally, these metals had a complex and toxic effect, with all metals found to be toxic above threshold concentrations (Gadd 1993). Above these concentrations, metals reduce the growth and reproduction of some groups of fungi in soil and aquatic environments. The impact of metals on many other groups of fungi remains unknown.

Zoosporic true fungi (chytrids) in the phyla Chytridiomycota and Blastocladiomycota are widely distributed in soil (Gleason et al. 2006; Sparrow 1960) as saprotrophs or parasites (Powell 1993; Sparrow 1960). They reproduce by motile spores (zoospores), with a single posteriorly directed whiplash flagella. Fungal zoospores lack a cell wall (Barr 2001). Zoospores of soil chytrids attach to, and grow saprotrophically on, many substrates of plant and animal origin, such as pollen, keratin and chitin (Sparrow 1960). Under these conditions, chytrids are likely to be directly exposed to metals both in soil solution and sequestered in organic matter. The lack of a cell wall may make the zoospore particularly sensitive to metals; therefore we predict that zoospore production and survival will be affected by heavy metals at lower concentrations than fungal growth. This research is important because chytrids are thought to have keystone roles in ecological functioning in both soil and freshwater ecosystems (Gleason et al. 2012).

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The purpose of this study is to determine the response of growth (biomass), reproduction (release of zoospores) and survival of (resumption of growth) chytrids during and after exposure to soluble copper (II), lead (II) and zinc (II). We predict that increasing concentrations of these metals will reduce the rates of growth and zoospore production rates of zoosporic true fungi.

3.3 Materials and methods

3.3.1 Selection and maintenance of isolates

Four species of zoosporic true fungi isolated from soils in New South Wales were selected for this study. The selected isolates represent four orders within the phylum Chytridiomycota; Terramyces sp. (AUS 3: Order Rhizophydiales) and Rhizophlyctis rosea (AUS 13: Order Rhizophlyctidales) from Sydney, Chytriomyces hyalinus (AUS 14 Order Chytridiales) from the Central Coast and Gaertneriomyces semiglobifer (Mar CC2: Order Spizellomycetales) from north-western NSW. These isolates were collected and previously described by Letcher et al. (2004a); Letcher et al. (2004b) and Commandeur et al. (2005) (Table 3) and were identified previously at the generic level and recorded with partial rDNA sequences in Genbank (Gleason et al. 2010). Stock cultures of all isolates were maintained on solid PYG agar containing peptone 1.25 gL-1, yeast extract 1.25 g L-1, glucose 3 gL-1 and agar 20 g L-1.

Table 3 – Zoosporic true fungi isolate description

Isolate Name Order Source Accession GPS Number Coordinates

A3 Terramyces sp. Rhizophydiales Lane Cove AY439045 S 33° 46.432ʹ NP Sydney DQ485633 E 151° 08.116ʹ

A13 Rhizophlyctis rosea Rhizophlyctidales University of EU379156 S 33° 53.168ʹ Sydney EU379199 E 151° 11.356ʹ

A14 Chytriomyces Chytridiales Ourimbah AB586078 S 33° 18.356ʹ hyalinus AB586083 E 151° 17.093ʹ

Mar. Gaertneriomyces Spizellomycetales Narrabri FJ827645 S 30° 20ʹ C/C2 semiglobifer FJ827701 E 149° 47ʹ FJ827738

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3.3.2 Preparation of inoculum

The fungi were subcultured on 25 ml solid PYG agar in petri dishes and incubated for 7 days at 20°C. Zoospore release was induced by flooding the cultures with 8 ml sterile de-ionised water at 20°C and further incubating for two hours. Zoospore numbers were counted using a haemocytometer. Each inoculum contained a minimum density of 2000 zoospores mL-1.

3.3.3 Growth in liquid media

Liquid PYG media contained peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 5 g

-1 -1 L and MgSO4.7H2O 0.12 g L . After sterilization, the pH of the media was adjusted to 5.5 with 10 mN KH2PO4 buffer using a pH meter (Eutech pH 510 bench meter, Eutech

Instruments Pty Ltd, Singapore). Stock solutions of copper (II) (CuSO45H2O; ), lead(II)

(PbCl2;) and zinc(II) (ZnSO4.7H2O; Riedel-de Haen) were prepared at a concentration of 1000 mg L-1 and filter-sterilised (0.22µm) before adding to liquid media at final concentrations of 0, 5, 10, 20, 30, 60 ppm for all metals and both 100 and 200 ppm for Pb (II) only. Control media contained PYG and buffer only. Five replicates of each isolate were inoculated with 0.2 ml of suspended zoospores into plastic 50 mL centrifuge tubes containing 25 mL of liquid solution. Tubes were incubated on a shaker at 200 rpm and 20°C for 7 days. The thalli were centrifuged in a Sorvall Super T21 (Sorvall Thermo) (2000 RPM, 10 mins, 20°C), washed with 25 mL de-ionized water and centrifuged again. The thalli were washed into aluminium dishes, dried at 65°C for 24 hours and weighed to determine the dry biomass. All experiments were repeated and data from one experiment has been included here.

3.3.4 Zoospore release

Solid PYG agar adjusted to pH 5.5 with the addition of 10mM K H2PO4 was placed into Petri dishes. Prior to solidification filter-sterilized stock solutions of metals were added at concentrations of 0, 10, 60 and 100 ppm. Control media lacked additional metals. Duplicate plates for each isolate at each metal concentration were prepared. One dish at each concentration of Cu (II), Pb (II) and Zn (II) and one control dish were inoculated with 1.6 mL of zoospores in suspension from each isolate. The cultures were incubated without shaking at 20°C for 7 days, and then flooded with 8 mL of de-ionized water adjusted to pH

5.5 with 10 mM KH2PO4 buffer. After 2 hours the number of zoospores released from the

43 cultures was counted using a haemocytometer. To calculate zoospore numbers, the haemocytometer surface was divided into 9 large squares. The volume above each square was 0.1 mm3. Three estimates of the number of zoospores in four of the large squares were recorded at each concentration for each isolate. The pH of the solutions on the surface of the solid media in the Petri dishes was estimated after 2 hours with pH paper. All experiments were repeated and data from one experiment included here.

3.3.5 Survival

The survival of the four isolates incubated with inhibitory concentrations of Cu (II), Pb (II) and Zn (II) was tested following zoospore release. Zoospores harvested from the fungi incubated in solid media containing metal concentrations of 60 (Cu and Zn) and 100 ppm (Pb only) were transferred to fresh solid PYG agar and the recovery of growth was microscopically determined. Those isolates that recovered growth within 7 days were determined to have survived the treatment. Experiments were repeated and data from one experiment has been included here.

3.3.6 Statistical analysis

Data were analysed using IBM SPSS at 5% significance, with dependent variable biomass and independent variable metal concentration. Where variance was homogeneous, data were analysed by ANOVA, and where variance was not homogenous, data were analysed using Welch’s robust test for equality of means. Significant differences among means were compared using the Tukey test. In cases where variance was not homogenous, significant differences among means were compared using the Games- Howell procedure. Means (±SEM) of each metal tested and labelled with the same letter are not significantly different (P<0.05).

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3.4 Results

3.4.1 Biomass production (Growth)

Biomass production of the four isolates when incubated with different concentrations of Cu (II), Pb (II) and Zn (II) (Figs. 4, 5 and 6) differed between fungi. Copper is the most toxic of the three metals tested, with significantly lower biomass (P< 0.05) at 30 ppm (0.47 mmol m-3) for three isolates and at 60 ppm (0.94 mmol m-3) for Terramyces sp. Pb (II) is the least toxic, with significant decline of biomass at 100 ppm (0.48 mmol m-3) for Terramyces sp. and G. semiglobifer and 200 ppm (0.96 mmol m-3) for C. hyalinus.

3.4.1.1 Copper

All isolates achieved a growth rate similar to the control at 5 ppm (0.08 mmol m-3) copper. Higher growth than the control is observed for 10 ppm (0.16 mmol m-3) Cu (II) for R. rosea (P= 0.001), with increases in biomass of 56 - 73%. Growth of R. rosea, C. hyalinus and G. semiglobifer is significantly (P<0.05) inhibited at 30 ppm (0.47 mmol m-3) Cu (II) and 60 ppm (0.94 mmol m-3) Cu (II) for Terramyces sp., with a reduction in biomass of 45 – 47% (R. rosea), 57 – 63% (C. hyalinus), 36 – 64% (G. semiglobifer) at 30 ppm. Growth decline is significant at 60 ppm Cu (II). In comparison to the control, biomass declined by 67 – 84% (Terramyces sp.) 45 – 89% (R. rosea), 66 – 89% (C. hyalinus) and 81 – 84% (G. semiglobifer), respectively.

3.4.1.2 Lead

All isolates achieved at least the growth rate of the control at 60 ppm (0.3 mmol m-3) Pb (II). Biomass increased 26 - 63% (Terramyces sp.), 69 - 80% (R. rosea) (sign P<0.05), 24 - 80% (C. hyalinus) and 28 - 244% (G. semiglobifer) in comparison to the control. In one experiment the highest mean biomass occurred at 30 ppm (0.14 mmol m-3) for C. hyalinus with a 45% increase and G. semiglobifer with a 131% increase.

Decline in growth occurred at 100 ppm (0.48 mmol m-3) Pb (II) for Terramyces sp., R. rosea and G. semiglobifer and 200 ppm (0.96 mmol m-3) Pb (II) for C. hyalinus. Mean biomass declined 32% (Terramyces sp.), 22 - 33% (R. rosea), 33 - 60% (G. semiglobifer) and 57% at 200 ppm for C. hyalinus. Declines were significant for isolates Terramyces sp. and C. hyalinus.

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3.4.1.3 Zinc

Growth declined for all isolates at 60 ppm (0.92 mmol m-3) Zn (II), with biomass declines of 47 – 63% (Terramyces sp.) 31 – 86% (R. rosea) 47 – 92% (C. hyalinus) and 69 – 70% (G. semiglobifer), respectively. This decline is significant (P<0.05) for Terramyces sp., C. hyalinus and G. semiglobifer. Higher biomass (74%) (P= 0.033) than the control is observed for G. semiglobifer in the presence of 10 ppm (0.16 mmol m-3) Zn (II).

Fig.4. Growth at different concentrations of copper (Cu (II)) for Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. Dry weights (mg: mean ± SE) were recorded after 7 d. Means with the same letter are not significantly different (P>0.05)

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Fig.5. Growth at different concentrations of lead (Pb (II)) for Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. Dry weights (mg: mean ± SE) were recorded after 7 d. Means with the same letter are not significantly different (P>0.05)

Fig.6. Growth at different concentrations of Zinc (Zn (II)) for Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. Dry weights (mg: mean ± SE) were recorded after 7 d. Means with the same letter are not significantly different (P>0.05)

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3.4.2 Zoospore production

Zoospore production of the four isolates after incubation in three concentrations of Cu (I), Pb (II) and Zn (II) (Figs. 7, 8 and 9) differed between fungi.

3.4.2.1 Copper

The zoospore production of each isolate in three concentrations of each metal indicates that Cu (II) is the most toxic metal, with all species declining at 60 ppm (0.94 mmol m-3). Terramyces sp. is the most sensitive, with zoospore production declining 43 – 60% at 10 ppm (0.16 mmol m-3) and 83 – 95% at 60 ppm Cu (II).

3.4.2.2 Lead

Zoospore production is reduced for three isolates incubated with 100 ppm (0.48 mmol m-3) Pb (II), by 62 – 70% (R. rosea), 70 – 77% (C. hyalinus) and 43 – 59% (G. semiglobifer). The isolate Terramyces sp. is least sensitive to Pb (II). Response to Pb (II) varied between and within some isolates. Terramyces sp. showed the greatest variation, increasing zoospore production rate by 68% at 100 ppm in one experiment and declining by 66% at 100 ppm in the other. G. semiglobifer increased by 58% at 10 ppm (0.05 mmol m-3) in one experiment only, however declined by 43 – 59% at 100 ppm Pb (II).

3.4.2.3 Zinc

Zoospore production declined for three isolates after incubation in media with 60 ppm (0.92 mmol m-3) Zn by 46 – 73% (Terramyces sp.), 63 – 87% (C. hyalinus) and 74 – 91% (G. semiglobifer). R. rosea increased zoospore production at 60 ppm Zn by 150 -450%. Terramyces sp. responded to incubation with 10 ppm (0.16 mmol m-3) Zn with a 50% increase in zoospore production. All other isolates did not increase at 10 ppm Zn and G. semiglobifer declined by 37 – 60%.

3.4.3 Survival - Recovery of growth after incubation with toxic levels of Cu (II) Pb (II) or Zn (II)

All isolates had recovered growth after incubation for 7 days with 60 ppm (0.92 mmol m-3) Zn (II) and 100 ppm Pb (II). R. rosea and G. semiglobifer, but not C. hyalinus and Terramyces sp., recovered growth after incubation in 60 ppm (0.94 mmol m-3) Cu (II).

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Fig.7. Release of zoospores from Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. after incubation on solid media containing different concentrations of copper (Cu (II)). Number of zoospores released (x 103: mean ± SE).

Fig.8. Release of zoospores from Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. after incubation on solid media containing different concentrations of lead (Pb (II)). Number of zoospores released (x 103: mean ± SE).

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Fig.9. Release of zoospores from Terramyces sp., Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces sp. after incubation on solid media containing different concentrations of zinc (Zn (II)). Number of zoospores released (x 103: mean ± SE).

3.5 Discussion

Copper, lead and zinc reduced the growth and zoospore production of the selected species of zoosporic true fungi. Under the experimental conditions, copper had a toxic effect on growth at comparatively low concentrations, while lead was the least toxic metal. Generally, patterns of zoospore production were similar to changes in biomass as metal concentrations increased. However, in some cases the concentration of metals at which biomass declined corresponded to an increase in zoospore production. The reasons for this disparity require further examination. The difference may be because toxic concentrations of metals stimulate a stress response in zoosporic fungi leading to dispersal of zoospores in soil to regions of lower metal concentration.

Processes other than toxicity may contribute to the observed patterns of growth and zoospore release. The effects of metals on other parts of the life cycle remain unclear; in particular, the ability of zoosporic fungi to complete their lifecycle in the presence of toxic levels of metals is not known. Also, very little is known about the effects of toxic metals on

50 survival of zoospores prior to encystment. Zoosporic fungi possibly survive high concentrations of metals as dormant encysted zoospores, accounting for slow growth. Elevated levels of metals may alternatively limit rhizoidal development disrupting colony formation and thereby reducing population densities. Morphological changes are known to occur in hyphae of filamentous fungi due to elevated metal levels (Fomina et al. 2005) and the same may occur among zoosporic true fungi.

Detrimental effects of Cu (II), Pb (II) and Zn (II) have been demonstrated in vitro; however, further work is required to determine the effects of these metals in soil. The nutritional components of complex media are likely to affect chytrid growth in the presence of metal. Toxicity of metals to decomposer basidiomycetes is known to decrease with increasing carbon concentrations in both soils and complex media (Fomina et al. 2005). Zoosporic fungal biomass and zoospore production are known to differ under different nutrient conditions (Lilje and Lilje 2008). It is also known that the pH of growth media significantly affects growth rate, with, for example, G. semiglobifer having higher biomass at neutral pH than at pH 5.5 (Gleason et al. 2010). Therefore G. semiglobifer, from soil of alkaline pH in north-western NSW, may have a different growth response in the field to Terramyces sp., Rhizophlyctis rosea and C. hyalinus which are from naturally acidic soils of coastal regions. The bioavailability of toxic metal in soil is mediated by pH and properties such as the concentrations of total soil cations and organic matter.

Contaminated soils in Australia may have free copper, lead and zinc in soil pore water at field capacity (Nolan et al. 2003) which is within the range or exceeding the range, examined within this study; and therefore growth and reproduction of zoosporic true fungi may be inhibited in these soils. Decline in biomass of chytrids in soil is likely to occur where elevated concentrations of these metals are available due to the consequent impact on growth and zoospore production. Zoosporic true fungi are common and widespread decomposer organisms within the soil (Sparrow 1960) and may be important in carbon cycling within the soil microbial loop. Chytrids degrade dissolved and particulate organic matter and are, in turn, consumed by protists and metazoans. Chytrids are also likely to play a role in the biogeochemical cycling of nitrogen and sulphur (Gleason et al. 2012). Decline in chytrid populations in metal-contaminated soil may lead to significant changes in ecological functions of soil.

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One hypothesis for the pattern of biomass increase and decline for some isolates in this study is the uptake of metalloporphyrins via the heme biosynthesis pathway. Heme, a cofactor for many proteins, is formed when ferrochelatase catalyses the insertion of ferrous iron into cellular protoporphyrin IX. Chlorophyll, a magnesium porphyrin, is known to undergo substitution of the central magnesium atom by copper, zinc and lead, with toxicity and metal assimilation increasing almost proportionally in the order: Cu (II) > Zn (II) > Pb (II) (Küpper et al. 1996). Ferrochelatase is able to use alternative substrates to iron in metalloporphyrin production, including Cu (II) and Zn (II) and although these metals cause substrate inhibition, this was masked by the presence of the buffers β-mercaptoethanol or imidazole, leading to accelerated initial ferrochelatase activity (Hunter et al. 2008). We predict that if the available levels of Cu (II), Pb (II) and Zn (II) are below the toxic threshold established here, these metals may be incorporated into cellular protoporphyrins within zoosporic true fungi; thus chytrid biomass will increase below the toxic threshold and decline when the threshold is exceeded.

An alternative hypothesis for patterns of growth increase and decline in this study is the effect of ionic strength (I) of the media. Biomass increase for R. rosea and Terramyces sp. occurred at I of 0.0124 (10 ppm) for CuSO4 and (60 ppm) PbCl2, while biomass increase for G. semiglobifer occurred at I of 0.0124 (10 ppm) for ZnSO4. Biomass reduction occurred at I of 0.0158 (60 ppm) for ZnSO4 and CuSO4 for three isolates and

0.0148 (100 ppm) for PbCl2 for three isolates. Ions may affect the attachment, germination, growth and morphological development of fungi (Shaw and Hoch 2007). For the aquatic fungus Blastocladiella emersonii cyst germination was induced by K+ , Na+ , Rb+ , Mg2+ and Ca2+ ions (Soll and Sonneborn 1972). Although this study does not attempt to address the effects of metals on the morphological development of zoosporic true fungi, it may be hypothesised that in the presence of certain metals germination rates may increase as ionic strength increases and that germination rates decline in the presence of toxic concentrations of the same metals.

Total concentration of ions in the pore water is expected to change as total soil water content changes. Total ion concentrations may increase as soils dry, leading to likely impacts on zoosporic fungi. If levels of free ions such as copper, lead and zinc exceed toxic limits for these organisms, germination, growth and zoospore release will decrease. In this way, cessation of fungal growth may be an avoidance response to metal polluted environments (Fomina et al. 2005). It is possible that zoosporic true fungi are r-adapted

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(Ruderal strategy) (Dix and Webster 1995) to take advantage of the short-term availability of soluble metal ions, such as the required micronutrients copper and zinc, by increasing growth over the short term and slowing or suspending growth as soil dries and soluble metal species increase to toxic concentrations.

In this study, growth and zoospore production of four isolates of zoosporic true fungi were sensitive to soluble metals with toxicity patterns generally corresponding to Cu (II)> Zn (II)> Pb (II) in the laboratory. The toxicity patterns differed with species of chytrid and with type of metal ion. This result is in broad agreement with the effects of these concentrations of metals on four Halophytophthora (zoosporic marine oomycete) isolates (Leano and Pang 2010). Zoosporic true fungi are an important part of the soil microbiota and their reduction in growth due to elevated toxic metal levels in soil may impact the composition of fungi, food webs dynamics and carbon cycling in the soil.

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Chapter 4: Copper (ll), Lead (ll) and Zinc (ll) reduce the rate of attachment in three zoosporic true fungi from soils of NSW, Australia.

The chapter has been submitted for publication in Nova Hedwigia.

The previous chapter determined the effect of copper (ll), lead (ll) and zinc (ll) on chytrid growth and zoospore production. This chapter examines a further important aspect of the effects of metals on the lifecycle of chytrids; the ability of the fungi to attach to organic substrates which are commonly encountered in the soil environment. Toxic metals may reduce the ability of chytrids to colonise and degrade organic materials, such as cellulose, keratin and chitin. Chytrids are considered to be an important component of the soil microbial loop (Gleason et al. 2012) as they are primary decomposers of organic detritus and are in turn consumed by protists and metazoans (Gleason et al. 2008). Reduction in attachment rates may detrimentally affect the role of chytrids in soil carbon and nutrient cycling.

4.1 Abstract

Zoosporic fungi (chytrids) are common soil saprotrophs and parasites and important organisms which decompose complex carbohydrates, yet little is known of their ability to colonise these substrates in the presence of toxic metals. Three saprotrophic isolates of zoosporic fungi from the soils of NSW, Australia are examined here for their ability to adhere to cellulose and chitin in the presence of various concentrations of the toxic metals Cu, Pb and Zn. Two isolates showed significant reduction in attachment rate at 60 ppm for Cu, while two declined significantly at 60 ppm Zn and two at 100 ppm Pb. Rhizoids of one isolate increased significantly both in number and length when incubated with 20, 30 and 60 ppm Pb. If these metals cause similar effects in soils, Cu, Pb and Zn are expected to reduce attachment rates of zoosporic fungi, thereby slowing the mineralisation of organic matter.

Keywords: chytrids, copper, lead, zinc, soluble metal, zoosporic true fungi

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4.2 Introduction

Toxic metals are common soil contaminants (Alloway 2012) and are derived from many agricultural, urban, and industrial activities (Adriano 2001) including pesticide applications, metal refining and vehicle exhaust. These metals may also be transported when attached to soil particles, to fresh water and marine ecosystems. Toxic metals may also have detrimental effects on human health (Jarup 2003), due to metal accumulation in soils and products such as vegetables (Islam et al. 2007; Kachenko and Singh 2006: Peralta-Videa et al. 2009). Therefore, it is essential to understand the effects of toxic metals on soil biota in order to effectively manage and remediate metal contaminated land.

Fungi are widespread and common in soil environments; as symbionts, pathogens and as decomposers of organic material. Fungi may dominate the microbiota in toxic metal-affected soils where pH is low; here metals are more likely to be present in biologically available forms (Baath and Anderson 2003). Some metals are necessary for fungal growth, metabolism and differentiation. Essential metals include K, Na, Mg, Ca, Mn, Fe, Cu, Zn, Co and Ni. Other metals, such as Rb, Cs, Al, Cd, Ag, Au, Hg and Pb have no known biological function; however all are expected to be toxic to fungi above threshold concentrations (Gadd 1993). When metals are present in soils at toxic levels, fungal colonisation, growth, reproduction and differentiation may be affected, fungi may not be able to complete their lifecycle and populations may decline. Also, metals such as Pb inhibit the rate of enzyme produced by fungi (Kähkönen et al 2008), potentially decreasing the degradation of organic matter. A decline in saprotrophic fungi may change the rate of degradation of organic matter with potential effects on soil carbon cycling. Although much work has been done on the effects of toxic metals on soil fungal communities, in general, the focus has predominantly been on ectomycorrhizal and arbuscular mycorrhizal fungi (plant mutualists), while little work has been done on fungi such as zoosporic true fungi or lower fungi (Henderson et al. 2017). However, metals such as Ag, Cu, Ni, Co and Zn are toxic at lower concentrations to zoospores of the oomycetes Phytophthora and Pythium (stramenopiles) (Slade and Pegg 1993) than at any other life cycle stage.

The response of soil fungi to metals is dependent on the range of tolerance and resistance mechanisms of each fungus (Formina et al 2005; Gadd 1993; Gadd and Griffiths 1980). These include both intracellular (sequestration by metallothioneins and

55 phytochelatins; increased melanin production) and extracellular mechanisms (absorbance to fungal cell walls, sequestration by ECM and precipitation by organic acids) that modify toxic metal effects on physiological parameters such as growth. Changes in distribution of growth temporally and spatially may also occur in the presence of toxic metals (Fomina et al. 2005).

Zoosporic true fungi are heterotrophs belonging to the phyla Chytridiomycota, Blastocladiomycota and Neocallimastigomycota (Gleason et al. in Press; Gleason et al. 2006; Sparrow 1960). Zoosporic fungi reproduce by motile zoospores (Barr 2001) which have a single posterior flagellum and which require free water for dispersal. The zoospores are chemically attracted to (Gleason and Lilje 2009) and encyst on (Mitchell and Deacon 1986) a wide range of detrital plant and animal substrates such as pollen, keratin and chitin (Sparrow 1960). Both zoospores and chytrid rhizoids are known to adhere tightly to both organic and inorganic substrates including glass, plastic, sand, cellulose, snakeskin, plant fibre, agar and other rhizoids (Sparrow, 1960; Barr, 1987, 2001; Gleason et al., 2011; Henderson and Lilje, unpublished observations). Attachment is thought to prevent the dislodgement of the developing sporangium by water currents (Gleason et al. 2012). As soil saprotrophs or parasites (Powell 1993) zoosporic fungi are expected to be exposed to toxic metals in soil solution and within organic matter in metal contaminated soil which may detrimentally affect their ability to colonise and degrade organic material.

Previous work has established that soluble copper Cu (ll), lead Pb (ll) and zinc Zn (ll) reduce the rate of growth (biomass) and reproduction (zoospore production rate) of zoosporic fungi (Henderson et al. 2015). The purpose of the current study is to determine the rate of attachment of isolates of three common soil zoosporic fungi (Rhizophlyctis rosea, Chytriomyces hyalinus, and Gaertneriomyces semiglobifer) during exposure to soluble Cu (II), Pb (II), and Zn (II). We predict that increasing concentrations of these metals will reduce the rates of attachment of zoosporic fungi to organic substrates.

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4.3 Materials and methods

4.3.1 Selection and maintenance of isolates

Three zoosporic fungal isolates were selected from those previously tested for growth and rate of zoospore production in the presence of various toxic metals by Henderson et al. (2015). The isolates were maintained on solid PYG (peptone 1.25 g L-1, yeast extract 1.25 g L-1, glucose 3 g L-1 and agar 20 g L-1) media and transferred at least once every seven days. The collection and characteristics of the isolates were previously described (Letcher et al. 2004a; Letcher et al. 2004b; Commandeur et al. 2005) (Table 4). Rhizophlyctis rosea (AUS 13: Order Rhizophlyctidales) from Camperdown, Sydney, Chytriomyces hyalinus (AUS 14: Order Chytridiales) from the Central Coast and Gaertneriomyces semiglobifer (Mar CC2: Order Spizellomycetales) from north-western NSW were previously identified at the generic level and recorded with partial rDNA sequences in GenBank (Gleason et al. 2010).

Table 4 –Location and Description of isolates of zoosporic true fungi Isolate Name Order Source Accession GPS Number Coordinates A13 Rhizophlyctis Rhizophlyctidales Camperdown, EU379156 S 33° rosea Sydney EU379199 53.168ʹ E 151° 11.356ʹ A14 Chytriomyces Chytridiales Ourimbah, AB586078 S 33° hyalinus Central Coast AB586083 18.356ʹ E 151° 17.093ʹ Mar. Gaertneriomyces Spizellomycetales Narrabri, FJ827645 S 30° 20ʹ C/C2 semiglobifer Western FJ827701 E 149° 47ʹ NSW FJ827738

4.3.2 Preparation of inoculum

Each isolate was subcultured on solid PYG agar at 20°C. After 7 days, zoospore release was induced with the addition of 8 mL de-ionised (DI) water with further incubation for 2 hrs at 20°C. Inoculum contained a minimum of 1000 zoospores ml-1.

4.3.3 Attachment to substrates in the presence of metal

Stock solutions of copper (II) (CuSO4.5H2O); lead (II) (PbCl2); and zinc (II)

-1 (ZnSO4.7H2O) were prepared at a concentration of 1000 mg L and filter-sterilised (0.22

57

µm). Solutions contained final concentrations of; 0, 5, 10, 20, 30, 60 ppm for all metals and both 100 and 200 ppm for Pb (II) only, and micronutrient solution (1 µM MnCl2.4H2O, 1

µM ZnCl2, 3 µM H3BO4, 0.1µM CuSO4.5H2O, 0.2 µM (NH4)6.Mo7O24, 0.2 µM CoCl2.6H2O).

The pH of the solutions was adjusted to 5.5 with 10 mN KH2PO4 buffer using a pH meter (Mettler Toledo FG2). Control media contained Milli-Q water, micronutrients and buffer only.

Rubber discs containing five evenly spaced holes were placed in sterile 100 mm diameter plastic petri dish and 15 ml of metal solution was added to a dish for each metal concentration. The experimental design is shown in Appendix 3. Petri dishes and rubber discs were first washed in 10% HCL then three times in DI water and dried overnight at 60°C. After 1 hour the central hole of each disc was inoculated with 0.2 ml zoospore suspension. A hole punch was used to create circular 3 mm diameter lens paper and snake skin baits, with one sterile bait placed in each of the five holes in the rubber disc. Each fungus was assessed using one bait type only. The specificity of attachment of R. rosea to cellulosic bait types has been previously determined (Mitchell and Deacon 1986). Lens paper was the bait type for R.rosea and snakeskin for C. hyalinus and G. semiglobifer. Dishes were incubated at room temperature (20 °C) in the dark for 48 hours. The numbers of attached sporangia were counted directly on the bait surfaces. Baits were removed from the petri dish and stained for 5 min in lacto phenol cotton blue, dipped into DI water to remove stain excess and then mounted onto glass slides. Slides were viewed using a light microscope (Motic BA310) at a magnification of x40. All sporangia were counted regardless of colour. All experiments were repeated and data from one experiment included here. Sporangial attachment was then photographed using a Leica DM6000 B light microscope and Leica DFC400 camera.

4.3.4 Measurement of R. rosea rhizoids after incubation with Pb

Morphological changes to the rhizoids of R.rosea, but not those of either C. hyalinus or G. semiglobifer, were observed during the Pb (ll) experiments described in Section 4.3.3. This may be because R.rosea rhizoids predominantly attached to the surface of the lens paper fibres, while C. hyalinus and G. semiglobifer rhizoids generally penetrated the snakeskin and were difficult to observe. Therefore, lens paper baits with the attached fungus were prepared for each experimental concentration of Pb (ll) as per Section 4.3.3 and rhizoids were counted and measured under the light microscope with x40 objective

58 and a stage micrometer. Five random fields of view were taken for each disk; the number of rhizoids attached to each sporangium was counted and the lengths of all rhizoids were measured. All experiments were repeated and data from one experiment included here.

4.3.5 Statistical analysis

Attachment data were analysed by ANOVA using IBM SPSS Ver 22 at 5 % significance, with attachment as dependent variable and metal concentration as independent variable. Significant differences between means were compared using

Tukey’s test. Data which were not normally distributed were log10 transformed for analysis. In cases where variance was not homogenous, significance was determined using Welch’s robust test for equality of means and significant differences between means were compared using Games-Howell’s procedure. Means (±SEM) labelled with the same letter were not significantly different (P < 0.05). Rhizoid measurement data were not normally distributed and were therefore analysed by Kruskal-Wallis ANOVA with significant differences between means determined using pairwise comparisons at 5% significance.

4.3.6 Preparation of fungi for SEM imaging

Fungi were prepared as per Section 4.3.3. Baits were then placed in individual specimen vials with 1 ml of 0.1 M phosphate buffer (pH 6.9). Baits were then fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer and incubated at room temperature for 1 hr. Following this, the baits were incubated three times for 5 m each time in 0.1 M phosphate buffer, then 1% Osmium tetroxide in 0.1 M phosphate buffer at room temperature for 1 hr. The baits were then washed in Milli-Q water (3 times, 5 m each), incubated in 50% ethanol (5 m twice); 70% ethanol (5 m twice); 95% ethanol (5 m three times); 100% ethanol (10 m three times). The baits were dehydrated using Critical Point Drying, mounted onto SEM stubs and sputter coated with gold and photographed using a JEOL Neoscope Tabletop SEM.

4.4 Results

4.4.1 Attachment to substrates in the presence of metal

Changes in attachment of fungi when incubated with different concentrations of Cu (ll), Pb (ll) and Zn (ll) (Figures 10, 11 and 12) were dependent on both metal and fungal species. All fungi were most sensitive to Cu (ll) and least sensitive to Pb (ll). Two fungi (R.

59 rosea and G. semiglobifer) declined significantly in attachment rate after incubation with Cu (ll). As Cu (ll) concentration increased to 60 ppm, R. rosea declined 17 – 50% (P < 0.02) compared to the control and G. semiglobifer 34 – 50% (P < 0.01). C. hyalinus attachment rate initially increased at 10 ppm 30 – 120% (P>0.05) and then declined to 75 – 80% (P > 0.05) of the control at 60 ppm Cu (ll).

Zn (ll) did not cause a significant decline in attachment for R. rosea as concentration increased to 60 ppm. However, at 20 ppm this fungus increased in rate of attachment by 50% (P = 0.06) to 90% (P = 0.001) compared to the control. Likewise, C. hyalinus increased in attachment rate by 10% (P=0.96) to 68% (P=0.03) at 10 ppm Zn (ll). In one case attachment rates then declined 50% (P = 0.01) at 60 ppm, however this decline did not occur when the experiment was repeated. Attachment rates for G.semiglobifer declined at 60 ppm Zn (ll) 44% (P = 0.27) to 49% (P = 0.005) compared to the control.

Attachment rates for R. rosea when incubated with Pb (ll) increased 12 – 20% (P >0.05) at 60 ppm. Attachment for this fungus then declined to 44 – 50% compared to the control at 200 ppm (P < 0.005). C. hyalinus attachment declined 27% (P = 0.0003) to 48% (P = 0.1797) compared to the control at 200 ppm Pb (ll). Attachment for G.semiglobifer declined 38% (P = 0.05) to 52% (P = 0.1797) compared to the control at 100 ppm Pb (ll).

Attachment rates were compared to biomass change data which was determined previously by Henderson et al. (2015) for all fungi and metals (Appendix 1). Attachment rate was significantly correlated to biomass in the presence of Pb (ll) for R. rosea (r = 0.321 and 0.503, p < 0.05) and for experiment two for C. hyalinus (r = 0.400, p < 0.05) and G. semiglobifer (r = 0.618, p < 0.01). G. semiglobifer attachment rate was significantly correlated to biomass for Cu (ll) (r = 0.498 and 0.561, p < 0.01). Attachment rate for all fungi and Zn (ll) was significantly correlated to biomass for experiment one only. No significant relationship was found between attachment and biomass for C. hyalinus and Cu (ll).

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Fig.10 Rates of attachment at different concentrations of copper Cu (ll) for Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces semiglobifer. Numbers of attached sporangia (mean ± SE).were recorded after 3d. Means with the same letter are not significantly different (P > 0.05).

Fig.11 Rates of attachment at different concentrations of lead Pb (ll) for Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces semiglobifer. Numbers of attached sporangia (mean ± SE) were recorded after 3 d. Means with the same letter are not significantly different (P>0.05).

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Fig.12 Rates of attachment at different concentrations of zinc Zn (ll) for Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces semiglobifer. Numbers of attached sporangia (mean ± SE) were recorded after 3 d. Means with the same letter are not significantly different (P>0.05).

4.4.2. Measurement of number and length of R. rosea rhizoids after incubation with Pb (ll)

Rhizoid numbers initially increased as Pb (ll) concentration increased and then declined, while rhizoid length increased as Pb (ll) concentration increased. Both length and number of rhizoids increased significantly compared to the control at 20, 30 and 60 ppm (p < 0.05) (Figure 13). Rhizoid length increased significantly from an average of 1.2 – 31.4 µm per sporangium in the control to 3.9 – 84.8 µm per sporangium at 100 ppm Pb (ll). Rhizoid numbers significantly increased from an average of 0.2 – 1.8 per sporangium in the control to 0.6 – 3.1 per sporangium at 30 ppm Pb (ll) (p < 0.05) and then declined to 0.1 – 2.1 per sporangium at 100 ppm Pb (ll). The maximum reached was an average of 3.6 rhizoids per sporangium in one experiment at 20 ppm.

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Fig.13 Number (mean ± SE) and length (mean ±SE) of rhizoids of Rhizophlyctis rosea recorded after 3 d attached to lens paper discs (3 mm diameter) grown in different concentrations of lead Pb (ll). * indicates rhizoid length is significantly different from the control (p<0.05),  indicates number of rhizoids is significantly different from the control (p<0.05).

4.4.3. Imagery of attached sporangia incubated with different concentrations of Pb (ll)

Rhizoids of R. rosea vary in morphology depending on whether they are unattached (Figure 14a and b) or attached to lens paper (Figure 14c). Polarised growth, defined here as the establishment of rhizoidal growth from one position only on the sporangium, typically at the point on the sporangium which is in contact with a substrate, appears to only occur in sporangia which are attached; however attached sporangia may also have non-polarised orientation. Rhizoids of R.rosea incubated with Pb (ll) (Figure 14d) have morphological differences compared to those incubated without Pb (ll). When incubated with 20 ppm Pb (ll) the fungus exhibits increased hyphal growth and increased branching; also extensive deposits of extracellular matrix are evident surround the attached fungal colony (Figure14d) and between the sporangium and substrate (Figure 14f).

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E F

Fig.14 Light microscope image (A) and SEM image (B) of unattached R. rosea sporangium incubated without lead Pb (ll); (C) light microscope image of R. rosea sporangium attached to lens paper (control - no lead Pb (ll)), arrow shows polar hyphal growth; (D) SEM image of attached R.rosea colony incubated with 20 ppm lead Pb (ll). Note the numerous small projections (arrow) extend from the rhizoids and abundant extracellular matrix (ECM) surrounding the colony; (E) SEM images of R. rosea with attachment appendages (arrow) incubated without Pb (ll); (F) incubated with Pb (ll). Note extensive ECM (arrow) located between the sporangium and substrate.

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4.4 Discussion

Copper and lead reduced the rate of attachment of R. rosea to lens paper, while copper, lead and zinc reduced rate of C. hyalinis and G. semiglobifer attachment to snakeskin. The rate of reduction was dependant on concentration, metal species and fungal species. Fungi declined in attachment at lower concentration (60 ppm) of copper in comparison to a decline at higher concentration (100 ppm) of lead. C. hyalinis and G. semiglobifer declined in the presence of zinc (60 ppm), while R. rosea did not decline. Generally, the patterns of increase and decline in attachment as metals increased were similar to the patterns previously determined for biomass change for these fungi (Henderson et al. 2015).

Change in attachment rates due to toxic metals were similar to zoospore production rates previously determined by Henderson et al. (2015). Zoospore production declined for all three fungi at 60 ppm copper and 100 ppm lead and for two fungi at 60 ppm zinc. R. rosea, however, increased in zoospore production by 150 – 450% at 60 ppm Zinc. R. rosea was also the only fungus which did not decline significantly in attachment in the presence of zinc. Previously, the increase in zoospore number in the presence of zinc was interpreted as an avoidance mechanism due to a toxic effect on zoospores (Henderson et al. 2015). However, our work here confirms that sporangial attachment does not decline at levels of zinc which cause biomass decline. Therefore, the zoospore response may be one of adaption to higher zinc levels, favouring increased reproduction rate over biomass, possibly by producing smaller sporangia.

The response of R. rosea rhizoids to increasing lead concentrations was similar to attachment rate. Rhizoids increased to maximum length at 30 ppm lead and rhizoid numbers increased to a maximum at 60 ppm lead. Increased hyphal extension in the presence of lead was also found to occur in the arbuscular mycorrhizal fungus Glomus intraradices (Pawlowska and Charvat 2004), which was interpreted by the authors as a metal avoidance strategy. Rhizoids are expected to be important in attachment because they are necessary for nutrient absorption and therefore require close contact with nutritive substrates (Jones 1994). The morphological change of rhizoids of R. rosea in the presence of lead may have caused the observed increase in attachment rate at concentrations of Pb below 60 ppm, which is below the threshold for toxicity and biomass

65 decline. Alternatively, the apparent increase in ECM observed for R. rosea incubated with Pb in SEM imagery may have increased attachment rates.

The correlation of zoosporic fungal attachment rate, biomass and rhizoid morphology may be explained in relation to colony building strategies of zoosporic fungi (Lilje and Lilje 2008). Changes in various nutrients promote colony growth. The formation of colonies may also mitigate toxic effects of metals, for example, by increasing extracellular matrix. Observations in the laboratory confirm that although sporangia grew on the sides of plastic falcon tubes in the absence of metal, during incubation with Pb (ll) there was no visible adherence to the sides of the tubes. Instead sporangia formed colonies which were suspended in media.

Growth patterns and rhizoid morphologies within colonies of Trichoderma viride and Rhizopus arrhizus were also affected by increasing Cu and Cd. Although length of fungal mycelium declined, hyphae were more densely branched as Cu and Cd levels increased. These changes in hyphal morphogenesis also included disruption to normal polarised growth (Gadd et al. 2001). Polarised growth in R. rosea rhizoids, where the rhizoids extend only from the base of the sporangium, appears to be initiated by attachment to lens paper. Unattached sporangia do not exhibit polarised growth (Figure 14a) and rhizoids extend from seemingly random positions on the sporangium. Attached sporangia, however, do not always have polarised growth morphology. Morphological changes due to copper in sporangia and rhizoids of R. rosea, including increased branching, have also been previously documented (Henderson et al. 2017).

Our work demonstrates that attachment of zoosporic fungi to organic substrates is detrimentally affected by toxic levels of copper, lead and zinc. This builds on previous work on toxic effects of metals on zoosporic fungi growth (biomass) and reproduction (Henderson et al. 2015). These results require confirmation in soil environments as attachment and growth of zoosporic fungi in soil may differ to the in vitro results presented here. In particular, our work was undertaken at one pH level (pH 5.5) and it is known that pH of growth media has a significantly effect on growth rate of zoosporic fungi (Gleason et al. 2010). Soil provides a heterogeneous environment for these fungi (Gleason et al. 2012) so levels of toxic metal exposure are expected to vary over spatial and temporal microscales.

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Zoosporic fungi are known to be widespread and common throughout soils world- wide (Sparrow 1960; Gleason et al. 2006). These fungi are thought to have keystone roles within the soil microbial loop (Gleason et al. 2012), in carbon cycling, as they degrade both dissolved and particulate organic matter (Sparrow 1960) and also in biogeochemical cycling of nitrogen and sulphur (Gleason et al. 2012). It is expected that toxic levels of copper, lead and zinc in soils will cause declines in soil zoosporic fungi populations and reduce colonisation of organic substrates, to the detriment of soil ecological function.

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Chapter 5: Metals, metal uptake and morphological change in fungi: A review

Introduction

The ability of microorganisms to function within environments with wide ranges of available metals is essential for their continued growth and reproduction. The availability and concentration of metals is expected to be an important factor in determining the ability of fungi to survive and reproduce in soil environments. All organisms are required to maintain micronutrient homeostasis; therefore all have a complicated system of metal uptake, chelation, trafficking and storage processes. Zinc is also required as a structural component in almost 10% of eukaryotic proteins, some of which are necessary for metal homeostasis. However soil saprotrophic fungi, such as the zoosporic fungi, may often lack utilizable nutrients (Gleason et al. 2012). Soil fungi also compete with other microorganisms, plants and animals for limited resources. Although the ability to grow and reproduce when metals concentrations are high, such as in polluted soils, is necessary for survival; conversely, the ability to outcompete other soil microorganisms for scarce resources is also essential for survival. The ability of fungi to upregulate metal transport systems, and morphological adaptations in response to metals, may enhance the uptake of essential nutrients and allow fungi to proliferate in low nutrient soil environments. Pathogenic fungi are able to upregulate metal transport and sequestration, particularly necessary as their hosts have strategies to withhold essential metals via the ‘nutritional immunity’ pathway (Kehl-Fie and Skaar 2010).

Metals and fungal morphology

Different fungi have different abilities to bind and transport metals. Metals may be bound to cell walls or transported into cells and sequestered within intracellular compartments (Gadd 1993). Fungi have a range of passive and active metal transport systems (Blaudez and Chalot 2011; Clemens et al. 2002) and a suite of antioxidant defence systems, including internally and externally expressed proteins (Gadd 2007; Das and Guha 2009). These systems may be expressed differentially depending on the fungus and metal species. The ability of each fungus to alter their lifecycle is also important, for

68 example, the fungus may increase reproduction or number of resistant spores at the expense of mycelial growth in response to metals. Differences in hyphal and rhizoidal morphologies may be a response to metal stress; increased length and decreased branching may be avoidance mechanisms, where hyphae extend without branching in order to traverse a toxic environment (Pawlowska and Charvat 2004). Rhizoids, like hyphae, are expected to be important in nutrient uptake and therefore morphological change may impact on this ability. In many instances metals appear to disrupt normal hyphal tip growth and branching. For example the filamentous fungi Trichoderma viride and Rhizopus arrhizus decreased in mycelial length in response to Cu or Cd and decreased in degree of branching in response to Cu (Gadd et al. 2001).

Fungi may survive toxic metals by increasing the formation of extracellular matrix (biofilms) (Paraszkiewisz and Dlugonki 2009; Paraszkiewicz et al. 2007). Yeast biofilm formation requires different cell types in certain configurations and therefore depends on morphological changes to the cell form (Shapiro et al. 2011). For example, the biofilm of Candida albicans is populated by yeast, pseudohyphae and hyphal forms. When C. tropicalis grows within a biofilm, the cells are able to withstand up to 65 times the concentration of metals when compared to planktonic cells (Harrison et al. 2006). Subinhibitory levels of metals may also control morphological change. The metals Co2+,

2+ + 2+ 2+ 2+ 2− 2− Cu , Ag , Cd , Hg , Pb , AsO and SeO3 inhibited yeast cells from changing to

2− 2+ hyphal cells in biofilms of C albicus and C. tropicalis; however CrO4 and Zn caused hyphal cell formation for C. tropicalis (Harrison et al. 2007). The absence of metals may also cause morphological change. When C. albicans, C.dubliniensis and C. tropicalis were grown in zinc deficient media, cells increased in size by more than four times the size of control cells, also cell adhesion to polystyrene increased (Malavia et al. 2017). It is clear that metals have a significant impact on the morphology and community structure of fungi.

Metal transport pathways and transcriptional regulation

The ability of fungal pathogens to sequester and transport iron and zinc is considered to be an important factor in virulence (Citiulo et al. 2012; Staats et al. 2013) and fungal uptake pathways for iron are relatively well understood (Haas et al. 2008). Copper uptake pathways have been determined as high and low affinity transporters for Saccharomyces cerevisiae and S. pombe (Ballou and Wilson 2016). However uptake pathways for other metals, particularly zinc, have only been explored more recently. In S. cerevisiae, zinc

69 uptake is mediated primarily by the high affinity (Zrt1) and low affinity (Zrt2) zinc uptake transporters (Gitan et al. 2003). In C. albicans the high affinity Zrt1 is regulated by the ZRT1- and IRT1-like protein (ZIP1) family (Huang et al. 2005) and functions under zinc- limiting conditions. Zinc efflux from the cell and cellular zinc storage is accomplished by the ZnT (zinc transporter) proteins (Huang et al. 2005). The ZIP1 family also co-regulates a secreted scavenger protein “zincophore” in C. albicans, Pra1 (pH regulated antigen), which binds large amounts of environmental zinc and also moderate amounts of copper (Huang et al. 2005). The protein is expressed under alkaline pH and zinc-limiting conditions. The process of Pra1 sequestration of zinc has been compared to iron chelation by secreted siderophores (Staats et al. 2013) and under zinc limiting conditions C. albicans requires Pra1 for normal hyphal growth. C. albicans also secretes an aspartic protease (Sap6) which actively chelates zinc extracellularly. Increases in zinc level cause increased cell to cell attachment, while deletion of zinc acquisition gene regulon ZAP1 result in lower aggregation (Kumar et al. 2017).

Both S. cerevisiae Zrt1 and Zrt2 transporters are able to utilise divalent iron and copper as substrates (Huang et al. 2005) and zinc-limited S. cerevisiae cells have the capacity to transport cadmium due to upregulation of Zrt1 (Gitan et al. 2003). In zinc- replete conditions the low-affinity zinc transporter Zrt2 is important in the uptake of zinc, copper, and iron (Zhao and Eide 1996). Zap1 is a transcription factor which regulates zinc homeostasis in C. albicans, C. gattii, Aspergillus fumigatus and S. cerevisiae (Staats et al. 2013). In S. cerevisiae, Zap1 regulates the expression of Dpp1, a farnesol synthesis protein which is important in cell to cell signalling during biofilm formation (Ganguly et al. 2011). The chytrid Spizellomyces punctatus has Pra1 and Zrt2 orthologues, however no Zrt1 orthologue (Citiulo et al. 2012). Evolutionary divergence suggests that many, if not all, fungi have Zap1orthologues (Ballou and Wilson 2016). It is likely that chytrids possess a Zap1 transcription factor which regulates Pra1 and Zrt2 in zinc transport and homeostasis.

Two highly conserved metal transporters are the cation diffusion facilitator (CDF) transporters and the Natural Resistance Associated Macrophage Proteins (NRAMP) family; both are conserved from prokaryotes to humans. In bacterial models the CDF ZitB has been established as an efflux pump for both inward and outward transfer of Cd2+ and is hypothesized to be part of a membrane bound metallotransporter complex driven by an a H+ diffusion (proton) gradient, coupling exchange of Cd2+ for H+ (Chao and Fu 2004).

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CDT transporters move Zn and other metal ions from the cytoplasm to either the extracellular space or into intracellular organelles.

NRAMP is divalent metal transporter which uses the transmembrane proton gradient, to move metals into the cytosol (Tamayo et al. 2014). The ATP-binding cassette (ABC) proteins are also a large (>10 000) protein family which is conserved from bacteria to eukaryotes (Kovalchuk and Driessen 2010). ABC proteins transport a broad range of substrates and perform numerous functions including as receptors and in cell protection against toxic metals. The ABC-B half-transporters are linked to heavy metal tolerance. They are vacuolar or mitochondrial membrane-associated and include Hmt1 from the vacuolar membrane of S. pombe which is required for cadmium tolerance (Kovalchuk and Driessen 2010).

The role of signalling pathways in fungi

Reactive oxygen species (ROS) are known to be cellular defence mechanisms. However, there is also a role for ROS in regulation of fungal populations, for example, suppression of germination and cell differentiation. There is evidence that in small quantities ROS are required for signalling within living cells (Breitenbach et al. 2015) in all stages of fungal and myxomycete development (Gessler et al. 2007). In the plant pathogenic fungus Sclerotium rolfsii sclerotia were the source of increasing peroxide levels in the presence of Fe2+ (Sideri and Georgiou 2000). Sclerotial differentiation in fungi is inhibited in the presence of numerous antioxidants (Georgiou 2000). ROS is an important determinant of the relationship between plant pathogenic fungi and the host in mycelial development (Gessler et al. 2007) and is initiated upon hyphal contact with the host organism. Peroxide may be synthesised by normal metabolic processes, as a by- product of electron transfer processes, and other processes which are specific to creation of ROS, including NADPH (Breitenbach et al. 2015). The target of peroxide signalling in fungi has not yet been elucidated (Breitenbach et al. 2015). However in the mammalian cell it is generally a phosphotyrosine phosphatase (PTP). NADPH oxidase (Nox) is required by the fungal cell for redox homeostasis as it reduces superoxide from the NADPH biological membrane electron transfer pathway (Takemoto et al. 2007). ) All fungi contain NoxA and NoxB (Scott et al. 2015) while some have NoxC (Takemoto et al.2007). NoxA is necessary for development of the sexual fruiting body in Aspergillus nidulansin (Lara-Ortíz et al. 2003), sexual reproduction in Podospora anserine (Malagnac et al. 2004)

71 and cell differentiation and growth in Neurospora crassa (Cano-Domínguez et al. 2008) and is also considered to be important in the development of multicellularity ( Lalucque and Silar 2003). NoxB is necessary for ascospore germination in P. anserina and N. crassa, while Magnaporthe oryzae requires both Nox A and NoxB to develop a penetration peg beneath the appressoria (Egan et al. 2007). The chytrid Allomyces macrogynus (Order Blastocladiomycota) contains NoxA, B C and R; while Batrachochytrium dendrobatidis (Order Chytridiomycota) contains NoxB and NoxR (Detry et al. 2014).

The Cpk1-MAPK cascade is a pheromone response pathway, sensing environmental factors to regulate gene expression changes including virulence via activating a pheromone receptor. The necrotrophic fungus Alternaria alternata mediates oxidative and osmotic stress via the HOG1 (high-osmolarity glycerol) MAP kinase and the SSK1 response regulator. This regulator is also required for invasive hyphal growth (Kumamoto 2005), mycelial growth, conidial germination, differentiation and protoplast formation (Yu et al. 2016). The HOG pathway was activated in Rhizopus microsporus during symbiosis establishment (Lastovetsky et al. 2016). Human parasitic fungi (C. albicans, C. neoformans, and A. fumigatus) utilize many signalling pathways (cascades) in order to sense environmental change and respond with morphological change (Shapiro et al. 2011). In C neoformans the Hog1 MAPK pathway is required for numerous environmental stressors including osmotic shock, UV irradiation, heat shock, oxidative damage, toxic metabolites, and antifungal drugs (Bahn et al. 2008).

Metalloproteins

For all fungi, superoxide dismutases (SOD) are essential enzymes in the detoxification of reactive oxygen species (ROS) generated by environmental stressors, including toxic levels of metals. SODs expressed by pathogenic fungi are thought to be involved in fungal virulence (Hwang et al. 2002; Frohner et al. 2009; Staats et al. 2013). Copper- and zinc-containing SOD1 (Cu/ZnSOD) is not just required for protection against oxidative stresses, it is also required for virulence in pathogenic fungi such as C. albicans (Hwang et al. 2002) and Cryptococcus neoformans (Cox et al. 2003). There is also evidence that upregulation of SOD is linked to morphological change in fungi. SOD1 activity increases in C. albicans during the morphological transition from yeast-like to hyphal growth (Gunasekaran et al. 1998). Also, SOD5 in C. albicans is uniquely hyphal- associated. Increased SOD5 is positively related to hyphal growth, adhesive proteins,

72 aggregation, adhesion and ECM formation (Heilmann et al. 2011). Reduced germ tube and hyphal growth occurred in Fusarium graminearum when SOD1 was disrupted (Yao et al. 2016). In AM fungi there is a high level of genetic diversity of SOD genes but the reason for this is unclear (Corradi et al. 2009). SOD1 is strongly expressed in the AMF Glomus intraradices during early stage symbiosis and could be needed, for example in signalling (Corradi et al. 2009). Further work is needed to fully understand the role of SODs in fungal development.

Metals in biofilm formation

Fungi may respond to increases in metals by increasing ECM production (Paraszkiewisz and Dlugonki 2009; Paraszkiewicz et al. 2007. This may increase the aggregation of cells to surfaces and to each other. In S. cerevisiae, Zap1 regulation is important in regulation of the signalling molecule farnesol (Ganguly et al. 2011). In C. albicans the zinc-response regulator Zap1 is upregulated at adherence and controls eleven adherence regulators and approximately one quarter of the predicted cell surface protein genes (Finkel et al. 2012). Along with the zinc acquisition gene regulon ZAP1, Sap6 regulates cell to cell attachment in C. albicans by chelating zinc (Kumar et al. 20017) indicating the protease may be essential in biofilm formation and nutrient supply of essential divalent metals to biofilm communities (Kumar et al. 2017).This is an important fungal community interaction in nutrient poor conditions where cell aggregation may allow sequestering of zinc or zinc-protein complexes.

Conclusions

The concept of nutritional immunity, the requirement of pathogenic fungi to upregulate metal sequestration and transport mechanisms, has been explored extensively in the literature. However, as pathogenic fungi require micronutrient trace metals, so also soil saprobes, such as chytrids and indeed fungi from all trophic levels. Competition for scarce metal resources in soil environments means soil organisms must secure essential nutrients such as zinc and copper in order to survive and proliferate. The mechanisms of iron uptake in fungi are well known, however, much more remains to be discovered about fungal strategies for assimilating other essential metals. The work undertaken so far on species of both pathogenic and non-pathogenic yeasts indicates roles for zinc transporter

73 genes, particularly the ZAP1 orthologue, in zinc assimilation. Recently, the importance of zinc in intracellular and extracellular signalling in mammals has been determined. Signalling is expected to be mediated by transient changes in free zinc in the nM to pM range (Liang et al. 2016). There are indications that zinc sensing and morphological change is important in colony development within biofilms for C. albicans and S. cerevisiae and may also be important in regulation of other fungal populations.

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Chapter 6: General Discussion

This study initially examined the important aspect of tolerance of high temperatures for two isolates of the zoosporic fungus G. semiglobifer. This data will allow further predictions of future distribution and abundance of the fungus. This study also examined the effects of metals on the growth and zoospore production of zoosporic fungi. Four isolates representing four orders within the phylum Chytridiomycota were sensitive to soluble metals with toxicity to growth increasing in the order Pb < Zn < Cu. The fungi responded similarly in sensitivity to the same metals in rates of attachment to organic substrates. These results may allow clarification of the physiological effect on chytrids due to toxic metal contamination in soils.

Although chytrids were expected to acclimate quickly to warming temperature due to their relatively short lifecycles, G. semiglobifer did not adapt to grow at higher temperatures in a semi-arid region compared to a mild temperate region. Maximum temperature for growth and maximum temperature for survival correlates well for numerous soil chytrid isolates (Gleason & McGee 2008). Therefore when soil temperature exceeds 37°C, G. semiglobifer will become quiescent or rely on heat resistant structures in order to survive. In this way, temperature is expected to modulate the abundance of G. semiglobifer within soil communities. A warming climate will reduce the growth of the fungus due to high temperatures more often in arid and semi-arid regions, where the upper threshold temperature for growth is more frequently exceeded. G. semiglobifer isolate from the warmer climate had consistently lower zoospore production which indicates a temperature-mediated shift in reproductive strategy.

Chytrids have a number of adaptive physiological responses which allow them to avoid exposure to metals. The increased production of ECM by attached R. rosea sporangia on exposure to Pb (ll) is likely to be an important defence against toxic metal and is an extracellular resistance mechanism commonly adopted by a variety of fungi (Gadd, 1993; Baldrian 2003). The polysaccharides and proteins in ECM are likely to precipitate metals and act as a physical barrier against toxic effect, including the precipitates described in chapters 1 and 4 on R. rosea rhizoids due to incubation with Cu (ll) and Pb (ll). Increased ECM in response to metal is also expected to increase chytrid-to- substrate and chytrid-to-chytrid attachments, thereby increasing colony formation and size. The benefits of colony formation include reduced exposure to toxic levels of metals.

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Increased zoospore production is a further adaptive response. Zoospores are sensitive to environmental changes (Gleason et al. 2012) which may trigger a response in zoospore behaviour. This may be crucial for the chytrid in seeking resources in soil environments. G. semiglobifer increased zoospore production two-fold after incubation at the upper temperature threshold for growth. This response may increase chytrid survival by allowing zoospores to disperse, perhaps to lower, cooler, soil horizons. Likewise, increased zoospore release occurred at low levels of Pb (60 mg L-1) and Zn (10 mg L-1) for Terramyces sp., R. rosea, C. hyalinus and G. semiglobifer. This response is not unique to chytrids. Thraustochytrids (Supergroup Straminopilia, Phyllum Labyrinthulomycota) also increase in spore production at similar low levels of Cu (ll) (2 – 64 mg L-1) (Pang et al. 2015). As these increases occurred below the threshold for toxicity we interpret this as a stimulatory effect due to an increase in osmotic potential or ionic strength. Chytrids respond rapidly to the short-term availability of soluble metals.

Metals cause numerous changes to rhizoid morphology, including increased branching, nodular protrusions and precipitates. Significant branching occurs when R. rosea is incubated with 60 ppm Pb (ll) (Appendix 4 Part D) and 60 ppm Cu (ll) (Appendix 4 Part E). Nodular protrusions (Appendix 4 Part C; Zn, Part E; Cu) and (Figure 14, Part D; Pb) and precipitations (Appendix 4, Part F) on rhizoid surfaces are evident. Morphological changes to the rhizoids in the presence of Pb (ll) are likely to impact the ability of chytrids to adhere to substrates. The correlation between R. rosea biomass increases from 60 to 80% when incubated with 60 ppm Pb (ll) and increased sporangial attachment rates reaffirms the fact that attachment is required for resource exploitation and growth and also that metals stimulate this cycle of growth. A significant amount of this growth is due to increased numbers and lengths of rhizoids. Total rhizoidal length produced on the lens paper baits increased by five to ten times from the control to 100 ppm Pb (ll). The calculated total volume of rhizoids produced per bait also increased from 0.53 to 0.89 mm3 in the absence of Pb (ll) to 2.41 to 8.78 mm3 in 100 ppm Pb (ll) (Appendix 5). R. rosea significantly increased in number of rhizoids per sporangium and then numbers declined at 100 ppm Pb (ll), which is also the concentration at which biomass declined. However, the average lengths of rhizoids per sporangium significantly increased at all concentrations of Pb (ll). The reason that chytrid rhizoid length did not reduce once the toxic limit was reached may be due to an exploratory growth phase, where rhizoid lengthening was an attempt to escape the toxic metal environment.

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Different species of fungi have different abilities to bind and transport metals. The sequestration and transport of metals, particularly iron and zinc, is considered to be an important factor in virulence. The low-affinity zinc transporter Zrt2 is also able to transport Zn, Cu and Fe (Zhao and Eide 1996). The chytrid Spizellomyces punctatus has a Zrt2 and also a Pra1 orthologue, which functions in extracellular zinc sequestration. This zinc homeostasis pathway is regulated in numerous fungi by the Zap1 transcription factor, which is conserved and may also be involved in metal uptake and homeostasis in chytrids. Interestingly, Zap1 also regulates the expression of a farnesol synthesis protein which is important in cell to cell signalling during ECM formation (Ganguly et al. 2011). Further investigation of potential metal transport pathways in chytrids is required.

This study provides insight into the effects of metals on the growth, zoospore production, attachment rates and morphology of zoosporic true fungi. The isolates examined represent four orders within the phylum Chytridiomycota. The levels of copper, lead and zinc determined here to be toxic to the lifecyles of these fungi in vitro may be applied in identifying inhibitory concentrations of metals in polluted soils. Metal toxicity in fungi may also be amelioration by a combination of metals (Hartley 1997). This study did not examine the effects on chytrids of combinations of different metals and chemical ameliorants, such as fertilizers, commonly added to agricultural soils, and this remains to be elucidated. The response of chytrids to metals, from sub-inhibitory to toxic levels, will affect chytrid population sizes and the rate at which soil organic material is degraded, it is therefore critical in the soil microbial loop. The effect of metals on chytrid populations within soil microbial communities should be further studied using community fingerprinting techniques, including RAPD, DGGE, RFLP, RISA and rep‐PCR in order to determine community phylogenetic changes and identify genes or gene families, including those encoding metal transport proteins, transcriptional regulators and enzymatic responses.

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Appendix 1

Pearson’s correlation coefficient (r) for regression of sporangial attachment rate with biomass yield at different concentrations of Cu (ll), Pb (ll) and Zn (ll). Attachment Correlation Fungal isolate Metal P N Experiment coefficient (r) Rhizophlyctis 1 .075 >0.05 25 rosea Cu (ll) 2 .402* .034 28

1 .503* .002 35 Pb (ll) 2 .321* .049 38

1 .532** .004 28 Zn (ll) 2 .140 >0.05 27

Chytriomyces 1 .273 >0.05 29 hyalinus Cu (ll) 2 .101 >0.05 28 1 .263 >0.05 35 Pb (ll) 2 .400* .001 39 1 .441* .021 27 Zn (ll) 2 .140 >0.05 27 Gaertneriomyces 1 .561** .002 29 semiglobifer Cu (ll) 2 .498* .007 28 1 .199 >0.05 35 Pb (ll) 2 .618** .000 35

1 .444* .018 28 Zn (ll) 2 .341 >0.05 28

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Appendix 2 - Growth of Gaertneriomyces semiglobifer isolate LEH1 at different concentrations of Cu (ll), Pb (ll) and Zn (ll). Dry weights (mg: mean ± SE) were recorded after 7 d. Means with the same letter are not significantly different (P>0.05)

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Appendix 3 – Methodology for measurement of rate of attachment at different concentrations of Cu (ll), Pb (ll) and Zn (ll) for Rhizophlyctis rosea, Chytriomyces hyalinus and Gaertneriomyces semiglobifer. Rubber discs containing five evenly spaced holes were placed in sterile 100 mm diameter plastic petri dish and 15 ml of metal solution was added to a dish for each metal concentration. After 1 hour the central hole of each disc was inoculated with zoospore suspension and one sterile circular 3 mm diameter bait was placed in each of the five holes.

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Appendix 4 – Additional morphological changes in R.rosea when incubated with metals.

A B

C D

E F

SEM image (A) and light microscope image (B) of unattached sporangium incubated without metal; (C) SEM image of sporangium incubated with 10 ppm Zn (ll) showing nodulated rhizoids; (D) light microscope image of sporangium attached to lens paper in the presence of 60 ppm Pb (ll), arrow shows hyphal swelling; (E) SEM image of sporangium incubated with 60 ppm Cu (ll), nodulations and numerous small projections extend from the rhizoids; (F) SEM image of a precipitate on a rhizoid incubated in 60 ppm Cu (ll).

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Appendix 5 – Rhizoid growth for Rhizophlyctis rosea attached to lens paper after incubation in different concentrations of Lead (ll) for 3 days.

Experiment 1 Experiment 2 Concentration of Pb (ppm) Total length of Total volume of Total length of Total volume of rhizoids rhizoids (L) (µm) rhizoids (mm3)* rhizoids (L) (µm) (mm3)*

0 283 .89 169 .53

5 1270 4.00 467 1.47

20 2393 7.52 149 .47

30 2675 8.40 208 .65

60 3313 10.41 384 1.21

100 2798 8.79 768 2.41

*Total volume is determined using the formula Ωr2L, with radius presumed to be 0.1µm

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