Conservation of a Critically Endangered Orchid Drakaea Elastica Lindl

Home , Caladenia, Diuris corymbosa, Drakaea, Drakaea elastica, Drakaea glyptodon, Kunzea ericifolia

Conservation of a critically endangered orchid Drakaea elastica Lindl. in the context of nutritional requirements and saprophytic competency of the mycorrhizal fungus and its propagation

Siti Nurfadilah

This thesis is presented for the degree of Master The University of Western Australia School of Plant Biology 2010

ABSTRACT

Drakaea elastica is a critically endangered orchid species that is a habitat specialist of nutrient deficient sandy soil and is dependent on a slow growing mycorrhizal fungus for its nutrient supply in its entire life cycle. In the conservation of this species, understanding the biology and ecology of the mycorrhizal fungus, especially the critical aspects determining the growth and the survival of the mycorrhizal fungus, is essential. The aim of this research is to investigate nutritional requirements, factors limiting the growth and survival, and saprophytic competency of the slow growing mycorrhizal fungus of Drakaea elastica in terms of its capacity to utilize a variety of nutrient sources (C, N, and P) relative to other sympatric faster growing orchid mycorrhizal fungi.

The assessment of the capacity of Drakaea elastica mycorrhizal fungus in axenic liquid media containing single nutrient sources showed that it utilised a variety of C, N, and P sources for its growth. A wide range of C sources that Drakaea elastica mycorrhizal fungus required for its growth encompassed glucose and mannose (monosaccharide), cellobiose (disaccharide), cellulose, pectin, starch, and xylan (polysaccharides). The mycorrhizal fungus did not grow on the other C substrates, galactose, rhamnose, arabinose (monosaccharide) and tannic acid (polysaccharides). Ammonium, a variety of amino acids (aspartic acid, glutamic acid, glutamine, asparagine, alanine, arginine, and glycine), and protein BSA are essential N sources required for the growth of Drakaea elastica mycorrhizal fungus. Drakaea elastica did not utilize nitrate, histidine, and proline as the N source for its growth. In terms of the requirement of P which is critically needed, Drakaea elastica mycorrhizal fungus utilised all forms of P sources including inorganic P (orthophosphate) and organic P (phytic acid and DNA). This investigation of nutritional requirements of Drakaea elastica is important to understand which nutrient sources required by Drakaea elastica mycorrhizal fungus for its robust growth. These data can also be a basis for an understanding of how Drakaea elastica mycorrhizal fungus utilizes different nutrient sources.

Drakaea elastica mycorrhizal fungus utilised the same C, N, and P sources with the sympatric faster growing orchid mycorrhizal fungi suggesting that it has to compete for the same nutrient sources. With its slow growing characteristic, Drakaea elastica mycorrhizal fungus presumably has lower competency to access nutrient sources

relative to faster growing fungi. This may explain the specialisation of Drakaea elastica mycorrhizal fungus in nutrient deficient sandy soil to avoid competition with many other fungi.

The growth and survival of Drakaea elastica mycorrhizal fungus was limited by the availability of an external source of the organic compounds thiamine and PABA. The ability of Drakaea elastica mycorrhizal fungus to utilize a wide range of C, N, and P sources was determined by the availability of these organic compounds which are important in the major metabolism for growth. Without these organic compounds, Drakaea elastica mycorrhizal fungus grew poorly on most C, N, and P sources.

The findings of this study can be incorporated into the biology and ecology of Drakaea elastica mycorrhizal fungus which is important in the conservation of Drakaea elastica. In the propagation of Drakaea elastica, understanding the biology and ecology for the seedlings development and establishment is important to optimise propagation of this critically endangered orchid species.

Table of contents

Abstract i Table of contents iii Acknowledgements v Declaration of candidate contribution vi Chapter I General introduction 1 1.1. Current status of Drakaea elastica 2 1.2. Distribution of Drakaea elastica 2 1.3. Taxonomy of Drakaea elastica 3 1.4. Biology and ecology of Drakaea elastica 4 1.4.1. Association with a specific pollinator 4 1.4.2. Association with mycorrhizal fungi 5 1.4.3. Habitat specialization of Drakaea elastica 7 1.5. Nutrient sources in orchid habitat 7 1.6. Utilization of nutrient sources by orchid mycorrhizal fungi 11 1.6.1. Media used to asses the capability to utilize nutrient sources 12 1.6.2. The assessment of the capacity to utilize nutrient sources 12 1.7. Saprophytic competency of Drakaea elastica mycorrhizal fungus 14 1.8. Propagation of Drakaea elastica 15 1.9. Project aims 15

Chapter II The capacity of Drakaea elastica mycorrhizal fungus to utilize carbon sources 19 2.1. Introduction 19 2.2. Methods 21 2.3. Results 24 2.4. Discussion 30

Chapter III The capacity of Drakaea elastica mycorrhizal fungus to utilize nitrogen sources 40 3.1. Introduction 40 3.2. Methods 42 3.3. Results 44 3.4. Discussion 48

Chapter IV The capacity of Drakaea elastica mycorrhizal fungus to utilize phosphorus sources 53 4.1. Introduction 53

4.2. Methods 54 4.3. Results 56 4.4. Discussion 61

Chapter V Optimization propagation of Drakaea elastica Lindl. 65 5.1. Introduction 65 5.2. Methods 65 5.3. Results 70 5.4. Discussion 75

Chapter VI General Discussion 78 6.1. Introduction 78 6.2. Nutritional requireents of Drakaea elastica mycorrhizal fungus 79 6.3. Factors limiting the growth and survival of Drakaea elastica mycorrhizal fungus 81 6.4. Saprophytic competency of Drakaea elastica mycorrhizal fungus 83 6.5. Propagation of Drakaea elastica 84 6.6. Conclusion 85

References 86

Appendix 1 100 Appendix 2 102

ACKNOWLEDGEMENTS

I am very grateful to my supervisors, Prof. Kingsley W. Dixon, Dr. Nigel D. Swarts, and Dr. Nigel J. Merritt for their excellent guidance, support, encouragement, and advice. I also wish to thank the orchid conservation group of Kings Park Botanic Gardens and Parks Authority, Dr. Ryan Phillips, Dr. Belinda Newman, and Myles Menz for their helps during my study in Perth. I am grateful to Dr. Eric Bunn, Keran Keys, and Beorn Harris that provided assistance and advice in the laboratory. I also would like to thank friends at Kings Park: Liann Smithson, Emma Dalziell, Clare White, Alison Ritchie, Adam Cross, Martha, Wolfgang Lewandrowski, Akshay Menon, Brinn, and other students doing their projects at Kings Park for the friendship during my study in Perth. Nedlanders: Wara, Nisa, mbak Yanti, Ing, Dewi, Heru Wibowo, Emielda Yusiharini, and other Indonesian students in Perth, Laily, Ristin, Tari, etc. thank you so much for the support.Also to ADS 9M friends; Umam, Zahid, Asep, Anto, Irwan, Mufid, Lora, Erna, Ekha, with you I learn how to be brave to reach dreams. I would like to thank The Government of Australia that awarded the scholarship that made it possible for me to study orhid conservation in Australia. My sincerest thanks for my family for their endless support and prayers for me.

CHAPTER I

General Introduction

The Orchidaceae is the largest plant family, comprising approximately 30,000 species classified into five subfamilies and containing 870 genera (Dressler, 1993). Orchids are cosmopolitan, occupying a diverse array of substrates and being found across continents in various climates and altitudes (Arditti, 1992). Despite the great diversity of orchids, many are under threat of extinction (IUCN, 2001). This is thought to be related to the unique biological characteristics of orchids, particularly their high dependency on the other organisms (mycorrhizal fungi and pollinators) to complete their life cycle. External threatening processes also place severe pressure on orchid habitat. Conservation programs are needed to prevent orchids from becoming extinct and the knowledge of the biology and ecology of orchids is important for the success of orchid conservation programs (Swarts and Dixon, 2009; Brundrett, 2007).

This thesis aims to increase the understanding of the biology and ecology of Drakaea elastica Lindl., a critically endangered terrestrial orchid endemic to Western Australia that is specialized on a nutrient deficient sandy soil habitat (Hopper and Brown, 2007). In its entire life cycle, Drakaea elastica relies heavily on a mycorrhizal fungus which is typically slow growing (in culture, relative to other terrestrial orchid mycorrhizal fungi) as the nutrient supplier (Ramsay et al, 1986). Thus, understanding factors determining the growth and the survival of the mycorrhizal fungus associate is substantial in the conservation programs of Drakaea elastica. The focal point of this study is to investigate the biological and ecological aspects influencing the fitness and the survival of Drakaea elastica mycorrhizal fungus, including its nutritional requirement, factors limiting its growth and survival, and its saprophytic competency in terms of its capacity to access a variety of nutrients (carbon, nitrogen, and phosphorus sources) relative to faster growing fungi from sympatric orchids. Furthermore, this study also investigated the biological and ecological requirements for seed germination and seedling development of Drakaea elastica for the optimization of propagation of this critically endangered orchid.

1.1. Current Status of Drakaea elastica

Drakaea is a terrestrial genus containing 10 other taxa endemic to the South-west Australian Floristic Region. Five Drakaea species are threatened with extinction and are legally protected under the Western Australian Wildlife Conservation Act and the Commonwealth Environment Protection and Biodiversity Conservation Act, including Drakaea elastica (Hopper and Brown, 2007). Drakaea elastica is declared as Rare Flora and ranked as Critically Endangered (Brown et al, 1998; IUCN 2001). Species classified as critically endangered have a high likelihood of becoming extinct without intervention as their habitats are usually under serious pressure from a variety of threatening processes. Habitat of Drakaea elastica is subject to a diverse array of threatening processes. Department of Environment and Conservation (2009) identified threatening processes towards habitat and populations of Drakaea elastica that include (i) land clearing for housing, industry, roads, other construction infrastructure, and agriculture that impact on the reduction in habitat size, quality and connectivity, (ii) degradation and fragmentation of habitat, (iii) increased density of ground-level native vegetation that has resulted in the disappearance of D. elastica in several populations, (iv) grazing of flower buds by rabbits, kangaroos, grasshoppers or caterpillars, (v) rubbish dumping that can introduce weeds and taking up habitat area, (vi) weeds, (vii) inappropriate fire regimes that may affect the viability of populations, (viii) poor recruitment where plant numbers continue to decline that may be associated with poor rainfall, decrease of the presence of the mycorrhizal fungus and the pollinator, (ix) recreational use of the areas, and (x) sand extraction. Apart from the threatening processes to Drakaea elastica habitat, the high specialization in terms of the symbiotic relationship with a specific mycorrhizal fungus and pollinator is contributing factors possibly limiting the survival of D. elastica. The lost of the mycorrhizal fungus and the associated pollinator would lead to the extinction of this species.

1.2. Distribution of Drakaea elastica

The distribution of this orchid is restricted to small areas of tall Kunzea ericifolia in deep sandy soil within a Banksia woodland between Cataby and Ruabon on the Swan Coastal Plain (Hoffman and Brown, 1998; Hopper and Brown, 2007). There has been a 2 marked decrease in suitable D. elastica habitat in that, of the 52 known locations of this orchid, in recent years, plants have only been found at 24 locations. A single population is known from the Moora District near Cataby and another 23 small populations in the Swan and Central Forest Regions, from Perth southwards to Ruabon Nature Reserve on the Swan Coastal Plain (Department of the Environment, Water, Heritage and Arts, 2007). The Swan Coastal Plain has a high degree of species diversity and a relatively high ecosystem diversity. However, much of this area is threatened by clearing for agricultural purposes, grazing, weed invasion, mining and other factors (Mitchel et al., 2002).

1.3. Taxonomy of Drakaea elastica

Historically, the name of Drakaea was given by Lindley in 1840 in respect to Miss Sarah Anne Drake, a botanical artist with her full dedication to orchid drawing and documentation. Two single-flowered Drakaeas were first collected in Swan River by James Drummond who was able to distinguish the two Drakaeas and named them D. elastica and D. lucida (which is now named D. livida). However, Lindley considered them as the same species, D. elastica and afterwards Drakaea was known as a monotypic genus consisting of one species, D. elastica. Since then, D. elastica which was also wrongly applied to D. livida has had a long history of confusion and misapplication of the name of new putative species members of Drakaea found by some other authors. This continued until Clements (1989) clarified the correct application of D. elastica and D. livida. With more findings of unidentified taxa within the genus, taxonomy of Drakaea has been revised by Hopper and Brown (2007) resolving separation the species members within the genus and made clear differentiation based on the thorough studies of morphology, habitat characteristics, geographic distribution and baiting male wasps experimentation to clarify distinct species within the genus. Drakaea elastica can be distinguished from other orchid species in the same genus by its specific characteristics with its shiny and light-green leaf, and the prominently hairy upper section to its labellum (Hoffman and Brown, 1998).

1.4. Biology and Ecology of Drakaea elastica

Drakaea elastica has typical morphology of Drakaea species in having a single leaf with heart shape held flat against the soil and a very short stem. The root is poorly developed penetrating in the soil and at the end of the root is a tuber as an adaptation to survive drought conditions. The formation of a tuber is a typical adaptation form of Western Australian terrestrial orchid species that allows survival in summer when mycorrhizal colonisation is devoid and access to water is limited (Dixon and Tremblay, 2009; Huynh et al, 2004). Tubers contain nutrient reserves and can persist in the soil for years (Batty et al, 2006).

After summer dormancy, the development of young plants occurs during the growing seasons of winter and spring, initiated by shoot development from the dormant tuber. In the moist conditions of winter, specific mycorrhizal fungi actively grow and centrally colonize the cortical cells of the stem forming a swollen structure which is called a collar. This mycorrhizal colonization is thought to be essential for nutrient supply. Anthesis occurs in late September to early November and often the leaf withers at this stage (Hopper and Brown, 2007).

The Drakaea elastica flower is bisexual with an anther and stigma joined together to form the column. The column is a characteristic structure in orchid flowers as one of the evolutionary forms in the orchid inflorescence, with the position of the anther containing pollen grains at the top of stigma essential for the success of insect-mediated pollination. Successful pollination is signified by a withered labellum and the swollen inferior ovary develops into a capsule containing a large number of seeds within four weeks. Pollination of Drakaea elastica is facilitated by a specific pollinator that is sexually attracted to its flower.

1.4.1. Association with a specific pollinator

Drakaea elastica achieves sexually deceptive pollination, attracting thynnine wasps (Zaspylothinnus spp.) by having floral morphology mimicking female wasps and a capacity to release a female pheromone-like scent (Peakall, 1990; Hopper and Brown, 2007). Evolutionary morphology of D. elastica flower with the labellum resembles female wasps and signals of a pheromones-like scent tricks male wasps which are sexually attracted to grasp the labellum. This male activity leads to the movement of

4 pollen into the stigma on the column which is positioned just in front of the labellum, resulting in pollination and the production of a fruit and seed set.

Like other orchid seeds, D. elastica seeds are dust-like. Orchid seeds are the smallest of any plant family and anatomically consist only of a mass of embryo and a layer of testa. The seeds do not contain endosperm as nutrient reserves (food storage essential for nutrient and energy supplies needed during the seed germination) (Arditti, 1967; 1992; Batty et al, 2000; Arditti and Ghani, 2000). With the lack of nutrient reserves, orchid seeds heavily rely on exogenous nutrients for the seed germination. The strategy to gain external nutrients is through symbiotic association with mycorrhizal fungi which transfer nutrients to the orchid seeds (Smith and Read, 1997).

1.4.2. Association with mycorrhizal fungi

Like other orchids, Drakaea elastica also forms a symbiotic association with mycorrhizal fungi for seed germination and seedling development. The mycorrhizal fungus infects the orchid seeds and forms pelotons, specific structures like coils in parenchyma cells of the embryo (Peterson, 1998). External hyphae of orchid mycorrhizal fungi absorb nutrients from the soil and deliver the nutrients to the internal hyphae colonizing orchid seeds and facilitating seed germination and seedling development (Smith and Read, 1997).

Drakaea elastica has a specialized symbiotic relationship with a mycorrhizal fungus which is characteristically very slowly growing and has a distinctive colour (pink) on Potato Dextrose Agar (PDA) culture (Ramsay et al, 1986). In this study, observation on the growth of Drakaea elastica mycorrhizal fungus from pelotons isolated from collars and plated on SSE media confirmed the slow growing characteristic of the mycorrhizal fungus associated with Drakaea elastica. Fungal hyphae of D. elastica grew out from the pelotons after one to three months plating the pelotons on SSE.

Figure 1.1. Drakaea elastica fungal culture with its distinctive colour (pink) on Potato Dextrose Agar (PDA)

In the taxonomy of orchid mycorrhizal fungi, all orchid mycorrhizal fungi belong to the Basidiomycota and most belong to the form-genus Rhizoctonia (Currah and Zelmer, 1992). The classification of orchid mycorrhizal fungal genera is divided into anamorphic and teleomorphic genera based on the state when the orchid mycorrhizal fungi occur. Most orchid mycorrhizal fungi occur as anamorphs, asexual state with no production of basidiospores and rarely occur in sexual state with the production of basidiospores (teleomorphs) (Currah and Zelmer, 1992; Hollick, 2004). Anamorphic genera include Ceratorhiza, Epulorhiza, and Moniliopsis), while teleomorphic genera consist of Ceratobasidium, Thanatephorus, Tulasnella, and Sebacina (Warcup and Talbot, 1967; Currah and Zelmer, 1992; Otero et al 2002). Mycorrhizal fungus associated with D. elastica belongs to the teleomorphic fungal genus Tulasnella (Ramsay et al, 1986).

Like other Australian terrestrial orchids, in the adult stage, D. elastica appears to adopt a mixotrophic model as a strategy to obtain nutrients effectively through dual nutritional capacity, photosynthetic capability and maintaining mycorrhizal association (Dearnaley, 2007; Julou et al, 2005; Tedersoo et al, 2007). This is related to its ineffective photosyntetic capacity with the poorly developed roots and the limited access to nutrients as they occur in nutrient deficient sandy soil.

1.4.3. Habitat specialization of Drakaea elastica

Drakaea elastica has a highly specialized habitat characterized by nutrient deficient sandy soils and is often found in association with Kunzea ericifolia in Banksia woodlands (Hopper and Brown, 2007). This species grows on bare patches of grey- white sand and with sparse organic matter in low-lying areas alongside winter-wet swamps and flats. Banksia menziesii, B. attenuata and B. ilicifolia are the typical Banksia taxa in Banksia woodlands of Drakaea elastica habitat. Drakaea elastica often occurs in sympatry with other orchid taxa sharing the same slow growing mycorrhizal fungus, such as Drakaea glyptodon (king-in-his-carriage), D. livida (warty hammer orchid), Paracaleana nigrita (flying duck orchid), Leporella fimbriata (hare orchid) and Pyrorchis nigricans (red beaks) (Carstairs and Coates 1994 cited in DEC, 2009; Hoffman and Brown 1998).

The habitat characteristic of D. elastica may reflect the habitat characteristic of the mycorrhizal fungi (Batty et al, 2002). The existence of orchids in this specific habitat shows the presence of the mycorrhizal fungus associated, the availability of specific nutrients promoting the growth, and the survival of the mycorrhizal fungus in this specialized habitat. Specialization of Drakaea elastica in nutrient deficient sandy soil may be related to the specialization of the mycorrhizal fungus associated in this specific habitat.

1.5. Nutrient sources in orchid habitat

In natural habitat, mycorrhizal fungi absorb nutrients from the soil and transfer a proportion of nutrients to the orchids with which they associate (Smith and Read, 1997). Available nutrients required for the growth and the survival of orchid mycorrhizal fungi are associated with organic matter which forms a variety of nutrient sources (Brundrett et al, 2003; Tuomi et al, 2009; Kuperman, 1999; Berg, 1984; Lindahl et al, 2002; Leake and Read, 1997). Plant litterfall together with animal and microbial residues form the top fraction of organic matter on the soil surface (Couteaux et al, 1995). Drakaea elastica habitat is characterized by sandy soil with sparse organic matter that can be from litterfall of Kunzea ericifolia or Banksias which potentially provide nutrients in this habitat.

Nutrients in the soil are available in a diverse form of carbon (C), nitrogen (N), phosphorus (P), organic compounds, vitamins and other minerals as a result of complex metabolism in soil by microbial processes and root-mycorrhizal fungi interaction (Kononova et al, 1961, Quastel, 1965; Taylor et al, 2009). a. Carbon

Carbon (C) is an essential nutrient for all living organisms and has two major functions (i) as the only source of chemical energy for cell function such as cellular metabolism and growth (ii) as a compound needed for the synthesis of essential biological materials, such as cell walls, food storage, nucleic acids, etc (Cochrane, 1958). In comparison to other nutrients such as N and P, C is the largest constituent in soil making up two thirds of the total nutrients in soil organic matter (Marschner et al, 2008; Rouifed et al, 2010). Soil analysis showed a variety of C sources present in soil organic matter including

(i) Monosaccharides

Monosaccharide is only present in a small amount in soil organic matter. This is a simple sugar that constitutes a complex polymer of polysaccharides and oligosaccharides. Its simple structure and low molecular weight allows for absorption directly from soil (Griffin, 1994). Two types of monosaccharide that naturally occur in soil are pentose with five C rings (arabinose) and hexose with six C rings including glucose, fructose, mannose, galactose, and rhamnose (Sparks, 1999; Cochrane, 1958).

(ii) Disaccharides

Disaccharides consist of two simple monomers of monosaccharide, such as cellobiose (two monomers of glucose), lactose (glucose and galactose), sucrose (glucose and fructose). They can be utilised directly or broken down first into simple sugar before absorption (Cochrane, 1958).

(iii) Polysaccharides

Polysaccharides are the main components of plant cell walls, including cellulose, hemicelluloses, pectin, and lignin comprise the highest proportion of C in soil (Norkrans, 1963; Cochrane, 1958; Sparks, 1999). Complex polysaccharides composed of a simple sugar (monosaccharide) need to be broken first into simple C before utilisation (Griffin, 1994). Cellulose is the major C in the soil organic matter (Cochrane, 8

1958). It is a homopolymer consisting of a chain of the same monomers as monosaccharide glucose and its utilisation requires extracellular enzymes cellulases (an array of enzymes working synergistically including 1,4-β endoglucanase, cellobiase and 1,4-β glucosidase) for complete hydrolysis process of complex polymer of cellulose into glucose (Valaskova and Baldrian, 2006, 2008; Cairney and Burke, 1998). Hemicelluloses are also an essential C in soil. Unlike cellulose, hemicelluloses are heteropolymers composed of different monomers such as mannan (glucose and mannose), arabinan (glucose and arabinose), rhamnan (glucose and rhamnose), xylan (xylose as the main components and a low proportion of arabinose). Utilisation of hemicelluloses is facilitated by specific enzymes, such as 1,4-β- mannosidase, 1,4-β- arabinosidase, in hydrolysis process to cleave complex polymer of hemicelluloses into simple sugars (mannose, arabinose, glucose, xylose etc.) (Steffen et al, 2007; Burke and Cairney, 1997). Pectin is also an important C source in soil widely utilised by a diverse range of mycorrhizal fungi. Orchid mycorrhizal fungi are reported to produce extracellular enzymes pectinase, pectate lyase, and polygalacturonase to breakdown complex polymer pectin into simple sugars which will subsequently be absorbed (Hadley and Perombelon, 1963). Starch and aromatic C, such as lignin also constitute essential elements of C sources in soil (Griffin, 1994). b. Nitrogen

Nitrogen (N), composed of organic N and inorganic N, is scarce in soil, present in small quantities relative to C (only 0.67% - 2.35% of total nutrients in organic matter) (Rouifed et al, 2010; Chalot and Brun, 1998; Adams et al, 2002). Organic N is comprised of amino acids and protein. Amino acids are simple organic N that can be absorbed directly by soil microorganisms including mycorrhizal fungi. A range of amino acids that can be found in soil including basic amino acids (arginine and histidine), acidic amino acids (aspartic acid and gibberelic acid) and neutral amino acids (alanine, glycine, proline, glutamine, etc.). Protein is a complex of organic N composed of several amino acids, which is also an essential N source in soil. Utilisation of protein requires extracellular enzymes, protease to depolymerize complex structure of protein to release amino acids before absorption (Leake and Read, 1990). Inorganic N (mineral N) + - in soil is present in the forms of ammonium (NH4 ) and nitrate (NO3 ) (Allen et al, 2003). Available inorganic N (mineral N) in soil is the result of the mineralization of + - organic N into (NH4 ) and subsequent oxidation to nitrate (NO3 ) (Hodge, 2005;

Colpaert and Tichelen, 1996). The mineralization of organic N in soil is well known to occur at low rate (Connell et al, 1995; Attiwill and Adams, 1993). c. Phosphorus

Phosphorus (P) is the scarcest element in soil organic matter relative to C and N representing 0.027% - 0.120% of total nutrient in soil organic matter. P comes in the form of organic and inorganic P (Rouifed et al, 2010; Adams, 1992). Organic P is composed of phosphomonoester and phosphodiester (Paul and Clark, 1996; McDowell et al, 2005). Phosphomonoester is present in the forms of inositol hexaphosphate and phytic acid (a major storage of P in plants) (McDowell et al, 2005; Lin et al, 1987; Hegeman et al, 2001; Murphy et al, 2008). Phosphodiester is present in soil in the form of DNA (deoxyribo nucleic acid) that are from the DNA of plants and other living organisms which contain P elements (Paul and Clark; 1996). Organic P (phosphomonoesther and phosphodiester) can be utilised after a complete hydrolysis process that is facilitated by the enzymes phosphomonoestherase and phosphodiesterase. Inorganic P (orthophosphate) is released from this process, which will subsequently be absorbed (Bartlett and Lewis, 1973; Margesin and Schinner, 1994; Gressel et al, 1996; Leake and Miles, 1996). Inorganic P in soil is available in the form of orthophosphate as the results of the mineralization of organic P (Colpaert and Tichelen, 1996). d. Organic compounds

Several organic compounds can be found in soil in trace amounts of less than 1 %, including vitamins, hormones, and other organic compounds (Nardi et al, 2000). The source of organic compounds can be from detrital plant materials in organic matter as plants contain thiamine, folate, PABA (para amino benzoic acid), and organic compounds (Gambonnet et al, 2001; Rapala-Kozik et al, 2009; Goyer, 2010) and from root exudates (Griffin, 1994). Plants are well known to release organic compounds, amino acids and vitamins through the roots into the rhizosphere (Hinsinger et al, 2006; Gunawardena et al, 2006). Roots of Kunzea ericifolia, may release several organic compounds to the rhizosphere, providing nutrients for D. elastica mycorrhizal fungus.

1.6. Utilisation of nutrient sources by orchid mycorrhizal fungi

The ability of orchid mycorrhizal fungi to utilise a variety of nutrient sources (C, N, and P, and other essential nutrients) is important to their saprophytic activity for their growth and survival in soil. This ability is also important to provide nutrient sources for the orchids associate that are completely reliant on the mycorrhizal fungi for their nutritional requirements (Smith and Read, 1997; Rasmussen, 2002).

Orchid mycorrhizal fungi can utilise a variety of nutrient sources. Midgley et al (2006) found that orchid mycorrhizal fungi associated with Pterostylis spp (Ceratobasidium) utilised a wide range of C sources, including arabinose, cellulose, CMC, cellobiose, pectin, and xylan in axenic culture. Orchid mycorrhizal fungi also take up glucose, cellulose, starch and transfer a proportion of C to the orchid seeds to induce seed germination and development of seedlings in symbiotic seed germination experiments on solid media (Hadley, 1969; 1984; Beyrle and Smith, 1993). Furthermore, orchid mycorrhizal fungi were demonstrated to be able to utilise cellulose in soil (Smith, 1966).

N is required by orchid mycorrhizal fungi for their growth. Orchid mycorrhizal fungi can utilise a variety of N sources such as ammonium, nitrate, and amino acids with varying capacity (Hadley and Ong, 1978; Stephen and Fung, 1971). For example, mycorrhizal fungi of the genus Tulasnella do not utilise nitrate, whereas mycorrhizal fungi from a fungal genus Ceratobasidum have a capacity to utilise nitrate effectively (Hadley and Ong, 1978).

The capacity of orchid mycorrhizal fungi to utilise organic P sources is largely unknown with only utilisation of inorganic P (orthophosphate) (Alexander et al, 1984) having been tested and thus still needs further research.

Organic compounds are also essential nutrients for some orchid mycorrhizal fungi. Some workers reported that some orchid mycorrhizal fungi require specific exogenous organic compounds to grow (Stephen and Fung, 1971; Hijner and Arditti, 1973; Hadley and Ong, 1978). The influence of organic compounds (biotin, thiamine, para amino benzoic acid (PABA), nicotinic acid, pyridoxine HCl supplemented in axenic liquid media on the growth of some orchid mycorrhizal fungi have been tested. The experimental designs were without organic compounds, single organic compounds, and combination of organic compounds in axenic liquid media. The results showed that orchid mycorrhizal fungi grew poorly in axenic liquid media without organic compounds or with single organic compounds, however these orchid mycorrhizal fungi 11 grew well in these media containing a combination of organic compounds (thiamine and PABA). Thiamine and PABA are known as essential compounds required as enzymatic cofactors in the universal metabolic pathways including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, and the metabolism of C and amino acids, which is important for growth (Goyer, 2010; Rapala-Kozik et al, 2009; Cossins, 2000; Stephen and Fung, 1971; Bucking et al, 2008; Jabrin et al, 2003; Bassett et al, 2004; 2005; Scott et al; 2000; Cossins and Chen, 1997). The function of enzyme cofactors derived from these organic compounds can not be replaced by amino acids or other nutrients (Webb and Smith, 2009). Many orchid mycorrhizal fungi associated with assignable Tulasnella require external source of thiamine and PABA to grow, whereas orchid mycorrhizal fungi associated with assignable Ceratobasidium do not require these external organic compounds to grow (Hadley and Ong, 1978). This suggests that Ceratobasidium has an ability to biosynthesise these organic compounds, while Tulasnella appears to be unable to biosynthesise these organic compounds (Hadley and Ong, 1978; IGEM, 2008).

Non-orchid mycorrhizal fungi are also widely demonstrated to require a wide range of nutrient sources including a variety of C, N, and P sources for their growth (Midgley et al, 2004; 2006; Sawyer et al, 2003a, 2003b, 2003c; Chen et al, 1999).

1.6.1. Media used to assess the capability to utilise nutrient sources

Assessment of the ability of a diverse array of mycorrhizal fungi to utilise a wide range of nutrient sources is often performed in axenic liquid media, with CN MMN formula (low Carbon Nitrogen Modified Melin and Norkrans) commonly used (Anderson et al, 1999; Chen et al, 1999; Sawyer et al, 2003a, 2003b; Midgley et al , 2004; 2006). A variety of C, N, and P sources potentially utilised by mycorrhizal fungi is investigated in CN MMN liquid media containing single type of C, N, and P sources tested. Previous investigation of forms of nutrients (C, N, and P) potentially utilised by mycorrhizal fungi for growth was performed in solid media. However, agar contains complex nutrients that can make interpretation difficult, whether C, N, and P from agar or from C, N, and P tested that support the growth of the mycorrhizal fungi (Midgley et al, 2006).

1.6.2. The assessment of the capacity to utilise nutrient sources

The capacity of orchid mycorrhizal fungi and non-orchid mycorrhizal fungi to utilise a variety of nutrient sources (C, N, and P sources) is usually assessed in the period of the exponential growth phase for each mycorrhizal fungi (Midgley et al, 2004; 2006; Sawyer et al, 2003a, 2003b). The exponential growth phase is the period of time when the fungi reach the highest metabolism rate and the highest rate of growth after inoculation into fresh media (Griffin, 1994).

Stationary phase

)

Exponential phase

Biomass of fungi of Biomass Biomass of fungi of Biomass

fungi orrhizal

Growth ( Growth fungi mycorrhizal Of Of myc Of ( Growth Lag phasephase

time Fig.1.2. Growth model of fungi over time (adapted from bacterial growth curve (Toddar, 2008)

Griffin (1994) described the growth and lifetime of fungi in nutrient-containing media. When fresh fungal inoculums are inoculated into nutrient – containing liquid media, the mycorrhizal fungi proliferate and the biomass of the mycorrhizal fungi increases with time. The growth rate of the mycorrhizal fungi is obtained by plotting the fungal biomass against time. Initially, the fungi have to adapt to the fresh environment and grow slowly, recognized as lag phase. After the fungi have adapted, the fungi grow rapidly resulting in a sharp increase of the fungal biomass during exponential phase. The highest metabolic rate occurs in this phase. After peak growth in the exponential phase, mycorrhizal fungi enter a stationary phase, when the rate of multiplication is very low resulting in the flat line (steady line). The growth model of fungi is described in Fig. 1.2.

In the assessment of the capacity of mycorrhizal fungi to utilise nutrient sources, liquid media which contain single type of each nutrient sources tested were inoculated with mycorrhizal fungi. Mycorrhizal fungi were harvested during the period

13 of their exponential growth phase. The growth in liquid media and biomass production on each single type of nutrient sources tested are the parameters to measure the capacity of the mycorrhizal fungi to utilise nutrient sources.

1.7. Saprophytic competency of Drakaea elastica mycorrhizal fungus

In soil, the hyphae of mycorrhizal fungi grow extensively to access a variety of nutrient sources in soil during their saprophytic activity. Many mycorrhizal fungi are reported to have a capacity to utilise a diverse array of nutrients including a variety of C, N, and P sources in liquid media (Midgley et al, 2004; 2006; Sawyer et al, 2003a, 2003b, 2003c; Chen et al, 1999). This reflects their saprophytic ability to access a diverse array of available nutrient sources in soil (Anderson et al, 1999). Competition for nutrient resources between mycorrhizal fungi at the same habitat prevails as most mycorrhizal fungi utilise the same common C, N, and P sources. However, they also appear to have a different niche in the utilisation of few certain nutrient sources. For example, ericoid mycorrhizal fungi from Woollsia pungens (Epacridaceae) and ectomycorrhizal Pisolithus spp. demonstrated to have an ability to utilise a diverse array of the same N sources (ammonium, glutamine, alanine, asparagines, glutamic acid, and other N sources) show different niche in the utilisation of few other N sources (histidine and arginine). Ericoid mycorrhizal fungi from Woollsia pungens utilise histidine and arginine as sole N sources effectively, however, ectomycorrhizal Pisolithus spp. from a similar dry sclerophyll forest habitat have a low capacity to utilise these N sources (Whittaker and Cairney, 2001; Anderson et al, 1999). Another example, orchid mycorrhizal fungi associated with Pterostylis spp and ericoid mycorrhizal fungi from Woollsia pungens and Leucopogon parviflorus (Epacridaceae) utilised the same some carbon sources (arabinose, cellulose, glucose, CMC, cellobiose, pectin, and xylan). They also showed different capacity to utilise a few other certain C sources (tannic acid and tryptophan). Mycorrhizal fungi of Pterostylis spp could not utilise tannic acid and tryptophan, while ericoid mycorrhizal fungi from Woollsia pungens and Leucopogon parviflorus (Epacridaceae) from the same habitat utilised both sources (Midgley et al, 2006). These differences in the utilisation of few certain nutrient sources are considered to be of ecological significance to minimize competition between mycorrhizal fungi for the same other nutrient sources (Whittaker and Cairney, 2001).

The slow growth characteristic of the mycorrhizal fungus becomes an important addition factor in the conservation of Drakaea elastica as the mycorrhizal fungus is potentially to be outcompeted by faster growing fungi in the competition for nutrient sources. Thus, the saprophytic competency of the slow growing mycorrhizal fungus in terms of its capacity to utilise a wide range of nutrient (C, N, and P sources) needs to be investigated. Specialization in nutrient deficient sandy soil may be a strategy to minimize competition with many other fungi to survive.

1.8. Propagation of Drakaea elastica

Given the rarity of D. elastica and population decline of D. elastica with many factors threatening its survival, optimization of propagation is expected to become the most reliable tool for providing a large number of seedlings for reintroduction and recovery of this critically endangered species. With limited information on effective propagation of this species, research on the optimization of propagation of D. elastica is needed to develop an effective and efficient means of generating robust seedlings for reintroduction back into suitable habitat.

Propagation includes a number of steps, with the first being seedling generation by seed germination. Seed germination should become a priority as it can maintain genetic variability of its species, which is important for the survival of the seedlings in the longer term when they are reintroduced to its natural habitat. Selection of the best substrates that support a high rate of growth and survival of seedlings prior to seedling establishment in natural habitat is also important for the preparation of the robust seedlings for reintroduction program.

1.9. Project aims

The growth and the survival of the mycorrhizal fungus determine the survival of Drakaea elastica. Thus, conservation of D. elastica needs to consider key aspects influencing the growth and the survival of the mycorrhizal fungus which is slow growing. This study investigated the growth, fitness and the survival of D. elastica mycorrhizal fungus. More specifically, this study focused on the nutritional requirements, factors promoting and limiting mycorrhizal growth and survival, and

15 saprophytic competency in terms of the capacity to utilise a wide range of nutrients relative to faster growing fungi. Understanding nutritional requirements gives insight into which forms of nutrients that D. elastica mycorrhizal fungus requires for its robust growth. With its characteristically slow growing saprophytic activity, distribution of D. elastica in soil can be limited by faster growing fungi. This can influence its existence in its natural habitat as it struggles to compete with more successful mycorrhizal fungi. Identifying factors that may limit the growth and the survival of the slow growing mycorrhizal fungus of Drakaea elastica can be a basis for possibile action to conserve the mycorrhizal fungus.

Data in the present study will provide knowledge of the biology and ecology of the D. elastica mycorrhizal fungus which is fundamental in the conservation of Drakaea elastica. Given the rarity of D. elastica and the declined population of D. elastica, a study optimizing the propagation of D. elastica for providing a large number of seedlings to support reintroduction and recovery of this critically endangered species is also incorporated into this project.

This study addresses the following three questions:

- What are the nutritional requirements of Drakaea elastica mycorrhizal fungus?

- What are factors limiting the growth and the survival of Drakaea elastica mycorrhizal fungus?

- How is the saprophytic competency of the slow growing mycorrhizal fungus relative to the sympatric faster growing fungi?

The structure of this thesis:

Chapter I general introduction of the biology and ecology of Drakaea elastica, factors related to the threatening process for this species and key aspects required in the conservation programs of Drakaea elastica in relation to the fitness and the survival of the mycorrhizal fungus.

Chapter II presents the capacity of the slow growing mycorrhizal fungus to access a variety of C sources, including monosaccharide, disaccharide, and polysaccahrides in axenic liquid media relative to that of faster growing fungi.

Chapter III provides the results of the comparison of the capability of the slow growing mycorrhizal fungus of D. elastica and faster growing orchid mycorrhizal fungi that were challenged with a diverse range of N sources.

The results of the ability of D. elastica mycorrhizal fungus to utilise various P sources (inorganic and organic P) in comparison to that of faster growing orchid mycorrhizal fungi is presented in Chapter IV.

Chapter V presents the optimization of propagation techniques for Drakaea elastica focusing on the generation of a large number of seedlings and the selection of the suitable substrates able to support the high rate of growth and the survival of D. elastica seedlings.

Chapter VI general discussion.

Figure 1.3. Drakaea elastica Lindl.

CHAPTER II

The capacity of Drakaea elastica mycorrhizal fungus to utilise carbon sources

2.1. Introduction

The habitat of Drakaea elastica is characterized by nutrient poor sandy soil and is often found in an association with Kunzea ericifolia in Banksia woodlands (Hoffman and Brown, 2007; Hopper and Brown, 2007). Much of the nutrients in this habitat are associated with organic matter from the litterfall of Kunzea ericifolia and Banksias as organic matter contains a variety of nutrient sources (carbon, nitrogen, phosphorus, and other nutrients) essential for growth (Tuomi et al, 2009; Kuperman, 1999; Berg, 1984; Lindahl et al, 2002; Sparks, 1999). Other orchids that have an association with faster growing fungi, such as Pterostylis recurva, Diuris corymbosa, and Caladenia flava also occur in Drakaea elastica habitat. In this habitat, these sympatric orchids grow on different types of substrates. Pterostylis recurva, Diuris corymbosa, and Caladenia flava grow with litter, while Drakaea elastica grows in bare patches sandy soil or in sandy soil with sparse organic matter ( Pers.comm Ryan Phillips).

Drakaea elastica forms a symbiotic association with a slow growing mycorrhizal fungus assignable to the teleomorphic genus Tulasnella, for which it relies on for its nutritional requirements (Ramsay et al; 1986). The growth of mycorrhizal fungi from a fungal genus Tulasnella is known to be limited by external specific organic compounds, thiamine and PABA (para amino benzoic acid). In their experiments, Hadley and Ong (1978) demonstrated that Tulasnella did not grow in the liquid media without thiamine and PABA, while other orchid mycorrhizal fungi from different fungal genus (Ceratobasidium) is reported to grow on the same media without these organic compounds (Hadley and Ong, 1978). Tulasnella only grew in the media supplemented with a combination of organic compounds, thiamine and PABA (Hadley and Ong, 1978). This suggests that Tulasnella highly depends on exogenous thiamine and PABA to survive. The organic compounds, thiamine and PABA in Drakaea elastica habitat is presumably associated with organic matter that contains a small proportion of organic compounds (Nardi et al, 2000) and root exudates of Kunzea ericifolia and Banksias as

19 plants are well known to release a small proportion of a variety of essential nutrients including sugar, amino acids, proteins, and organic compounds into the rhizosphere (Hinsinger et al, 2006; Gunawardena et al, 2006). This study investigated factors determining the growth and the survival of the slow growing mycorrhizal fungus of Drakaea elastica including what forms of nutrients required by Drakaea elastica mycorrhizal fungus for its growth, and compared its capacity to utilise nutrient sources with faster growing fungi. If Drakaea elastica mycorrhizal fungus utilises the same nutrient sources with faster growing orchid mycorrhizal fungi, the slow growing mycorrhizal fungus of Drakaea elastica will have to compete for the same nutrient sources. It would therefore be very likely that the slow growing mycorrhizal fungus of Drakaea elastica would be outcompeted by faster growing fungi. This may explain its microniche specialization in nutrient deficient sandy soil.

Carbon, nitrogen, and phosphorus are main nutrients required for growth (Griffin, 1994). This chapter focuses on the carbon requirements of Drakaea elastica mycorrhizal fungus and its capacity to utilise a variety of carbon sources relative to faster growing fungi. Carbon is the largest pool of nutrient in soil organic matter (Marschner et al, 2008; Rouifed et al, 2010). It is an essential element as the source of chemical energy for cellular metabolism and growth and a basic compound for the synthesis of essential biological materials, such as cell walls, food storage and nucleic acids (Cochrane, 1958). In soil, it is present in a variety forms including glucose, mannose, galactose, rhamnose, and arabinose (monosaccharide), cellobiose (disaccharide), cellulose, hemicellulose, pectin, starch, xylan (polysaccharides), and other carbon sources (Sparks, 1999). Simple C sources, such as monosaccharide and disaccharide can be absorbed directly, while polysaccharides need to be broken down first into simple sugar by specific extracellular enzymes including cellulase, pectinase, amylase, xylanase, and other extracellular enzymes (Cairney and Burke, 1998). Other orchid mycorrhizal fungi have previously been reported to utilise a variety of these carbon sources (Midgley et al, 2006; Hadley, 1969; 1984; Beyrle and Smith, 1993). Here, we investigated forms of carbon sources required by the slow growing mycorrhizal fungus of Drakaea elastica and its capacity to utilise a variety of carbon sources relative to faster growing fungi. In addition, we examined the role of exogenous organic compounds thiamine and PABA on the growth of Drakaea elastica mycorrhizal fungus on a variety of carbon sources.

It was hypothesized that:

- Drakaea elastica mycorrhizal fungus utilises the same carbon sources with faster growing sympatric orchid mycorrhizal fungi

- The capacity of D. elastica mycorrhizal fungus to utilise a wide range of carbon sources is limited by the availability of external organic compounds (thiamine and PABA).

2.2. Methods

Isolation of D. elastica mycorrhizal fungus

Mycorrhizal fungi were isolated from the collars of Drakaea elastica from D. elastica plants sourced from their natural habitat in Paganoni Reserve (32° 26’ 447” S ; 115° 48’ 5.7” E). The collars were surface sterilized with sterile water three times. Pelotons were teased out from the collars under a microscope using micropipettes and single pelotons were plated onto SSE media + streptomycin as a bactericide for inhibiting bacterial growth and incubated at 19° C. When the fungal hyphae grew out from the pelotons, the hyphal tips were cut and transferred onto PDA (Potato Dextrose Agar) (6.8 g/l). The other orchid mycorrhizal fungi used in this study were faster growing mycorrhizal fungi isolated from orchids co-exist with Drakaea elastica in the habitat of D. elastica including Pterostylis recurva, Caladenia flava, and Diuris corymbosa. Orchid mycorrhizal fungi associated with Pterostylis recurva, Caladenia flava, and Diuris corymbosa belong to different fungal genera, Ceratobasidium, Sebacina, and Tulasnella, respectively. These orchid mycorrhizal fungi were obtained from Kings Park’s fungal collection.

Preparation of inoculums

For the preparation of inoculums, the fungi grown on PDA were first subcultured onto low carbon and nitrogen of modified Melin and Norkrans media (CN MMN) agar. The -1 composition of CN MMN media (L ): glucose, 5.0 g; KH2PO4, 0.30 g; (NH4)2HPO4,

0.25 g; MgSO4.7H2O 0.14 g; CaCl2, 50 mg; NaCl, 25 mg; ZnSO4, 3 mg; ferric EDTA

(C10H12FeN2NaO8), 12.5 mg and thiamine, 0.13 mg (Marx and Bryan, 1975 cited in Midgley et al, 2006). The pH was adjusted to 5.0-5.5 before autoclaving. As the fungal

21 inoculums, two plugs of agar were excised from the leading edge of the actively growing colonies on CN MMN agar with a cork borer with a diameter of 5 mm.

Determination of exponential growth phase of the mycorrhizal fungi The capacity of D. elastica mycorrhizal fungus and other orchid mycorrhizal fungi to utilise carbon sources was assessed in the period of the exponential growth phase for each mycorrhizal fungus. To determine the exponential phase of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi, they were grown in nutrient liquid media containing glucose as a carbon source. Pterostylis recurva mycorrhizal fungus and C. flava mycorrhizal fungus grew in CN MMN liquid media, while Drakaea elastica and Diuris corymbosa mycorrhizal fungi that belong to the same fungal genus Tulasnella did not grow in these nutrient media that only contain one organic compound, thiamine (see formula of CN MMN media above). Drakaea elastica and Diuris corymbosa mycorrhizal fungi grew in liquid media containing a combination of organic compounds thiamine and PABA (para amino benzoic acid) (CN MMN + PABA liquid media), with the concentration of PABA (0.20 mg/l). Therefore, for the determination of exponential phase of Drakaea elastica and Diuris corymbosa mycorrhizal fungi, CN MMN+PABA liquid media was used, whereas exponential phase of Pterostylis recurva mycorrhizal fungus and C. flava mycorrhizal fungus was determined using CN MMN liquid media. Pterostylis recurva and Diuris corymbosa mycorrhizal fungi were harvested every two days as they are fast growing fungi, while slower growing fungi of C. flava and D. elastica were harvested every four days. To harvest the mycorrhizal fungi, the fungal mycelia grown in liquid media were filtered with nylon mesh and transferred onto alumunium foil, then dried for about one hour at 80ºC. The fungal growth curve was obtained by plotting biomass of the mycorrhizal fungi against the time and the exponential phase of the mycorrhizal fungi was determined based on the growth curve.

Assessment of the capacity of Drakaea elastica mycorrhizal fungus and other mycorrhizal fungi to utilise carbon sources

To investigate whether thiamine and PABA limit the growth and the capacity of Drakaea elastica mycorrhizal fungus to utilise a wide range of carbon sources, the assessment of the capacity of Drakaea elastica mycorrhizal fungus and other mycorrhizal fungi to utilise various carbon sources was performed using two types of 22 liquid media (CN MMN and CN MMN + PABA) containing the individual carbon sources tested. To assess the capacity of D. elastica mycorrhizal fungus and other mycorrhizal fungi to utilise carbon sources, these mycorrhizal fungi were grown in the liquid media containing a single type of carbon sources in 30-ml screw-capped containers. The assessment of the capacity of mycorrhizal fungi to utilise carbon sources was based on the biomass production of mycorrhizal fungi on a single carbon source in the liquid media. The carbon sources tested were mannose, arabinose, rhamnose, galactose, cellobiose, cellulose, carboxymethylcellulose (CMC), starch, pectin, xylan and aromatic carbon (tannic acids) (from Sigma Chemical Co.). Each type of carbon sources tested was added into the liquid media from which the carbon source (glucose) was omitted, so the liquid media only contain one type of carbon source tested. A treatment with glucose as a carbon source was also included. For all carbon treatments, the final concentration of single carbon sources added into the liquid media was 2 g C l-1. A control treatment free of carbon sources was included.

There were four replicates for each carbon treatment. Two plugs of agar were excised from the leading edge of actively growing colonies on CN MMN agar using a cork borer with a diameter of 5 mm and inoculated into the liquid media containing a single type of carbon source. As crystalline cellulose is undissolved in the liquid media, growth on this carbon substrate was determined by visual comparison to carbon-free controls. For the other carbon treatments, the growth on the other carbon sources was based on the biomass production on each carbon substrate. Mycelia of mycorrhizal fungi were harvested as described above during the exponential growth phase of the mycorrhizal fungi (see the result of the determination of exponential phase of each mycorrhizal fungus below). Raw data of biomass on each carbon treatment was substracted by mean data of carbon-free treatment to obtain data of net biomass and growth of mycorrhizal fungi only from carbon sources tested.

1. The net biomass of mycorrhizal fungi in on single carbon source in CN MMN liquid media A = B - C

A= net biomass of mycorrhizal fungi on single carbon source in CN MMN liquid media

B= biomass of mycorrhizal fungi in CN MMN liquid media containing single type of carbon source

C= biomass of mycorrhizal fungi in CN MMN liquid media without a carbon source

2. The net biomass of mycorrhizal fungi on single carbon source in CN MMN+ PABA liquid media

X = Y - Z

X= net biomass of mycorrhizal fungi on single carbon source in CN MMN+PABA liquid media

Y= biomass of mycorrhizal fungi in CN MMN+PABA liquid media containing single type of carbon source

Z= biomass of mycorrhizal fungi in CN MMN+PABA liquid media without a carbon source

Statistics Analysis

All data were analysed for normal distribution and where required, were normalized using logarithmic transformations before analysis using Analysis of Variance (ANOVA) with Genstat 12th Edition. Significant differences between treatments were determined by Fisher’s PLSD 5%. The influence of the types of media on the capacity of orchid mycorrhizal fungi to utilise carbon sources was analysed by a two way ANOVA.

2.3. Results

- Growth of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi

stationary phase

exponential phase ase

lag phase

Fig. 2.1. Growth curve of Drakaea elastica mycorrhizal fungus

stationary phase

exponential phase

lag phase

Fig. 2.2. Growth curve of Diuris corymbosa mycorrhizal fungus

stationary phase

exponential phase

lag phase

Fig. 2.3. Growth curve of Caladenia flava mycorrhizal fungus

stationary phase

exponential phase

lag phase

Fig. 2.4. Growth curve of Pterostylis recurva mycorrhizal fungus

Fungal growth curves are presented in (Fig.s 2.1-2.4). Lag phase, exponential phase, and stationary phase were observed from these growth curves. Periods of these phases varied between mycorrhizal fungi (Fig.s 2.1-2.4). The exponential phase of each mycorrhizal fungus which is revealed in the period of the highest rate of mycorrhizal fungal growth varied between each mycorrhizal fungus. The time to harvest each mycorrhizal fungus was standardized based on the time required by each mycorrhizal fungus to reach biomass of ca. 5 mg (during the exponential phase of all mycorrhizal fungi). Harvest times were 8 days for P. recurva mycorrhizal fungus, 16 days for Diuris corymbosa, 20 days for C. flava mycorrhizal fungus, and 28 days for Drakaea elastica mycorrhizal fungus (Fig. 2.1–2.4). These incubation times were used for the subsequent experiments to assess the capacity of orchid mycorrhizal fungi to utilise carbon, nitrogen and phosphorous sources (in this chapter and in the next chapters, Chapter III, and Chapter IV, respectively).

Utilisation of carbon sources

Biomass of Drakaea elastica mycorrhizal fungus on a range of C sources 7 g efg 6 g efg fg 5 fg efg def 4 mycorrhizal fungus de d

(mg) CN MMN 3 C CN MMN + PABA

D. elastica D. 2 c c b b ab ab ab 1 a ab

biomass of 0

xylan CMC pectin starch glucose mannose galactose arabinose rhamnose cellobiose tannic acid carbon sources

Fig. 2.5. Mean biomass production (± SE) of D. elastica mycorrhizal fungus in liquid media containing a wide range of carbon sources. Letters on columns indicate significant differences (P<0.05).

In CN MMN liquid media, Drakaea elastica mycorrhizal fungus and Diuris corymbosa mycorrhizal fungus produced very low biomass on most carbon sources, with the exception of xylan, pectin, and starch (Fig. 2.5 and Fig. 2.6). They produced significantly (P<0.001) higher biomass on a wide range of carbon sources, including glucose, mannose, xylan, cellobiose, CMC, pectin, and starch in CN MMN+PABA liquid media than in CN MMN liquid media (Fig. 2.5 and Fig.2.6). However, biomass production on arabinose, rhamnose, and galactose remained poor in CN MMN+PABA liquid media. There was no growth on tannic acid in both CN MMN and CN MMN+PABA liquid media (Fig. 2.5 and Fig. 2.6). Biomass produced by Drakaea elastica and Diuris corymbosa mycorrhizal fungi on cellulose in CN MMN+PABA liquid media was greater than in carbon-free controls. Poor growth was observed on cellulose in CN MMN liquid media.

Biomass of Diuris corymbosa mycorrhizal fungus on a range of C sources

12 j 10 i i

mycorrhizal 8 i hi h hi 6 g CN MMN g 4 CN MMN + PABA

fungus (mg) fungus de ef cde f D. corymbosa D. 2 bcd abcd abc a ab bcd c d 0

biomass of -2 xylan CMC pectin starch glucose mannose galactose arabinose rhamnose cellobiose tannic acid carbon sources

Fig. 2.6. Mean biomass production (± SE) of D. corymbosa mycorrhizal fungus in liquid media containing a wide range of carbon sources. Letters on columns indicate significant differences (P<0.05). Similar to Drakaea elastica and Diuris corymbosa mycorrhizal fungi, Caladenia flava and P. recurva mycorrhizal fungi also produced high biomass on glucose, mannose, xylan, cellobiose, pectin, and starch and produced low biomass on arabinose, rhamnose, and galactose, with the exception of P. recurva that produced high biomass on galactose (Fig. 2.7 and Fig. 2.8). Another similarity is that they did not grow on tannic acid (Fig. 2.7 and Fig. 2.8). However, there was a difference in the types of media that support for growth of mycorrhizal fungi. Drakaea elastica and Diuris corymbosa mycorrhizal fungi grew well in CN MMN+PABA liquid media and they grew poorly in CN MMN liquid media (Fig. 2.5 and Fig. 2.6). The types of media significantly (P<0.001) influenced biomass production of Drakaea elastica and Diuris corymbosa mycorrhizal fungi on a wide range of carbon sources for growth (Table 2.1 and Table 2.2). In contrast, the growth of Caladenia flava and P. recurva mycorrhizal fungi was not influenced by the types of media (Table 2.3 and Table 2.4). Their biomass production on both CN MMN and CN MMN+PABA liquid media did not differ significantly (Fig. 2.7 and Fig. 2.8). Biomass production of C. flava and P. recurva mycorrhizal fungi on cellulose in both CN MMN and CN MMN+PABA liquid media was higher than on carbon-free control.

Biomass of Caladenia flava mycorrhizal fungus on a range of C sources

9 h h 8 gh 7 fg

mycorrhizal fg 6 fg ef def 5 CN MMN cde bcd bc 4 cde bc b CN MNN + PABA

fungus (mg) fungus 3 Caladeniaflava 2 a a 1 a a a a

0 biomassof

xylan CMC pectin starch glucose mannose galactose arabinose rhamnose cellobiose tannic acid carbon sources

Fig. 2.7. Mean biomass production (± SE) of C. flava mycorrhizal fungus in liquid media containing a wide range of carbon sources, Letters on columns indicate significant differences (P<0.05).

Biomass of Pterostylis recurva mycorrhizal fungus on a range of C sources

12 j 10 ij j hij ghij hij hij 8 efgh fghi mycorrhizal efgh efg efg de ef 6

cd CN MMN

4 c fungus (mg) fungus P. recurva P. abc CN MMN + PABA bc ab 2 a

0 biomass of

xylan CMC pectin starch glucose mannose galactose arabinose rhamnose cellobiose tannic acid carbon sources

Fig. 2.8. Mean biomass production (± SE) of P. recurva mycorrhizal fungus in liquid media containing a wide range of carbon sources. Letters on columns indicate significant differences (P<0.05). Table 2.1. Two way ANOVA of the influence of PABA on the capability of Drakaea elastica mycorrhizal fungus to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Carbon sources 9 5.34740 0.59416 52.01 <.001 Types of media 1 1.70089 1.70089 148.88 <.001 Residual 60 0.68547 0.01142 Total 79 9.31323

Table 2.2. Two way ANOVA of the influence of PABA on the capability of Diuris corymbosa mycorrhizal fungus to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Carbon sources 9 29.13843 3.23760 141.03 <.001 Types of media 1 7.12564 7.12564 310.40 <.001 Residual 60 1.37738 0.02296 Total 79 48.97977

Table 2.3. Two way ANOVA of the influence of PABA on the capability of Caladenia flava mycorrhizal fungus to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. carbon_sources 9 386.5279 42.9475 46.83 <.001 Types of media 1 1.1281 1.1281 1.23 0.272 Residual 60 55.0242 0.9171 Total 79 467.4711

Table 2.4. Two way ANOVA of the influence of PABA on the capability of Pterostylis mycorrhizal recurva fungus to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Carbon sources 9 453.062 50.340 40.54 <.001 Types of media 1 0.221 0.221 0.18 0.675 Residual 60 74.503 1.242 Total 79 534.599

2.4. Discussion

Carbon sources for Drakaea elastica mycorrhizal fungus

Presented data showed forms of carbon sources required and utilised by Drakaea elastica mycorrhizal fungus to grow. It utilised glucose, mannose, cellobiose, xylan, pectin, CMC, starch, and cellulose as carbon sources for growth, however, it had limited access to arabinose, rhamnose and galactose. Drakaea elastica mycorrhizal fungus could not utilise tannic acid. Its capacity to access a variety of carbon sources in liquid media may reflect its ability to access available various carbon sources in its natural habitat. Carbon sources in nutrient deficient sandy soil habitat of Drakaea elastica are associated with organic matter from litterfall of Kunzea ericifolia and Banksias, or from root exudates of these trees as plants are well known to release sugars

30 and polysaccharides, and other essential compounds into the rhizophere (Hinsinger et al, 2006).

It has been demonstrated in this study that the sympatric faster growing orchid mycorrhizal fungi utilised the same carbon sources with Drakaea elastica mycorrhizal fungus. The pattern of carbon utilisation and carbon sources required by other orchid mycorrhizal fungi for growth were similar to those of Drakaea elastica mycorrhizal fungus. Utilisation of glucose and mannose by Drakaea elastica mycorrhizal fungus and other orchid mycorhizal fungi may be related to the low molecular weight and simple structure of glucose and mannose that can be directly absorbed and metabolized for growth (Nehls and Hampp, 2000; Guzman et al, 2005; Arnosti, 2003, Griffin, 1994). Utilisation of cellobiose as a carbon source by D. elastica fungus and other mycorhizal fungi for growth suggests that they produced extracellular enzymes cellobiase to hydrolise cellobiose into glucose before absorption (Griffin, 1994; Burke and Cairney, 1998). Alternatively, they absorbed directly cellobiose as it can also be utilised directly.

An array of extracellular enzymes (cellulase, carbaxymethylcellulase, amylase, xylanase and pectinase) is required to depolymerize complex polymer of polysaccharides (cellulose, CMC, starch, xylan and pectin, respectively) into simple sugar before absorption (Valaskova and Baldrian, 2006, 2007; Cairney and Burke, 1998; Warren, 1996; Steffen et al, 2007; Perotto et al , 1997, Jafra et al, 1999; Hadley and Perombelon,1963). Good growth of D. elastica mycorrhizal fungus and other orchid mycorrhizal fungi on cellulose, CMC, starch, xylan and pectin implies their capacity to release a range of extracellular enzymes to facilitate the utilisation of these polysaccharides as carbon sources for growth. The other orchid mycorrhizal fungi have previously been demonstrated to utilise a variety of these polysaccharides (Smith, 1966; Hadley, 1969; 1984; Beyrle and Smith, 1993; Midgley et al, 2006).

In the establishment process of symbiotic association, Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi have to be able to degrade cell walls of orchid seeds which consist of complex polysaccharides (cellulose, hemicelluloses, pectin, starch and other polysaccharides) (Norkrans, 1963). The ability of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi to utilise a range of polysaccharides demonstrated in this study confirms that they were able to hydrolyze polysaccharides, which is important for the degradation of cell wall of the orchid associated during the formation of symbiotic association with the orchid

31 associate. Furthermore, for the carbon transfer to the seedlings in the development of the seedlings, Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi must be able to take up a range of carbon sources from soil and deliver them to the orchids associate. Their capacity to utilise a variety of carbon sources demonstrated in this study also reflects their fundamental role to provide carbon for the orchids for the function of the symbiotic association.

Arabinose, rhamnose, and galactose were less efficiently utilised by Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi as carbon sources, although arabinose, rhamnose, and galactose are also simple sugar (monosaccharide), like glucose and mannose. Metabolism pathways of rhamnose, arabinose and galactose may be different from glucose and mannose metabolic pathways (Jennings, 1995). Interestingly, P. recurva mycorrhizal fungus utilised galactose efficiently suggesting that it has an effective system to take up and metabolize galactose.

Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi could not utilise tannic acid as a carbon source. The inability of orchid mycorrhizal fungi to utilise tannic acid has previously been shown in the other studies (Midgley et al, 2006; Hollander, 1932 and Burgeff, 1936 cited in Midgley et al, 2006).

Saprophytic competency of Drakaea elastica mycorrhizal fungus to utilise carbon sources

The pattern of carbon utilisation of Drakaea elastica mycorrhizal fungus was similar to that of sympatric faster growing orchid mycorrhizal fungi. This implies that Drakaea elastica mycorrhizal fungus will have to compete for the same carbon sources with faster growing fungi in soil. Non-orchid mycorrhizal fungi and other soil fungi also have been reported to utilise a diverse array of carbon sources (Midgley et al, 2004; 2006; Daynes et al, 2008). Specialization of Drakaea elastica mycorrhizal fungus in nutrient deficient sandy soil with sparse organic matter may be a strategy to avoid competition with many other fungi (Hollick, 2004). Drakaea elastica is often found in bare sandy soil or with sparse organic which appears to be preferable habitat of the mycorrhizal fungus (Department of Environment and Conservation, 2009). A rich nutrient environment with much organic matter may not support the growth and the survival of Drakaea elastica mycorrhizal fungus as this environment is colonized by

32 many fungi and soil microorganisms that will overgrow Drakaea elastica mycorrhizal fungus (Dickie et al, 2002; Rossling et al, 2003).

Although Drakaea elastica mycorrhizal fungus has a capacity to utilise a wide range of carbon sources, like other orchid mycorrhizal fungi, its slow growing characteristic will affect its saprophytic competency to access carbon sources in the competition for the same carbon sources with faster growing fungi. The slow growing characteristic of Drakaea elastica mycorrhizal fungus may be an intrinsic factor limiting its growth and its distribution in soil. With its slow growing characteristic, the growth and the distribution of Drakaea elastica mycorrhizal fungus may be restricted to certain patches or areas which are not occupied by faster growing fungi to avoid competition with many faster growing fungi. There is a greater likelihood for Drakaea elastica mycorrhizal fungus to be outcompeted by faster growing fungi in the competition for carbon sources. Its patchy distribution in soil may reflect its search for a space to grow without interference from faster growing mycorrhizal fungi. This study provides data that in part explain the reason of the microniche specialization of Drakaea elastica in bare sandy soil with limited nutrient sources in relation to the slow growing characteristic of the mycorrhizal fungi. Unlike Drakaea elastica that is often found in bare sandy soil or with sparse organic matter, the sympatric orchids grow with litter as the mycorrhizal fungi associate are faster growing fungi that can compete with other fungi to access a wide range of nutrients in litter (organic matter).

Organic compounds (thiamine and PABA) limit the growth and the survival of Drakaea elastica mycorrhizal fungus

Data in the present study also showed that the growth and the survival of D. elastica mycorrhizal fungus were limited by the availability of organic compounds (vitamins) thiamine and PABA. Drakaea elastica mycorrhizal fungus grew poorly on most carbon substrates in CN MMN liquid media that only contain one organic compound (thiamine), while it grew well on most carbon substrates in CN MMN + PABA liquid media. This suggests that Drakaea elastica mycorrhizal fungus required external sources of thiamine and PABA to grow. Thiamine, PABA, and other organic compounds in soil are associated with detrital plant materials in organic matter as plants contain thiamine, folate, PABA, and other organic compounds (Gambonnet et al, 2001; Rapala-Kozik et al, 2009; Goyer, 2010; Chaikelis, 1946; Hanson and Gregory III, 33

2000). The source of organic compounds in Drakaea elastica habitat can be from detrital plant materials in sparse organic matter from litterfall of Kunzea ericifolia and Banksias. Another source of organic compounds can be from root exudates of Kunzea ericifolia and Banksias to the rhizosphere as plants are well known to release organic compounds, amino acids and vitamins through the roots into the rhizosphere (Griffin, 1994; Hinsinger et al, 2006; Gunawardena et al, 2006).

Thiamine and PABA are only required in a small amount, however they have a massive impact in the regulation of vital metabolism (Hanson and Gregory, 2002). Thiamine and PABA are known as essential compounds and their function are indispensable by other nutrients. They are required as enzymatic cofactors in the major metabolism for growth (Goyer, 2010; Rapala-Kozik et al, 2009; Cossins, 2000; Stephen and Fung, 1971; Bucking et al, 2008; Jabrin et al, 2003; Basset et al, 2005; Scott et al; 2000; Cossins and Chen, 1997). It was also demonstrated that Diuris corymbosa mycorrhizal fungus had the same trend as Drakaea elastica mycorrhizal fungus in the dependency on thiamine and PABA to be able to utilise a wide range of C sources for growth, although they have different growth rate and characteristics. Interestingly, both Drakaea elastica and Diuris corymbosa mycorrhizal fungi belong to the same fungal genus, Tulasnella. This may be a characteristic of mycorrhizal fungi belong to Tulasnella that require external organic compounds, thiamine and PABA to grow.

The results demonstrated here, are in agreement with other studies using Tulasnella mycorrhizal fungi isolated from other orchids that are dependent on exogenous organic compounds thiamine and PABA. Hadley and Ong (1978) reported that Tulasnella isolated from other orchid species (Platanthera bifolia, Diuris longifolia Arachnis cv Maggie Oei and Dactylorhiza purpurella) and from different regions and continents (Asia, Australia and Europe) were not able to grow in the absence of thiamine and PABA. Hijner and Arditti (1973) demonstrated a similar result that orchid mycorrhizal fungi they used in their study required exogenous thiamine and PABA for growth. Stephen and Fung (1971) also reported that orchid mycorrhizal fungi associated with Arundina that presumably associated with Tulasnella required external organic compounds (thiamine and PABA) to grow. Each of these studies had similar experimental designs investigating the importance of organic compounds (biotin, nicotinic acid, thiamine, folic acid, PABA) on mycorrhizal fungi nutrition. They found that single organic compound did not affect the growth of Tulasnella, however, in a 34 combination of external thiamine and PABA a significantly increased in fungal biomass production was observed. In contrast, the fungal genus (Ceratobasidium) which is also a common orchid mycorrhizal fungus was found to grow without these exogenous organic compounds suggesting that they have the ability to biosynthesise these organic compounds. Tulasnella may be incapable of biosynthesis of these organic compounds and requires an exogenous source thiamine and PABA (Hadley and Ong, 1978; IGEM, 2008). Unlike mycorrhizal fungi that belong to Tulasnella, P. recurva and C. flava mycorrhizal fungi which have associations with the fungal genera, Ceratobasidium and Sebacina respectively were able to grow in both CN MMN and CN MMN+PABA liquid media. Hadley and Ong, 1978 reported that Ceratobasidium isolated from the orchid (Goodyera repens (L.) R. Br.) is self sufficient for organic compounds and did not require external organic compounds, such as thiamine, PABA, and other organic compounds (Hadley and Ong, 1978). Orchid mycorrhizal fungi associated with Ceratobasidium may be able to biosynthesise organic compounds (Hadley and Ong, 1978; IGEM; 2008). Further study is warranted to investigate the organic compounds requirements of diverse orchid mycorrhizal fungi from various orchid fungal genera as these external organic compounds are a key determinant of the growth and the survival of some of the mycorrhizal fungi.

Pictures of growth or no growth orchid mycorrhizal fungus in liquid media containing a particular nutrient tested

a b

Fig.2.9. No growth or poor growth of D. elastica mycorhizal fungus in CN MMN liquid media containing alanine after 28 days incubation (a) growth of orchid mycorrhizal fungus in CN MMN + PABA liquid media containing alanine after 28 days incubation (b)

Fig.2.10. Growth of P. recurva mycorhizal fungus in liquid media containing xylan as a C source after 8 days incubation

Fig.2.11. Growth of P. recurva mycorhizal fungus in liquid media containing pectin as a C source after 8 days incubation

Fig.2.12. Growth of P. recurva mycorhizal fungus in liquid media containing cellobiose as a C source after 8 days incubation

Fig.2.13. Growth of P. recurva mycorhizal fungus in liquid media containing glucose as a C source after 8 days incubation

Fig.2.14. Growth of P. recurva mycorhizal fungus in liquid media containing CMC (carbaxymethylcellulose) as a C source after 8 days incubation

Fig.2. 14. Poor growth of D. elastica mycorhizal fungus in liquid media after 28 days incubation

Fig.2.14. No growth of P. recurva mycorhizal fungus in liquid media containing tannic acid after 8 days incubation

CHAPTER III The capacity of Drakaea elastica mycorrhizal fungus to utilise nitrogen sources

3.1. Introduction

Like other orchids, the survival of Drakaea elastica is determined by the survival of the mycorrhizal fungus as a critical component to complete Drakaea elastica’s life cycle (Rasmussen, 2002). The role of the mycorrhizal fungus is indispensable as the nutrient supplier for Drakaea elastica in almost entire of its lifetime. Thus, in the conservation of Drakaea elastica understanding factors influencing the growth and the survival of the mycorrhizal fungus is substantial. The mycorrhizal fungus associated with Drakaea elastica is a slow growing member of the teleomorphic genus Tulasnella. Some key aspects influencing the growth and the survival of the slow growing mycorrhizal fungus of Drakaea elastica, such as its nutritional requirements, factors limiting its growth and survival, and its saprophytic competency to access nutrients in the competition with faster growing fungi, needs to be understood.

The previous chapter has elucidated nutritional requirements of Drakaea elastica mycorrhizal fungus with the focus on a variety of carbon sources required by Drakaea elastica mycorrhizal fungus for its growth and its capacity to utilise carbon sources relative to the sympatric faster growing mycorrhizal fungi. This chapter will continue investigation of nutritional requirements of Drakaea elastica mycorrhizal fungus with the focus on nitrogen. Nitrogen is one of the essential nutrients required by orchid mycorrhizal fungi and other living organisms for the synthesis of amino acids, proteins, enzymes, nucleic acids and other biological compounds critically needed for growth (Hodge, 2005).

Detailed information of nitrogen in nutrient deficient sandy soil habitat of Drakaea elastica is largely unknown. There is a report that sand plain habitat of Ericaceae which is similar to D. elastica habitat is limited by nitrogen (Bell et al, 1994) and much of the nitrogen in these typical sandy soils is associated with organic matter (Blair and Crossley, 1988; Kuperman, 1999). Organic matter typically contains a low proportion of nitrogen relative to carbon (only 0.67% to 2.35% of the total nutrient

40 content in soil organic matter relative to carbon) (Knox et al, 1995; Hodge, 2005; Rouifed et al, 2010). The form of nitrogen in organic matter is organic nitrogen including proteins and a variety of amino acids (basic amino acids, acidic amino acids, and neutral amino acids). Mineralization of organic nitrogen will release ammonium that will subsequently be oxidized into nitrate. This process contributes to the availability of inorganic nitrogen in soil in Drakaea elastica habitat (Connell et al, 1995; Hodge, 2005). The association of Drakaea elastica with Kunzea ericifolia may also provide some benefit in terms of the availability of amino acids and proteins as root plants release a low proportion of exudates containing amino acids, proteins, and organic compounds to the rhizosphere (Hopper and Brown, 2007; Jones et al, 2009; Hinsinger et al, 2006; Jackson et al, 2008).

Forms of nitrogen required and utilised by Drakaea elastica mycorrhizal fungus that are essential for its growth is largely unknown. Other orchid mycorrhizal fungi belong to a fungal genus Tulasnella isolated from other orchid species (Platanthera bifolia; Diuris longifolia; Arachnis cv Maggie Oei and Dactylorhiza purpurella) have been reported to require and utilise a variety of nitrogen sources including inorganic nitrogen (ammonium) and organic nitrogen (a variety amino acids) in axenic culture (Hadley and Ong, 1978). The ability of these orchid mycorrhizal fungi to utilise a variety of these nitrogen sources is determined by the availability of external specific organic compounds, thiamine and PABA, as Tulasnella may not be able to biosynthesise these organic compounds (Hadley and Ong, 1978; IGEM, 2008). In the previous chapter, it was demonstrated that Drakaea elastica mycorrhizal fungus required external source of thiamine and PABA to be able to utilise a wide range of carbon sources. In this chapter, we investigated nitrogen sources required and utilised by Drakaea elastica mycorrhizal fungus for its growth and examined the influence of thiamine and PABA on the ability of Drakaea elastica mycorrhizal fungus to utilise a wide range of nitrogen sources. Furthermore, we compared its capacity to utilise nitrogen sources with the sympatric faster growing orchid mycorrhizal fungi (mycorrhizal fungi associated with Pterostylis recurva, Caladenia flava, and Diuris corymbosa) to investigate saprophytic competency of the slow growing mycorrhizal fungus of Drakaea elastica to access nitrogen sources relative to the sympatric faster growing orchid mycorrhizal fungi.

3.2. Methods

Preparation of fungal inoculums

The protocol for the preparation of fungal inoculums of Drakaea elastica, Pterostylis recurva, Caladenia flava, and Diuris corymbosa was provided in Chapter II.

The capacity of Drakaea elastica mycorrhizal fungus and other mycorrhizal fungi to utilise nitrogen sources

The capacity of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi to utilise nitrogen sources naturally occur in soil was assessed in two types of media CN MMN and CN MMN+PABA liquid media containing single type of nitrogen sources. The composition of CN MMN liquid media (L-1) was glucose, 5.0 g;

KH2PO4, 0.30 g; (NH4)2HPO4, 0.25 g; MgSO4.7H2O 0.14 g; CaCl2, 50 mg; NaCl, 25 mg; ZnSO4, 3 mg; ferric EDTA (C10H12FeN2NaO8), 12.5 mg and thiamine 0.13 mg. PABA 0.20 mg was added into CN MMN + PABA liquid media. For nitrogen treatments containing single type of nitrogen source, individual nitrogen sources were supplemented into basal media CN MMN and CN MMN + PABA liquid media from which N source, ammonium (NH4)2HPO4 was omitted. For each nitrogen treatment, final nitrogen concentration was added at the same level based on the concentration of total N in the formula of CN MMN liquid media (53 mg/l). A range of nitrogen sources tested were in the form of inorganic nitrogen (ammonium and nitrate) and organic nitrogen including simple organic nitrogen (amino acids) and complex organic nitrogen (protein) in the form of BSA (Bovine Serum Albumin, from Sigma). Amino acids used in this experiment included acidic amino acids (l-glutamic acid and l-aspartic acid), basic amino acids (l-arginine and l-histidine) and neutral amino acids (l-alanine, l- asparagine, l-glutamine, glycine and l-proline) (all are from Sigma). Organic nitrogen sources were sterilized through filter Millipore with a diameter of 0.22 µm into autoclaved basal media as organic nitrogen is unstable to heat sterilization (autoclaving), and the pH was adjusted to 5.0. Inorganic nitrogen sources were sterilized by heat sterilization (autoclaving). Two plugs of active fungal inoculums were excised from the leading edge of the colony using a cork borer with a diameter of 5 mm. The inoculums were inoculated into each nitrogen treatment (liquid media containing single type of nitrogen sources in 30 ml tubes). There were four replicates for each treatment.

The time to harvest each mycorrhizal fungus was in time of the exponential phase of each fungi (see chapter II), P. recurva mycorrhizal fungus (8 days), Diuris corymbosa mycorrhizal fungus (18 days), C. flava mycorrhizal fungus (20 days) and Drakaea elastica mycorrhizal fungus (28 days). To harvest the mycorrhizal fungi, the fungal mycelia grown in liquid media were filtered with nylon mesh and transferred onto tarred alumunium foil, then dried for about one to two hours at 80ºC. A free- nitrogen treatment was included. Raw data for each nitrogen treatment were substracted by mean data of nitrogen-free treatment to obtain net biomass of mycorrhizal fungi only from nitrogen sources tested.

1. The net biomass of mycorrhizal fungi on single type of nitrogen source in CN MMN liquid media A = B - C

A= net biomass of mycorrhizal fungi on single type of nitrogen source in CN MMN liquid media

B= biomass of mycorrhizal fungi in CN MMN liquid media containing single type of nitrogen source

C= biomass of mycorrhizal fungi in CN MMN liquid media without nitrogen sources

2. The net biomass of mycorrhizal fungi on single type of nitrogen source in CN MMN+ PABA liquid media X = Y - Z

X= net biomass of mycorrhizal fungi on single type of nitrogen source in CN MMN+PABA liquid media

Y= biomass of mycorrhizal fungi in CN MMN+PABA liquid media containing single type of nitrogen source

Z= biomass of mycorrhizal fungi in CN MMN+PABA liquid media without nitrogen sources

Statistics Analysis

All data were analysed for normal distribution and where required, were normalized using logarithmic transformations before analysis using Analysis of Variance (ANOVA) Genstat 12th. Significant differences between treatments were determined by

Fisher’s PLSD 5%. The influence of the type of media on the capacity of orchid mycorrhizal fungi to utilise nitrogen sources was analysed by a two way ANOVA.

3.3. Results

Utilisation of nitrogen sources

Results showed that Drakaea elastica mycorrhizal fungus produced relatively high biomass on a variety of nitrogen sources including ammonium, aspartic acid, glutamic acid, arginine, alanine, asparagine, glutamine, and glycine in CN MMN + PABA liquid media (Figure 3.1). Drakaea elastica also produced biomass on BSA in some extent. Poor biomass production was observed on nitrate, histidine, and proline.

In CN MMN liquid media, Drakaea elastica mycorrhizal fungus produced low biomass on most nitrogen sources with an exception with aspartic acid, glutamic acid, arginine on which significant biomass production was produced in both CN MMN and CN MMN + PABA (Figure 3.1). Overall, the types of media significantly influenced (P < 0.001) biomass production of D. elastica mycorrhizal fungus on most nitrogen sources (table 3.1), with CN MMN + PABA liquid media induced relatively high biomass production on most nitrogen sources, while poor biomass production was observed in CN MMN liquid media.

Biomass of Drakaea elastica mycorrhizal fungus on a range of N sources

5 g g 4.5 fg 4

3.5 ef ef ef ef 3 de 2.5 mycorrhizal (mg) fungus CN MMN cd bc cd 2 bc bc CN MMN + PABA 1.5

D elastica D 1 0.5 a ab a a a a a a

0 biomass of

BSA nitrate alanine glycine proline argininehistidine ammonium asparagine glutamine aspartic glutamicacid acid nitrogen sources

Figure 3.1. Mean biomass production (± SE) of D. elastica mycorrhizal fungus in liquid media containing a wide range of nitrogen sources. Letters on columns indicate significant differences (P<0.05). 44

Diuris corymbosa mycorrhizal fungus had a similar trend to D. elastica mycorrhizal fungus in that it produced relatively high biomass production on the same nitrogen sources as Drakaea elastica mycorrhizal fungus in CN MMN+PABA liquid media. Poor growth was observed on most nitrogen sources in CN MMN liquid media with the exception on aspartic acid, glutamic acid, and arginine (Figure 3.2). Like Drakaea elastica mycorrhizal fungus, the types of media had a significant (P < 0.001) effect on the biomass production of Diuris corymbosa mycorrhizal fungus on nitrogen sources (table 3.2).

Biomass of Diuris corymbosa mycorrhizal fungus on a range of N 16 sources j 14

10 mycorrhizal mycorrhizal i 8 hi hi CN MMN

6 fgh gh fgh CN MMN + PABA fungus(mg)

D.corymbosa fgh 4 efg ef de a 2 bc

a ab a a abc c biomass of 0

-2 BSA nitrate alanine glycine proline arginine histidine ammonium asparagine glutamine aspartic acid glutamic acid nitrogen sources Figure 3.2. Mean biomass production (± SE) of D. corymbosa mycorrhizal fungus in liquid media containing a wide range of nitrogen sources. Letters on columns indicate significant differences (P<0.05).

Biomass of Caladenia flava mycorrhizal fungus on a range of N sources

5 f 4.5 4 3.5 ef ef ef ef ef ef fg 3 ef

mycorrhizal fungus ef e e 2.5 CN MMN

(mg) 2 cd de CN MMN+PABA

1.5 de 1 abc bc

Caladeniaflava abc abc abc abc 0.5 a ab 0

biomassof -0.5

nitrate BSA arginine histidine alanine glycine proline ammonium asparagine glutamine aspartic acidglutamic acid nitrogen sources

Figure 3.3. Mean biomass production (± SE) of C. flava mycorrhizal fungus in liquid media containing a wide range of nitrogen sources. Letters on columns indicate significant differences (P<0.05).

Caladenia flava mycorrhizal fungus and Pterostylis recurva mycorrhizal fungus produced relatively high biomass on the same nitrogen sources as Drakaea elastica and Diuris corymbosa mycorrhizal fungi, including ammonium, aspartic acid, glutamic acid, arginine, alanine, and asparagine. All orchid mycorrhizal fungi also grew on BSA in some extent and produced relatively low biomass on proline. However, there was a large difference in the biomass production on few certain nitrogen sources including nitrate, glycine, histidine, and glutamine between these orchid mycorrhizal fungi (Figures 3.1-3.4). Pterostylis recurva mycorrhizal fungus produced high biomass on nitrate, while low biomass production on nitrate was observed in other orchid mycorrhizal fungi. Caladenia flava mycorrhizal fungus grew well on histidine, while other orchid mycorrhizal fungi grew poorly on it. However, poor growth of C. flava mycorrhizal fungus was observed on glutamine, while other orchid mycorrhizal fungi grew well on this amino acid. Another stark difference was that Drakaea elastica and Diuris corymbosa mycorrhizal fungi utilised glycine, while C. flava and P. recurva mycorrhizal fungi inefficiently utilised this nitrogen source resulting in their poor growth on this nitrogen source.

Biomass of Pterostylis recurva mycorrhizal fungus on a range of N sources 35

30 j ij j hij

25 ghij ghij

20 fghi fghi CN MMN efgh defgh

mycorrhizal(mg) fungus 15 CN MMN + PABA cdefg bcdef 10 abcde abcde abcd

P. recurva P. abcd abcd abc abc 5 abc abc ab a

biomassof 0

BSA -5 nitrate alanine glycine proline arginine histidine ammonium asparagine glutamine aspartic acidglutamic acid nitrogen sources Figure 3.4. Mean biomass production (± SE) of P. recurva mycorrhizal fungus in liquid media containing a wide range of nitrogen sources. Letters on columns indicate significant differences (P˂0.05).

Noticeable difference was also observed in the types of media on which these orchid mycorrhizal fungi grew. Caladenia flava and Pterostylis recurva mycorrhizal fungi grew and produced relatively high biomass on a wide range of nitrogen sources in both CN MMN and CN MMN+PABA liquid media (Figure 3.3 and Figure 3.4), while Drakaea elastica and Diuris corymbosa mycorrhizal fungi produced relatively high biomass on most nitrogen sources only in CN MMN+PABA liquid media. Drakaea elastica and Diuris corymbosa mycorrhizal fungi grew poorly on most nitrogen sources in CN MMN liquid media. Biomass production of C. flava and P. recurva mycorrhizal fungi in both types of media (CN MMN and CN MMN+PABA liquid media) did not differ significantly (table 3.3 and table 3.4).

Table 3.1. Two way ANOVA of the influence of PABA on the capability of D. elastica fungi to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Nitrogen sources 11 74.9886 6.8171 22.69 <.001 Types of media 1 28.1667 28.1667 93.76 <.001 Residual 72 21.6308 0.3004 Total 95 183.4247

Table 3.2. Two way ANOVA of the influence of PABA on the capability of D. corymbosa fungi to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Nitrogen sources 11 339.245 30.840 17.84 <.001 Types of media 1 259.767 259.767 150.25 <.001 Residual 72 124.481 1.729 Total 95 926.059

Table 3.3. Two way ANOVA of the influence of PABA on the capability of C. flava fungi to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Nitrogen sources 11 118.8278 10.8025 <.001 19.03 Types of media 1 1.6977 1.6977 2.99 0.088 Residual 72 40.8625 0.5675 Total 95 175.0312

Table 3.4. Two way ANOVA of the influence of PABA on the capability of P. recurva fungi to utilise a wide range of carbon sources

Source of variation d.f. s.s. m.s. v.r. F pr. Nitrogen sources 11 4985.32 453.21 <.001 14.65 Types of media 1 75.91 75.91 2.45 0.122 Residual 72 2227.29 30.93 Total 95 7790.74

3.4. Discussion

Nitrogen sources for Drakaea elastica mycorrhizal fungus

It was demonstrated that Drakaea elastica mycorrhizal fungus and other orchid mycorhizal fungi utilised a wide range of nitrogen sources including inorganic nitrogen and organic nitrogen (a variety of amino acids and protein BSA) they required for their growth. Growth of orchid mycorrhizal fungi was measured on a single nitrogen source at one concentration, therefore, applying these data to what takes place in situ requires a degree of consideration as in the soil nitrogen sources can be mixed and present at different concentration (Whittaker and Cairney, 2001; Abuarghub and Read, 1988). However, data in the present study can be a basis of understanding the ways in which Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi utilise various

48 nitrogen sources for their growth. These data also identified which nitrogen sources that are essential for the best growth of orchid mycorrhizal fungi and which nitrogen sources that are less useful for their growth.

Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi effectively utilised inorganic nitrogen in the form of ammonium and they had a low ability to utilise nitrate with the exception of Pterostylis recurva mycorrhizal fungus that had a high capability to utilise ammonium and nitrate. Other orchid mycorrhizal fungi have previously been reported to utilise ammonium and did not utilise nitrate as a nitrogen source (Hadley and Ong, 1978). Inorganic nitrogen in the macrohabitat of Drakaea elastica can be from the mineralization of sparse organic matter that release ammonium and subsequently can be oxidized into nitrate (Hodge, 2005; Kemmitt et al, 2008; Connell et al, 1995). Fire incidence also contributes to the availability of nitrate (Stewart et al, 1993) which is important for mycorrhizal fungi that had a capacity to utilise nitrate, such as P. recurva mycorrhizal fungus. The ability of Pterostylis recurva mycorrhizal fungus to utilise nitrate benefits this mycorrhizal fungus to utilise available nitrate as a nitrogen source.

A variety of amino acids including aspartic acid, glutamic acid, arginine, alanine, asparagine, and glutamine are essential amino acids for the growth of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi. Other orchid mycorrhizal fungi are previously reported to utilise these amino acids for their growth (Hadley and Ong, 1978). Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi also utilised protein BSA to some extent, suggesting that they produced protease to hydrolise protein BSA into amino acids before absorption (Leake and Read, 1990).

Like other nutrients, organic nitrogen (amino acids and proteins) in soil is also associated with organic matter as the source of a variety of nutrients (Blair and Crossley, 1988; Kuperman, 1999). Organic matter from litterfall of Kunzea ericifolia and Banksias can be the important source of amino acids and proteins in Drakaea elastica habitat. Decomposition of organic matter is an important process in soil that can release amino acids for the availability of amino acids in soil (Rothstein, 2009). The availability of amino acids and proteins in Drakaea elastica habitat can also be from root exudates of Kunzea ericifolia that release amino acids, proteins, and other essential

49 nutrients (Hopper and Brown, 2007; Jones et al, 2009; Hinsinger et al, 2006; Jackson et al, 2008).

It was previously shown that Drakaea elastica mycorrhizal fungus and the sympatric orchid mycorrhizal fungi (faster growing fungi) required and utilised the same nitrogen sources including ammonium, aspartic acid, glutamic acid, arginine, alanine, asparagine, and glutamine. Consequently, Drakaea elastica mycorrhizal fungus will have to compete with faster growing orchid mycorrhizal fungi for nitrogen sources. Faster growing mycorrhizal fungi may overgrow Drakaea elastica mycorhizal fungus. It is the same condition, where Drakaea elastica mycorrhizal fungus utilised the same carbon sources with faster growing fungi and consequently, it will have to compete for the same carbon sources (Chapter II). This is an increased evidence for the specialization of Drakaea elastica mycorrhizal fungus to avoid competition with many other fungi especially faster growing fungi. Many other fungi were also reported to utilise a wide range of nitrogen sources (Finlay et al, 1992; Abuzinadah and Read, 1988; Bajwa et al, 1985; Littke et al, 1984; Tibbett et al, 1988). Drakaea elastica is often found in bare patches sandy soil or in sandy soil with sparse organic matter which may be related to the favor spaces of the slow growing mycorrhizal fungus as these areas may not be occupied by many other fungi. In comparison to the sympatric orchids that have an association with faster growing mycorrhizal fungi, they grow on litter which is rich of nutrients as they may be able to compete with other fungi for nutrients. Fungi typically prefer areas with rich organic matter. Bare patches sandy soil as the preferable patches of Drakaea elastica mycorrhizal fungus presumably also contains nutrients including amino acids and other essential nutrients from the leaching of organic matter (Rothstein, 2009) during rainy growing season in winter that will provide Drakaea elastica mycorrhizal fungus nutrition to grow.

Apart from the utilisation of the same nitrogen sources, it was noticed in the presented data that there was remarkable difference between Drakaea elastica mycorrhizal fungus and the sympatric orchid mycorrhizal fungi in the utilisation of few other nitrogen sources including nitrate, histidine, glycine, and glutamine. Pterostylis recurva mycorrhizal fungus efficiently utilised nitrate, while the other orchid mycorrhizal fungi had little ability to utilise nitrate. The other remarkable difference was noticed that C. flava mycorrhizal fungus utilised histidine as a nitrogen source, while other orchid mycorrhizal fungi did not utilise it. Caladenia flava mycorrhizal fungus was observed had low capacity to utilise glutamine, while this amino acid is an 50 important nitrogen source for the growth of other orchid mycorrhizal fungi. Another stark difference was that D. elastica and Diuris corymbosa mycorrhizal fungi were able to utilise glycine, while C. flava and P. recurva mycorrhizal fungi inefficiently utilised this amino acid resulting in their poor growth on this nitrogen source. The other studies also showed that mycorrhizal fungi co-exist at the same habitat utilised the same nitrogen sources and had different niche in the utilisation of few certain other nitrogen sources. Ectomycorrhizal fungi Pisolithus spp. and ericoid mycorrhizal fungi from Woollsia pungens (Ericaceae) from dry schlerophyll forest habitats utilised the same nitrogen sources and also utilised different few certain other nitrogen sources (histidine and arginine). Ericoid mycorrhizal fungi from Woollsia pungens (Ericaceae) had an ability to utilise basic amino acids (histidine and arginine) as sole nitrogen sources effectively, however, ectomycorrhizal Pisolithus spp. from a similar dry schlerophyll forest habitat has low capacity to utilise these amino acids (Anderson et al, 1999; Whittaker and Cairney, 2001). Apart from the competition for the same nitrogen sources, there were also differences in the utilisation of few other nitrogen sources. Differences in the utilisation of few nitrogen sources between all these orchid mycorrhizal fungi are a form of niche partitioning between mycorrhizal fungi which is of ecological significance for their coexistence at the same habitat to minimize competition for nutrients that are scarce in soil, such as nitrogen (Whittaker and Cairney, 2001; Dickie et al, 2002; Adams et al; 2002). Differences in the nitrogen utilisation between Pterostylis recurva, Caladenia flava, and Diuris corymbosa that grow on the same substrates (litter) may be to minimize competition for nitrogen sources for their coexistence, while Drakaea elastica mycorrhizal fungus had a strategy to specialize in nutrient deficient sandy soil to avoid competition with other fungi for its existence. Differentiation of Drakaea elastica mycorrhizal fungus in the utilisation of few nitrogen sources can also provide some benefit for its survival.

Thiamine and PABA are factors limiting the growth and the survival of Drakaea elastica mycorrhizal fungus

It is evident from presented data that the growth of Drakaea elastica mycorrhizal fungus was limited by the availability of external source of thiamine and PABA. Without these organic compounds, Drakaea elastica had poor ability to utilise most nitrogen sources. The ability of Drakaea elastica mycorrizal fungus to utilise a wide range of nitrogen sources was determined by the availability of these organic compounds. This confirms the dependency of Drakaea elastica mycorhizal fungus on 51 these organic compounds to be able to utilise a wide range of nutrient sources. Previously, it was also demonstrated that Drakaea elastica mycorrhizal fungus required external source of thiamine and PABA to be able to utilise another nutrient source, carbon sources (chapter II).

Diuris corymbosa mycorrhizal fungus that belongs to the same fungal genus, Tulasnella, also had the same requirement for external thiamine and PABA in nitrogen source utilisation. Present data confirms the reliance of orchid mycorrhizal fungi that belong to the same fungal genus, Tulasnella on these organic compounds to utilise a wide range of nitrogen sources previously reported by other study (Hadley and Ong, 1978). (See the discussion of thiamine and PABA in Chapter II).

Caladenia flava and Pterostylis recurva mycorrhizal fungi showed that they grew equally well in both CN MMN and CN MMN + PABA liquid media on most nitrogen sources. It shows their consistency to grow equally well in both liquid media as it was previously been demonstrated in the experiment of the utilisation of carbon sources (Chapter II).

CHAPTER IV

The capacity of Drakaea elastica mycorrhizal fungus to utilise phosphorus sources

4.1. Introduction

In this chapter, the nutritional requirement of Drakaea elastica mycorrhizal fungus was further investigated with the focus on phosphorus. Phosphorus is considered as the primary limiting factor for growth (Read and Perez-Moreno, 2003; Hinsinger, 2001). It is one of essential nutrients critically needed for growth, as a cell component, a constituent of nucleic acids (DNA), an integral part of plasma membrane, and for generation of chemical cellular energy (ATP - adenosin triphosphate) essential for the majority of cellular metabolism for gowth (Raghotama and Karthikeyan, 2005; Karandashov and Bucher, 2005).

Information of the ability of orchid mycorrhizal fungi to utilise a wide range of phosphorus (inorganic phosphorus and organic phosphorus) is limited. There is one published report of the capability of orchid mycorrhizal fungi (mycorrhizal fungi associated with Goodyera repens) that were demonstrated to take up phosphate (inorganic phosphorus) from soil and deliver it to the orchids associate (Alexander et al, 1984). The ability of orchid mycorrhizal fungi to utilise organic phosphorus is largely unknown. Much information of the ability of mycorrhizal fungi to utilise phosphorus is derived from other mycorrhizal fungi that have widely been reported to utilise a wide range of phosphorus including inorganic phosphorus and organic phosphorus (Leake and Miles, 1996; Sharples and Cairney, 1997; Chen et al, 1999; Sawyer et al, 2003b; Midgley et al, 2004; Bieleski, 1973). The ability of mycorrhizal fungi to utilise organic phosphorus lies on its capacity to produce extracellular enzymes phosphomonoesterases and phosphodiesterases to hydrolise organic phosphorus (phosphomonoesthers and phosphodiesthers) into inorganic phosphorus (orthophosphate) which is subsequently absorbed. With this ability, mycorrhizal fungi facilitate to enhance phosphorus nutrition for improved growth of the associated plants (Antibus et al, 1997; Bartlett and Lewis, 1973; Jayachandran et al, 1992; Joner et al, 2000; Dighton, 1983).

Like other nutrients, phosphorus sources in soil are also associated with organic matter. Organic matter contains phosphorus in the forms of organic phosphorus

(phosphomonoesthers and phosphodiesthers) and inorganic phosphorus. DNA (deoxyribonucleic acid) that one of the building blocks is phosphate is one of forms of phosphodiesthers in soil organic matter. Detrital materials from plants and other living organisms contribute for the availability of phosphorus in soil (Paul and Clark, 1996) as they contain DNA in their cell nuclei. Phytic acid is a common form of phosphomonoesthers in soil. It is known as a major storage of phosphorus in plants (McDowell et al, 2005; Lin et al, 1987; Hegeman et al, 2001; Murphy et al, 2008).

The availability of phosphorus sources in nutrient deficient sandy soil habitat of Drakaea elastica is presumably associated with organic matter from litterfall of Kunzea ericifolia and Banksias as organic matter contains phosphorus and other nutrients (Blair and Crossley, 1988; Kuperman, 1999; Rouifed et al, 2010). Forms of phosphorus that the slow growing mycorrhizal fungus of Drakaea elastica required and utilised and its capacity to utilise a wide range of phosphorus sources were investigated in this study. Furthermore, its capacity to utilise phosphorus sources was compared with the sympatric faster growing orchid mycorrhizal fungi (mycorrhizal fungi associated with Pterostylis recurva, Caladenia flava, and Diuris corymbosa). In the previous Chapters (Chapter II and Chapter III), it was demonstrated that Drakaea elastica mycorrhizal fungus required external source of thiamine and PABA to be able to utilise a wide range of carbon and nitrogen sources. The influence of thiamine and PABA on the capability of Drakaea elastica mycorrhizal fungus to utilise a wide range of phosphorus sources was also investigated in the present study.

4.2. Methods

Preparation of inoculums

For the preparation of inoculums, orchid mycorrhizal fungi from Drakaea elastica, Pterostylis recurva, Caladenia flava, and Diuris corymbosa grown on Potato Dextrose Agar (PDA) were first subcultured onto low carbon nitrogen modified Melin and

Norkrans (CN MMN) media agar from which P sources (NH4)2HPO4 and KH2PO4 were omitted to deplete P stored in mycelia. NH4NO3 (in double concentration of 0.606 g) and 164 mg l-1 KCl were added to activate growth of inoculum in CN MMN without P sources, and to provide K source into the media (Sawyer et al, 2003b; Midgley et al, 2004). The composition of CN MMN media agar from which phosphorus excluded for -1 preparation of inoculums for phosphorus treatments (l ) was glucose, 5.0 g; NH4NO3,

0.606 g; MgSO4.7H2O 0.14 g; CaCl2, 50 mg; NaCl, 25 mg; 164 mg KCl; ZnSO4, 3 mg; ferric EDTA (C10H12FeN2NaO8), 12.5 mg, thiamine, 0.13 mg, agar 8 g. pH was adjusted to 5.0-5.5. Two plugs of agar were excised from the leading edge of the colony grown on CN MMN agar as active inoculums with a cork borer (diameter of 5 mm).

The capacity of orchid mycorrhizal fungi to utilise phosphorus sources There were two types of media used to assess the capacity of Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungi to utilise phosphorus sources, CN MMN and CN MMN + PABA liquid media containing single type of phosphorus source. For CN MMN + PABA liquid media, amount of PABA used was 20 mg/l. For phosphorus treatments, either NaH2HPO4.2H2O (0.3 g/l), phytic acid (0.2115 g/l), and DNA supplied as sodium salt of salmon testis from Sigma (0.6605 g/l) was supplemented into basal media (CN MMN and CN MMN +PABA liquid media from which phosphorus source omitted). This amount of phosphorus sources added into basal media was at the same final total P concentration in the media (60 mg P l-1). As DNA contains carbon and nitrogen, the amount of glucose was reduced to 4.02 g and NH4NO3 was excluded from DNA treatment. However, nitrogen in DNA exceeded that in the other treatments by c. 25 mg l-1 (based on the estimates of carbon and nitrogen concentration in salmon DNA) (Zubay, 1993 cited in Midgley et al, 2004). DNA was sterilized by immersion in 3 ml of 70 % ethanol solution for 48 hours and added into autoclaved basal media. Basal media was kept at a maximum temperature of 80°C on a heating block and stirred with a magnetic stirrer until DNA was completely dispersed (Leake and Miles, 1996). Phytic acid was dissolved in basal media and the pH was adjusted to pH 5.00 before filter sterilisation using a - 0.22 µm Millipore membrane filter into the autoclaved basal media.

Two plugs of fungal inoculums with a diameter of 5 mm were added into each treatment in 30 ml tubes containing 25 ml liquid media in unshaken culture. There were four replicates in each treatment. Mycorrhizal fungi were harvested according to each of their growth rates, P. recurva fungi (8 days), D. corymbosa fungi (18 days), C. flava fungi (20 days) and D. elastica fungi (28 days). To harvest the mycorrhizal fungi, the fungal mycelia were filtered through nylon mesh and transferred onto tarred alumunium foil, then dried for about one hour at 80ºC. A free-phosphorus treatment was included. Raw data for each phosphorus treatment were substracted by mean data of phosphorus- free treatment to obtain net biomass of mycorrhizal fungi only from phosphorus sources tested.

1. The net biomass of mycorrhizal fungi on individual phosphorus source in CN MMN liquid media containing single type of phoshorus source

A = B - C

A= net biomass of mycorrhizal fungi on single phosphorus source in CN MMN liquid media

B= biomass of mycorrhizal fungi in CN MMN liquid media containing single type of phosphorus source

C= biomass of mycorrhizal fungi in CN MMN liquid media without phosphorus source

2. The net biomass of mycorrhizal fungi on individual phosphorus source in CN MMN+ PABA liquid media containing single type of phosphorus source

X = Y - Z

X= net biomass of mycorrhizal fungi on single phosphorus source in CN MMN+PABA liquid media

Y= biomass of mycorrhizal fungi in CN MMN+PABA liquid media containing single type of phosphorus source

Z= biomass of mycorrhizal fungi in CN MMN+PABA liquid media without phosphorus source

Statistics Analysis

Data were analysed for normal distribution, and where data were found not normally distributed, the data were normalized using logarithmic transformations before analysis using ANOVA with GenStat 12th Edition. Significant differences between treatments were determined by Fisher’s PLSD 5%. The influence of the types of media (CN MMN and CN MMN+PABA liquid media) on the capability of orchid mycorrhizal fungi to utilise a wide range of phosphorus sources was analysed by a two way ANOVA (Analysis of Variance).

4.3. Results

Drakaea elastica mycorrhizal fungus produced biomass on all forms of phosphorus sources (orthophosphate, DNA, and phytic acid) in CN MMN + PABA

liquid media, whereas in CN MMN liquid media, its growth was limited to certain phosphorus sources (DNA and phytic acid and it grew poorly on orthophosphate (Fig. 4.1). Types of media had a significant (P < 0.001) influence on the biomass production of Drakaea elastica mycorrhizal fungus (table 4.1), with CN MMN + PABA liquid media significantly increased the biomass production of Drakaea elastica mycorrhizal fungus on a range of phosphorus sources. In CN MMN+PABA liquid media, biomass c production of Drakaea elastica mycorrhizal fungus on orthophosphate and phytic acid was significantly higher than on DNA (Fig. 4.1).

Biomass of Drakaea elastica mycorrhizal fungus on a range of P sources

3 c c 2.5

Drakaea 2 b

mycorrhizal mycorrhizal CN MMN 1.5 b ab CN MMN+PABA

biomass of 1 elastica elastica

0.5 a 0 phosphate DNA phytic acid phosphorus sources

Fig. 4.1. Mean biomass production (± SE) of D. elastica mycorrhizal fungus in liquid media containing a range of phosphorus sources. Letters on columns indicate significant differences (P < 0.05).

Biomass of Diuris corymbosa mycorrhizal fungus on a range of P sources

5 c 4.5 4 ab bc 3.5 3 a CN MMN

D. corymbosa 2.5 CN MMN + PABA 2 1.5

1 biomass biomass of mycorrhizal fungus (mg) 0.5 0 orthophosphate DNA phytic acid phosphorus sources

Fig. 4.2. Mean biomass production (± SE) of D. corymbosa mycorrhizal fungus in liquid media containing a range of phosphorus sources. Letters on columns indicate significant differences (P < 0.05). Diuris corymbosa mycorrhizal fungus showed the same trend as Drakaea elastica mycorrhizal fungus in terms of the type media that support its biomass production on phosphorus sources. In CN MMN media its growth was limited only on DNA and there was no biomass production on orthophosphate and phytic acid (Fig. 4.2). It produced biomass on all forms of phosphorus sources in CN MMN + PABA liquid media (Fig. 4.2). The types of media significantly influenced the biomass production of Diuris corymbosa mycorrhizal fungus (P < 0.001) (table 4.2), with significantly higher biomass was produced in CN MMN+PABA liquid media than in CN MMN liquid media. In CN MMN +PABA liquid media, biomass production of Diuris corymbosa mycorrhizal on all forms of phosphorus was not significantly different. It grew well on orthophosphate, DNA, and phytic acid in CN MMN + PABA liquid media (Fig. 4.2).

Biomass of Caladenia flava mycorrhizal fungus on a range of P sources 5 c c 4.5 4 3.5 3 2.5 b CN MMN Caladenia 2 ab a 1.5 a CN MMN+PABA

mycorrhizal fungus 1

0.5 biomassof

flava 0 orthophosphate DNA phytic acid phosphorus sources

Fig. 4.3. Mean biomass production (± SE) of C. flava mycorrhizal fungus in liquid media containing a range of phosphorus sources. Letters on columns indicate significant differences (P<0.05). Caladenia flava and Pterostylis recurva mycorrhizal fungi utilised a variety of phosphorus sources (orthophosphate, DNA, and phytic acid) in both CN MMN and CN MMN + PABA liquid media (Fig. 4.3 and Fig. 4.4). The biomass production of Caladenia flava and Pterostylis recurva mycorrhizal fungi in both types of media (CN MMN and CN MMN + PABA) was not significantly different (table 4.3 and table 4.4). Biomass production of C. flava mycorrhizal fungus on orthophosphate was significantly higher than on DNA and phytic acid (Fig. 4.3). Pterostylis recurva mycorrhizal fungus produced higher biomass on orthophosphate and phytic acid than on DNA (Fig. 4.4).

Biomass of Pterostylis recurva mycorrhizal fungus on a range of P sources

4.5 d 4 cd bc 3.5 3 recurva ab

P. P. 2.5 CN MMN 2 ab a CN MMN + PABA 1.5 b

1 biomass of

mycorrhizal(mg) fungus 0.5 0 orthophosphate DNA phytic acid phosphorus sources

Fig. 4.4. Mean biomass production (± SE) of P. recurva fungi in liquid media containing a range of phosphorus sources. Letters on columns indicate significant differences (P<0.05).

Table 4.1. Two way ANOVA of the influence of types of media on the capability of Drakaea elastica mycorrhizal fungus to utilise phosphorus sources

Source of variation d.f. s.s. m.s. v.r. F pr. Phosphorus sources 2 0.8404 0.4202 1.26 <.001 types of media 1 10.6223 10.6223 31.83 <.001 Residual 18 6.0067 0.3337 Total 23 22.7963

Table 4.2. Two way ANOVA of the influence of types of media on the capability of Diuris corymbosa mycorrhizal fungus to utilise phosphorus sources

Source of variation d.f. s.s. m.s. v.r. F pr. Phosphorus sources 2 2.9108 1.4554 7.07 0.005 types of media 1 46.4817 46.4817 225.82 <.001 Residual 18 3.7050 0.2058 Total 23 65.7233

Table 4.3. Two way ANOVA of the influence of PABA on the capability of Caladenia flava mycorrhizal fungus to utilise phosphorus sources

Source of variation d.f. s.s. m.s. v.r. F pr. Phosphorus sources 2 41.2353 20.6176 40.91 <.001 Types of media 1 0.0067 0.0067 0.01 0.910 Residual 18 9.0708 0.5039 Total 23 51.2681

Table 4.4. Two way ANOVA of the influence of PABA on the capability of P. recurva mycorrhizal fungus to utilise phosphorus sources

Source of variation d.f. s.s. m.s. v.r. F pr. Phosphorus sources 2 14.7925 7.3962 16.01 <.001 Types of media 1 3.3750 3.3750 7.30 0.015 Residual 18 8.3175 0.4621 Total 23 31.6775

4.4. Discussion

Utilisation of phosphorus sources

Presented data showed that the slow growing mycorrhizal fungus of Drakaea elastica and other orchid mycorrhizal fungi (faster growing orchid mycorrhizal fungi) utilised a variety of phosphorus sources (orthophosphate, DNA, and phytic acid). Orthophosphate was the best phosphorus source for Drakaea elastica mycorrhizal fungus and other orchid mycorrhizal fungus as this is a simple form of phosphorus and readily utilised (Colpaert et al, 1999; Ezawa et al, 2002; Bucking, 2004). Orthophosphate is also the only one phosphorus form that can be directly absorbed, while organic phosphorus must be hydrolized first by extracellular enzymes phosphatases (phosphomonoesterase and phosphodiesterase) into orthophosphate, which subsequently will be absorbed (Bartlett and Lewis, 1973; Margesin and Schinner, 1994; Gressel et al, 1996).

The capability of D. elastica fungi and other orchid mycorrhizal fungi to utilise phytic acids demonstrated in the present study indicates the production of phosphomonoesterase, an extracellular enzyme facilitating the hydrolysis of phytic acid into orthophosphate before absorption (Bartlett and Lewis, 1973; Hilger and Krause, 1989; Antibus et al, 1992; Margesin and Schinner, 1994; Gressel et al, 1996). So far, there has not been published data on the ability of orchid mycorrhizal fungi to produce phosphomonoesterase. Knowledge of extracellular enzyme phosphomonoesterase is derived from the other mycorrhizal fungi that release phosphomonoesterase (phytase) into the media when they were grown on phytic acid as the sole phosphorus source (Hilger and Krause, 1989; Antibus et al, 1992). Drakaea elastica mycorrhizal fungus in the present study presumably produced similar extracellular enzymes to mediate phytic acid utilisation.

Several other mycorrhizal fungi, such as ericoid mycorrhizal fungi have previously been demonstrated to utilise DNA as a phosphorus source, with their capacity to produce extracellular enzymes phosphodiestherase (Leake and Miles, 1996; Chen et al, 1999; Midgley et al, 2004). This enzyme facilitates the hydrolysis of DNA that will release orthophosphate which then will be absorbed (Leake and Miles, 1996; Maccheroni and Azevedo, 1998). The capability of Drakaea elastica mycorrhizal fungus to utilise DNA demonstrated in the present study suggests that they are able to produce extracellular enzymes phosphodiesterases.

It is evident from presented data that Drakaea elastica mycorrhizal fungus required and utilised a variety of phosphorus sources including inorganic phosphorus (orthophosphate) and organic phosphorus (phytic acid and DNA) for growth. Organic matter from litterfall of Kunzea ericifolia and Banksias can be a source of phosphorus and other nutrients in sandy soil habitat of Drakaea elastica. Detailed data of the composition and proportion of phosphorus sources in Drakaea elastica habitat is lacking. Further study is required to investigate the proportion and composition of phosphorus source as well as other nutrients in Drakaea elastica habitat.

Data in the present study also demonstrated that all orchid mycorrhizal fungi utilised the same phosphorus. It is the same situation where Drakaea elastica mycorrhizal fungus and faster growing orchid mycorrhizal fungi utilised the same carbon sources and nitrogen sources (Chapter II and Chapter III). This is an increased evidence that Drakaea elastica mycorrhizal fungus will have to compete for the same essential nutrient sources (carbon, nitrogen, and phosphorus). All data from the experiments of the utilisation of carbon, nitrogen, and phosphorus sources has led to the better understanding of the reasons why Drakaea elastica mycorrhizal fungus is specialized in nutrient deficient sandy soil (bare sandy soil or sandy soil with sparse organic matter). This may be the strategy of Drakaea elastica mycorrhizal fungus to avoid competition with many other fungi for nutrient sources (C, N, and P sources). In this bare sandy soil of habitat Drakaea elastica, nutrients are presumably present from the leaching of simple nutrients (such as glucose, amino acids, and orthophosphate, and organic compounds) from organic matter as the results of nutrients run-off or nutrients flow during rainy winter season (growing season). The variation in the degree of the capacity of orchid mycorrhizal fungi to utilise phosphorus sources demonstrated in the present study, physiologically, may be associated with the different degree of extracellular enzymes phosphatases activities or

different capacity to absorb orthophosphate between orchid mycorrhzal fungi (Ho and Zak, 1979; Antibus et al, 1986; 1997; Kieliszewska-Rokicka, 1992; Colpaert et al; 1999). Different preferences of phosphorus sources between orchid mycorrhizal fungi is similar situation with the different degree of the capacity of orchid mycorrhizal fungi to utilise nitrogen sources previously demonstrated in Chapter III. This is thought to be of ecological significance to minimize competition between orchid mycorrhizal fungi for the nutrient sources that are scarce in the soil, such as nitrogen and phosphorus for their coexistence in the habitat of Drakaea elastica. Different preference for nitrogen and phosphorus sources between orchid mycorrhizal fungi in the present study also extends different niche of mycorrhizal fungi in the utilisation of scarce resources in soil that have been previously demonstrated in several other studies (Leake and Miles, 1996; Chen et al, 1999; Sawyer et al, 2003b; Whittaker and Cairney, 2001; Anderson et al, 1999).

Thiamine and PABA are factors limiting the growth of Drakaea elastica mycorrhizal fungus It was demonstrated that Drakaea elastica mycorrhizal fungus had limited ability to utilise a range of phosphorus sources in CN MMN liquid media that only contain thiamine. It had a capacity to utilise a variety of phosphorus sources for its growth in media containing organic compounds thiamine and PABA (CN MMN + PABA liquid media). This suggests that the ability of Drakaea elastica mycorrhizal fungus to utilise a wide range of phosphorus sources is determined by the availability of external source of organic compounds thiamine and PABA. This confirms the importance of these external organic compounds for Drakaea elastica mycorrhizal fungus to be able to utilise a wide range of the other nutrient sources, carbon and nitrogen sources (Chapter II and Chapter III respectively) (see the discussion of thiamine and PABA in Chapter II). It is clear that the growth and the survival of Drakaea elastica mycorrhizal fungus were limited by the availability of external source of thiamine and PABA. The potential availability of exogenous thiamine and PABA in Drakaea elastica habitat is yet to be investigated as these organic compounds determine the growth and the survival of the mycorrhizal fungus.

Diuris corymbosa mycorrhizal fungus that belong to the same fungal genus as Drakaea elastica (Tulasnella) also showed its dependency on the external source of thiamine and PABA to be able to utilise a broad range of nutrient sources. The reliance

on thiamine and PABA appears to be a typical characteristic of orchid mycorrhizal fungi that belong to Tulasnella.

In contrast, Caladenia flava mycorrhizal fungus and Pterostylis recurva mycorrhizal fungus showed their consistency to grow either in CN MMN or in CN MMN+PABA liquid media on a variety of nutrient sources (carbon, nitrogen, and phosphorus).

Chapter V Optimisation propagation of Drakaea elastica Lindl.

5.1. Introduction The Orchidaceae is the most diverse species-taxa that contains approximately 30,000 species distributed across the world in a wide range of climates, altitudes, and habitats (Dressler, 1993; Arditti, 1992). Despite its richness, many members of Orchidaceae are threatened with extinction. Anthropogenic processes are considered as the main factor threatening the survival and the existence of orchids in their natural habitat, such as land clearance for housing and industry leading to habitat loss and population decline (Coates and Atkins, 2001). Reliable methods of propagation are essential to orchid conservation programs to provide seedlings for reintroduction programs. Orchid propagation is initiated by seedling generation through tissue culture or seed germination (Batty et al, 2006; Wright et al; 2009; Diaz and Alvarez, 2009; Avila-Diaz et al, 2009). Seed germination is often the first priority for propagation to maintain the genetic diversity of seedlings and ensure high survival when reintroduced to the natural habitat (Fay and Krauss, 2003). Terrestrial orchids often become a priority for optmising propagation as these orchids are more vulnerable to habitat alteration and are often more difficult to propagate than their epiphytic counterparts. This may be, in part, due to the requirement of terrestrial orchids for compatible mycorrhizal fungi in their entire life cycle, from the early development of orchids for seed germination and to the subsequent stages of orchids’ life (Rasmussen, 2002; Smith and Read, 1997). Thus, colonization of the compatible mycorrhizal fungi into orchid seedlings is essential for effective propagation of terrestrial orchids (Hadley and Williamson, 1971). Drakaea elastica is a critically endangered terrestrial orchid species with its distribution restricted to small areas of tall Kunzea ericifolia in deep sandy soil within a Banksia woodland between Cataby and Ruabon on the Swan Coastal Plain (Hoffman and Brown, 1998; Hopper and Brown, 2007). There has been a marked decrease in suitable D. elastica habitat in that, of the 52 known locations of this orchid, plants have only been found at 24 locations in recent years. A single population is known from the Moora District near Cataby and another 23 small populations in the Swan and Central Forest Regions, from Perth southwards to Ruabon Nature Reserve on the Swan Coastal Plain (Department 65 of the Environment, Water, Heritage and Arts, 2007). The Swan Coastal Plain has a high degree of species diversity with relatively high ecosystem diversity within the subregion, however, much of this area is threatened by clearing for agricultural purposes, grazing, weed invasion, mining and other factors (Mitchel et al, 2002). Given the rarity of D. elastica and with many factors threatening its survival, propagation is expected to become the most reliable tool for providing a large number of seedlings needed in the conservation program for reintroduction and recovery of this critically endangered species. Understanding the biological and ecological requirement for the seedlings’ development and establishment is one of keys for the success orchid propagation (Swarts, 2007). Temperature, light intensity, and fungal isolates are some of the biological and ecological requirements influencing seed germination and seedling development (Harvais and Hadley, 1966; Huynh et al, 2009). Some orchid mycorrhizal fungi (e.g. Rhizoctonia solani) are reported to enhance the percentage of seed germination at low temperatures (Harvais and Hadley, 1966). High percentage of seed germination is also associated with the most effective fungal isolates to germinate the seeds (Huynh et al, 2009). Huynh et al (2009) noted there was variation in the effectiveness of mycorrhizal fungi isolated from Caladenia formosa from leafing stage to fruiting stage in the germination percentages and the seedlings growth. This suggests that selection of the most effective isolates for germination and seedling development is important in the orchid propagation program. The aim of this study was to optmise propagation of Drakaea elastica based on the biological and ecological requirements for seedling development and establishment. We investigated the influence of temperature, light intensity, and D. elastica fungal isolates on the symbiotic seed germination and development of D. elastica seedlings. Furthermore, we investigated substrate types that support for the highest seedling survival in soil.

5.2. Methods - Mycorrhizal fungi isolation Mycorrhizal fungi were isolated from the mycorrhizal organ of Drakaea elastica (the collar) from different stages of orchids grown in a glass house (leafing stage, budding stage, flowering stage, and fruiting stage) and from plant from the natural habitat, Paganoni

Reserve (leafing stage, budding stage, and flowering stage). The collars were surface sterilized with sterile water three times. Pelotons were teased out from the collars of the orchids under microscope using micropipets and single pelotons were transferred onto SSE media + streptomycin as a bactericide for inhibiting bacterial growth and incubated at 19° C. There were 30 pelotons isolated from each of leafing, budding, flowering stages (natural habitat) and from each of leafing, budding, flowering, and fruiting stages (glass house) and plated onto SSE media that made up for the total of 210 pelotons isolated from plants across different stages of the plants from the glass house and the natural habitat. The fungal hyphae grew from 34 of total 210 pelotons from plants in the glass house and natural habitat were acquired during one to three months after plating. Hyphae were not obtained from pelotons originating from plants in the act of anthesis in the glass house. The hyphal tips of the mycorrhizal fungi that grew out from each peloton were cut and transferred onto PDA (Potato Dextrose Agar) and prepared as isolates for testing the effectiveness of the mycorrhizal fungal isolates for the seed germination and seedlings development.

- Symbiotic Germination Trials Symbiotic germination trials were conducted to (a) investigate the most suitable temperature and light intensity for symbiotic seed germination and seedling development, and (b) to test the effectiveness of isolates of D. elastica mycorrhizal fungi on the symbiotic seed germination and seedling development.

- The effect of temperature and light intensity on the symbiotic seed germination and seedling development With the lack of Drakaea elastica seeds we used seeds from a related species, Drakaea glyptodont which is a common species to investigate the most suitable temperature and light intensity for symbiotic seed germination and seedling development. Drakaea glyptodon seeds were placed in a nylon mesh package, surface sterilized in 1 % sodium chloride for 25 minutes and then washed three times in sterile water. The seeds were sown on 2.5 % oat meal agar (OMA) and two plugs of fungal inoculums excised from the leading edge of the fungal colony of Drakaea glyptodon (from Kings Park collection) were inoculated onto the same plates. Five replicate plates were placed at different temperatures (5°C, 10°C, 15°C, 20°C, 25°C) under a 12/12h light/dark regime, or in the dark (covered by

67 aluminium foil) for eight weeks, and then all plates were incubated under the 12/12 h light/dark regime for an additional four weeks before scoring of seed germination. Scoring of seed germination is adapted from Ramsay et al (1986). Stage 1 (ungerminated seeds), stage 2 (testa of the seeds ruptured), stage 3 (protocorms with rhizoids), and stage 4 (protocorms with the leaf) (Fig. 5.1). Seed germination was assigned from stage 2 to stage 4.

2 3

Fig. 5.1. Scoring of seed germination adapted from Ramsay et al (1986).

- Testing of the effectiveness of isolates of D. elastica mycorrhizal fungi on symbiotic seed germination and seedling development Thirty four isolates of D. elastica mycorrhizal fungi isolated from different stages of plants and grown either in the glass house or the natural habitat were tested for their effectiveness in symbiotic seed germination (see the protocol for the fungal isolation above). Drakaea elastica seeds in a package of nylon mesh were surface sterilized in 1 % sodium chloride for 25 minutes and then washed three times in sterile water. The seeds were sown on 2.5 % oat meal agar (OMA) and two plugs of fungal inoculums taken from each of D. elastica mycorrhizal fungal isolates were inoculated into the same plate. The symbiotic plates were placed at 20 °C in the dark (covered by aluminium foil). There were three replicates in this experiment. Scoring of seed germination was conducted after 12 weeks with the same protocol described above.

- Transfer of seedlings onto sand over agar in growth containers Transfer of seedlings onto sand over agar before transplanting into soil has been found to increase the survival of seedlings in soils (Batty et al, 2006; Swarts, 2007). For this transfer, 0.25% oat meal agar (OMA) was sterilized in the autoclave at 121°C for 20 minutes. About 1 cm layer of OMA was poured into sterilized growth containers enclosed by a ventilated lid covered with an adhesive Millipore disc. Once the OMA had solidified, about 1 cm sterilized white silica sand was added under sterile conditions. To test if fungal isolates affect seedling survival, seedlings infected by different isolates were transferred into different growth containers with each of growth containers contained seedlings infected by the same fungal isolates. There were three replicates in this experiment.

Fig. 5.2. Drakaea elastica seedlings on sand over agar

- Transfer of seedlings onto the soils in the glass house To investigate suitable substrates for a high rate of seedling survival, Drakaea elastica seedlings (after 12 weeks in the growth containers) were transferred into different types of substrates with different content of potting mix,(i) sandy soil (ii) ¾ sandy soil and ¼ potting mix (iii) ½ sandy soil and ½ potting mix (iv) ¼ sandy soil and ¾ potting mix (v) potting mix (vi) soil collected from Drakaea elastica habitat. There were three replicates for each treatment and each replicate consisted of 10 seedlings. Seedlings were monitored and recorded fortnightly for survival until the end of growing season. 69

5.3. Results Symbiotic seed germination trials - The effect of temperature and light intensity on the symbiotic seed germination Data in the present study showed that after 12 weeks incubation no seeds germinated under the light/dark regime condition across a range of temperatures (5°C, 10°C, 15°C and 25°C) and only very low percentage of seed germination (0.6%) occurred at 20°C in the light (Fig. 5.3). For seeds incubated first in the dark condition for eight weeks incubation (prior to transfer to the light/dark regime for the next four weeks), the percentage of seed germination was higher with the increase of temperature from 5°C to 25°C. At temperatures of 5°C and 10°C, the percentage of seed germination was less than 1 % (0.13% and 0.67%, respectively). At 15°C and 20°C, the percentage of seed germination was higher, around 3 % and 13%, respectively. The highest percentage of seed germination was observed at temperature of 25 °C (38%). However, seedling development (seed germination to the “green leaf” stage stage, protocorms with primordium leaf and first leaf; stage 3 and stage 4) only occurred at 15°C and 20°C, while at temperatures of 5°C, 10°C, and 25°C seeds germinated to stage 2 (the testa ruptured) but did not develop into seedlings (Fig. 5.4).

The percentage of Drakaea elastica symbiotic seed germination under the light condition after 12 weeks incubation

1.2 1

0.8 Stage 2 0.6 Stage 3 0.4 Stage 4

0.2 % of seed germination seed of % 0 5º C 10º C 15º C 20º C 25º C temperatures

Fig. 5.3. Means of the percentage of D. elastica symbiotic seed germination (±SE) at different temperatures under light condition after 12 weeks incubation

The percentage of Drakaea elastica symbiotic seed germination in the dark

45 d 40 35 30 Stage 4 25 c Stage 3 20 Stage 2 15 10 b 5 a a

percentagegerminationseed of (%) 0 5º C 10º C 15º C 20º C 25º C temperature

Fig. 5.4. Means of the percentage of D. elastica symbiotic seed germination (±SE) at different temperature after 8 weeks incubation in the dark and transferred to under light condition for the next four weeks

- Seed germination of Drakaea elastica infected with different D. elastica fungal isolates

Percentage of symbiotic seed germination of Drakaea elastica 80 70 60 50 40 30 20 10

percentage of seed germination (%) germination seed of percentage

2a 2c 4a 5a 6a 3a 5a 6a 1a 2a 3a 3c 1a 2a 2c 3a 4c 5a 1a 1c 3c 5a

4b 5b 2b 3b 1b 2b 2d 5b 1b 3d -10 2b Leafing budding fruiting leafing budding flowering

Glass house Paganoni reserve fungal isolates from different stages of plants from glass house and Paganoni reserve

Fig. 5.5. the mean percentage of D. elastica symbiotic seed germination (±SE) infected by different fungal isolates from different stages of Drakaea elastica (from glass house and from natural habitat).

All fungal isolates from different plant stages (leafing, budding, flowering and fruiting from glass house and from natural habitat) were effective at germinating the seeds, with the percentages of seed germination ranging from around 1 % to 58%. Pooled data of the percentage of seed germination showed that the percentage of seed germination of most isolates was ≤ 10%, with only four of 34 isolates inducing a higher percentage of seed germination (20% - 58%) (Fig. 5.5). Fig. 4 demonstrates that most fungal isolates induced seed germination to the “green leaf” stage (seedlings), with the percentages of seed germination to the “green leaf” stage of most isolates ≤ 5 % with only four of 34 isolates induced higher percentage of seed germination to the “green leaf” stage (20%-38%). A few isolates did not induce seed germination to the “green stage” (Fig. 5.6.).

- Seed germination to green stage of D. elastica seedlings

Percentage of symbiotic seed germination to the green stage of Drakaea elastica

greenstage (%) 10

percentageseedof germination to the -10

leafing

fruiting

Leafing

budding budding flowering Glass house Paganoni reserve fungal isolates

Fig. 5.6. the mean percentage of D. elastica symbiotic seed germination to the green leaf stage (±SE) infected by different fungal isolates from different stages of Drakaea elastica (from glass house and from natural habitat).

- Survival of D. elastica seedlings on sand over agar in the growth containers All seedlings infected by different isolates of D. elastica fungi survived and grew on sand over agar.

- Survival of D. elastica seedlings in soils (different substrates)

a f n a f n a f n a f n a f a f n n z yz

rx rx r

Fig. 5.7. the mean percentage of the survival of D. elastica seedlings (±SE) on different subsrates

I sandy soil II ¾ sandy soil and ¼ potting mix III ½ sandy soil and ½ potting mix IV ¼ sandy soil and ¾ potting mix V potting mix VI soil collected from Drakaea elastica habitat

All seedlings survived (100 %) after 1 month transfer to soils with different substrates. Seedling survival on all substrates declined after 2 months transfer, however, there was no significant difference of seedlings survival on different substrates, with seedling survival ranges from 87%-97%. After 3 months, at the end of the growing season, the survival of seedlings in all substrates declined, and seedling survival on different substrates was significantly different (Fig. 5.7). A higher percentage of seedling survival was observed on sand (70%) and on substrates with less potting mix (3/4 sand and ¼ potting mix; ½ sand and ½ potting mix) (53% and 35, respectively), whereas on substrates with relatively rich potting mix (1/4 sand and ¾ potting mix; and 100% potting mix) the seedling survival was 13% and 0%, respectively suggesting that seedlings mortality was very high. Seedling survival on the soil collected from natural habitat of Drakaea elastica was also low.

Fig. 5.8. Drakaea elastica seedlings after three months on different substrates.

5.4. Discussion Data in the present study demonstrate the biological and ecological requirements for Drakaea elastica seedling development and establishment. Almost no seeds germinated under the light condition across temperatures. Seed germination only occurred when seeds were first incubated in darkness (in the first eight weeks and transfer to a light/dark regime in the next four weeks) with the variation of the percentage of seed germination between temperatures. The temperature affected the development of the seeds into the seedlings with the temperatures of 15°C and 20°C induced the seed germination to the “green leaf” stage (seedlings). These data suggest that D. elastica requires temperature range of 15°C to 20°C for the development of its seeds into protocorms (in the dark) and the development from protocorms to the seedlings (under light). This may be related to the growing season of D. elastica (growth occurs during winter) with the average temperature during winter being in the range of 15°C - 20°C. Seed germination to the “protocorm” stage takes place underground and the leaves will develop from protocorms induced by sunlight. This also suggests that for generation of a large number of seedlings for the conservation of Drakaea elastica, symbiotic seed germination needs to be conducted at between 15°C to 20°C the darkness to initiate protocorms development and, and protocorms then need to be transferred to light conditions for the development of robust seedlings. The effectiveness of seed germination and seedling development varied between mycorrhizal fungal isolates, with only a few isolates having a high capability to germinate the seeds to the “green leaf” stage (seedlings). This suggests that selection of the best 75 fungal isolates that have a high capability to germinate the seeds to the “green leaf” stage is important to generate a large number of seedlings for the effective propagation. A similar result was previously showed in another study using the other orchid species Caladenia formosa with the variation of the efficacy of the mycorrhizal fungal isolates to germinate the seeds to the “green leaf” stage (Huynh et al, 2009). In the soil, the survival of seedlings after one month and two months transfer was relatively high across all substrates (100% and 87-97%, respectively). After three months transfer, at the end of growing season, the survival of seedlings declined in all substrates, and seedling survival varied between substrates. A higher percentage of seedling survival was observed on sand and substrates with less potting mix (3/4 sand and ¼ potting mix; ½ sand and ½ potting mix), whereas substrates with relatively rich potting mix experienced a high rate of seedling loss. These data indicate that deficient nutrient substrates are the most suitable substrates for the establishment of D. elastica seedlings in the soil. This may be associated with the specialization of Drakaea elastica in deficient nutrient sandy soil habitat.

This study reported the environmental conditions that induce a relatively high percentage of D. elastica seeds to germinate to the green stage (seedlings) and indicated suitable substrates for the relatively high survival of D. elastica based on the biological and ecological requirement of D. elastica seedling development and establishment. Optmisation of propagation of Drakaea elastica in the longer term involving translocation of D. elastica to its natural habitat is needed for the recovery of this critically endangered orchid species. It is worthwhile to investigate further the biological and ecological requirement of Drakaea elastica for the effective conservation of this species.

CHAPTER VI General Discussion

6.1. Introduction

This study has provided insight into some aspects of the biology and ecology of Drakaea elastica through research into its mycorrhizal associate. With the high dependency of Drakaea elastica on its mycorrhizal fungus for nutrient supply, understanding factors influencing the growth and the survival of its slow growing mycorrhizal fungus is an essential component in the conservation of this critically endangered terrestrial orchid species. The present study elucidates key factors for the growth and the survival of D. elastica’s mycorrhizal fungus, including its nutritional requirements and saprophytic competency in terms of its capacity to access essential nutrient sources (C, N, and P) relative to that of faster growing fungi. Furthermore, biological and ecological requirements for seedling development and establishment were investigated to advance the propagation of D. elastica for the production of a large number of seedlings for the future conservation of this threatened orchid.

Present study has demonstrated forms of nutrient sources (C, N, and P sources) required and utilised by the slow growing mycorrhizal fungus of D. elastica for its growth and survival (Chapter II, III, and IV respectively). It was also revealed that D. elastica mycorrhizal fungus had a capacity to utilise a wide range of C, N and P sources (Chapter II, III, and IV respectively).

The growth and survival of D. elastica mycorrhizal fungus was limited by exogenous organic compounds (thiamine and PABA-para amino benzoic acid). Drakaea elastica mycorrhizal fungus belongs to a fungal genus Tulasnella which is highly dependent on external organic compounds (thiamine and PABA) to utilise a wide range of C, N, and P sources for growth (Chapter II, III, and IV respectively). Without these external organic compounds, it grew poorly on most C, N, and P sources.

Data from this study suggest that D. elastica mycorrhizal fungus utilised the same C, N, and P sources as faster growing fungi (although there was variation in the utilisation of N and P sources) (Chapter II, III, and IV, respectively). This suggests that Drakaea elastica mycorrhizal fungus competes for the same nutrient sources as sympatric faster growing orchid mycorrhizal fungi (from other orchids such as Diuris 78 corymbosa, Caladenia flava, and Pterostylis recurva that co-occur in the macrohabitat of D. elastica). In the saprophytic activity in soil, faster growing fungi can overgrow the slow growing mycorrhizal fungus of D. elastica. Saprophytic competency of D. elastica mycorrhizal fungus can be limited by faster growing fungi. This may in part explain why D. elastica mycorrhizal fungus has a narrow distribution and microniche specialization in nutrient deficient sandy soil to minimize competition with many other fungi.

Understanding the requirement for seedling development and establishment is essential for the successful propagation of orchids (Swarts, 2007). This study elucidated factors such as temperature, light intensity, mycorrhizal fungal isolates, substrates that were required for seed germination and development.

6.2. Nutritional requirements of Drakaea elastica mycorrhizal fungus

Presented data showed a variety of nutrients including C, N, and P sources that D. elastica mycorrhizal fungus were required for its growth (chapter II, III, and IV). Although there is no soil nutrient composition data in the sandy soil habitat of Drakaea elastica, it is clear from other studies that organic matter present in all terrestrial ecosystems contains a variety of nutrient sources. C, N, and P sources in Drakaea elastica habitat are most likely litterfall of Kunzea ericifolia and Banksia. Plant roots are well known to release a variety of nutrients including sugar, amino acids, proteins, vitamins organic compounds to the rhizosphere. Kunzea ericifolia and Banksias presumably release nutrients that may be nutrient sources in the Drakaea elastica habitat. The capacity of Drakaea elastica mycorrhizal fungus to utilise a wide range of nutrient sources in its habitat is important for its survival.

The ability of Drakaea elastica mycorrhizal fungus to utilise a wide range of nutrient sources in culture may reflect its ability to access a variety of nutrient sources in its natural habitat. Interpreting these laboratory data in the field condition needs a degree of consideration as growth was measured in optimal conditions on only single nutrient sources and at one single concentration where in the field condition nutrient sources can be mixed at different concentrations (Whittaker and Cairney, 2001; Abuarghub and Read, 1988). However, presented data give a basis for understanding the way Drakaea elastica mycorrhizal fungus may utilise different types of nutrient sources. Furthermore, these data can be implemented for further research to optimise seed germination of Drakaea elastica. Implementing these data on the further study of 79 seed germination also needs a degree of caution. The robust growth of mycorrhizal fungus on certain nutrient sources at high concentration can be problematic as the mycorrhizal fungi may become parasitic to the orchids by killing the seeds and developing protocorms (Hadley, 1970; Beyrle et al, 1991). Sparse growth of mycorrhizal fungi in moderate nutrient concentrations may more likely support the development of healthy seedlings (Beyrle et al, 1991). The outcome of the interactions (parasitic or symbiotic association) between the mycorrhizal fungi and the orchids can be determined by the concentration and type of nutrients (Beyrle et al, 1991; Beyrle and Smith, 1993). Further study is needed to optimise conditions, types and concentrations of nutrients required for the formation of the healthy symbiotic association between Drakaea elastica seed and the mycorrhizal fungus for seedling recruitment.

In the symbiotic association between orchids and the mycorrizal fungus, it is believed that the mycorrhizal fungus takes up nutrient from soil and transfer a proportion of nutrient to the orchids (Alexander et al, 1984). However, the forms of nutrients that orchid mycorrhizal fungi take up from soil and transfer to the orchids is incompletely understood. Data from this study indicated a variety form of C, N, and P that Drakaea elastica mycorrhizal fungus could absorb from soil (Chapter II, III, and IV). Further study is required to investigate the forms of nutrients (forms of C, N, and P) that Drakaea elastica mycorrhizal fungus delivers to the plant to enhance our understanding of the symbiotic association between orchids and the mycorrhizal fungi associates. The capability of orchid mycorrhizal fungi to transport nutrient using isotopically labelled sources of C, N, and P (13C and 14C, 15 N, and 33P, respectively) has been well demonstrated (Smith, 1967; Alexander and Hadley, 1985; Alexander et al, 1984; Cameron et al, 2006; 2007). However, the forms of C, N, and P sources that orchid mycorrhizal fungi deliver to its host plant are largely unknown. - Utilisation of C sources

This study demonstrated that Drakaea elastica mycorrhizal fungus utilised a wide range of C sources including glucose, mannose (monosaccharide), cellobiose (disaccharide), cellulose, carbaxymethylcellulose (CMC), starch, pectin, and xylan (polysaccharides). The capacity of D. elastica mycorrhizal fungus to decompose a variety of polysaccharides reflects that it is capable to produce extracellular enzymes (cellulose, amylase, pectinase, and xylanase) to degrade a range of polysaccharides (cellulose, starch, pectin, and xylan, respectively), which is substantial in its saprophytic activity in soil. This ability is also thought to be substantial in the establishment of 80 symbiotic association with the orchids associate to degrade the cell walls of the orchids that are composed of a high proportion of polysaccharides (cellulose, pectin, xylan, and other polysaccharides).

- Utilisation of N sources

Drakaea elastica utilised a range of N sources including inorganic N (ammonium), organic N including amino acids (aspartic acid, glutamic acid, alanine, asparagine, arginine, glutamine, glycine), and protein (bovine serum albumin-BSA). The ability of D. elastica mycorrhizal fungus to utilise protein suggests the production of extracellular enzymes protease to breakdown protein into amino acids that will subsequently be absorbed.

- Utilisation of P sources

Drakaea elastica mycorrhizal utilised a variety of P sources such as inorganic P (orthophosphate) and organic P (phytic acid and DNA) (chapter IV). The utilisation of organic P needs extracellular enzymes phosphatases (phosphomonoestherase and phosphodiestherase) to degrade phytic acid and DNA, respectively into orthophosphate which subsequently will be absorbed. The ability of Drakaea elastica mycorrhizal fungus to utilise organic P reflects its capability to produce extracellular enzymes phosphatases to hydrolyze organic P.

6.3. Factors limiting the growth and the survival of D. elastica mycorrhizal fungus

Organic compounds (thiamine and PABA) were identified in this study as factors limiting the growth and the survival of Drakaea elastica mycorrhizal fungus. Drakaea elastica mycorrhizal fungus grew poorly on most C, N, and P sources in nutrient media supplemented with only one organic compound (thiamine) (CN MMN). Drakaea elastica mycorrhizal fungus grew well on most C, N, and P sources in the nutrient media supplemented with a combination of organic compounds, thiamine and PABA (CN MMN + PABA). This suggests that Drakaea elastica mycorrhizal fungus depends on the availability of exogenous organic compounds (thiamine and PABA) to utilise each nutrient source for growth. Other orchid mycorrhizal fungi that belong to the same fungal genus Tulasnella are reported to require a combination of these external organic compounds to grow (Hadley and Ong, 1978).

Organic compounds such as amino acids, proteins, sugar, and other essential nutrients into the soil rhizosphere can be from organic matter, soil microorganisms and plants roots that release a variety of organic compounds. The source of thiamine and PABA in Drakaea elastica habitat can be from organic matter and root exudates of Kunzea ericifolia. This may explain why Drakaea elastica is often found in an association with these trees. There is a need to further investigate the composition and proportion of the root exudates of Kunzea ericifolia and its effect on the growth and the survival of Drakaea elastica mycorrhizal fungus.

Diuris corymbosa mycorrhizal fungus also belongs to the same fungal genus as Drakaea elastica mycorrhizal fungus. This mycorrhizal fungus also required exogenous thiamine and PABA to utilise each nutrient source. This dependency on external thiamine and PABA may be a specific characteristic of Tulasnella. This study supports findings of previous studies that document the requirement of external thiamine and PABA for nutrient uptake in Tulasnella. Other orchid mycorrhizal fungi (from Pterostylis recurva and Caladenia flava) which belong to different fungal genera, Ceratobasidium and Sebacina, respectively grew in both CN MMN and CN MMN + PABA liquid media and their growth in both media on each nutrient source was not significantly different. This suggests that these orchid mycorrhizal fungi were independent from external PABA, however it is not yet clear if nutrient uptake is independent from exogenous thiamine. Further study of organic compounds requirement of orchid mycorrhizal fungi is needed to increase understanding of organic compounds required by orchid mycorrhizal fungi for their growth and survival. This study can be performed by screening organic compounds required by a diverse range of orchid mycorrhizal fungi across orchid mycorrhizal fungal genera.

Table 1. Growth of orchid mycorrhizal fungi from different fungal genera in nutrient media containing thiamine and in nutrient media containing a combination of thiamine and PABA

No orchid mycorrhizal fungal genus organic compounds fungi species thiamine thiamine + PABA 1 Drakaea elastica Tulasnella Poor growth on most C, Good growth on most mycorrhizal fungus N, and P sources C, N, and P sources 2 Diuris corymbosa Tulasnella Poor growth on most C, Good growth on most mycorrhizal fungus N, and P sources C, N, and P sources 3 Caladenia flava Sebacina Good growth on most C, Good growth on most mycorrhizal fungus N, and P sources C, N, and P sources 4 Pterostylis recurva Ceratobasidium Good growth on most C, Good growth on most mycorrhizal fungus N, and P sources C, N, and P sources

6.4. Saprophytic competency of Drakaea elastica mycorrhizal fungus

Presented data showed that the slow growing mycorrhizal fungus of Drakaea elastica and faster growing fungi (mycorrhizal fungi associated with Diuris corymbosa, Caladenia flava, and Pterostylis recurva) utilised the same C, N, and P sources. This implies that Drakaea elastica mycorrhizal fungus will have to compete for the same nutrient sources with faster growing fungi. Although Drakaea elastica mycorrhizal fungus had a capacity to utilise a wide range of C, N, and P sources, its slow growing characteristic could impede its saprophytic competency in the competition for nutrient sources with other fungi. The slow growing mycorrhizal fungus of Drakaea elastica has the potential to be outcompeted by faster growing fungi and must evolve a strategy to survive in this competition. Specialization in nutrient deficient sandy soil may be one of the strategies of Drakaea elastica mycorrhizal fungus to minimize competition with many other fungi. Much study is required to understand specialization of Drakaea elastica in this habitat. If the microhabitat of Drakaea elastica mycorrhizal fungus is characterized by slow growing fungi and slow growing microorganisms, do they then have the same chance to access nutrient sources in Drakaea elastica habitat? Further study in situ is also required to investigate the distribution of the slow growing mycorrhizal fungus of Drakaea elastica in its natural habitat as faster growing fungi from other orchid species (Diuris corymbosa, Caladenia flava, and Pterostylis recurva) also exist in the habitat of Drakaea elastica. Do faster growing fungi inhibit the distribution of Drakaea elastica mycorrhizal fungus in soil? Understanding habitat characteristic of Drakaea elastica is also one of essential components in its conservation. Analysis of microbial community structure, soil analysis related to 83 proportion and composition of nutrients in Drakaea elastica habitat and soil profiling, analysis of vegetation surround Drakaea elastica are essential to be performed to add information of the habitat (microhabitat and macrohabitat) characteristic of Drakaea elastica.

Our data demonstrated different preferences in the utilisation of certain N and P source between Drakaea elastica mycorrhizal fungus and faster growing orchid mycorrhizal fungi growing in sympatry with Drakaea elastica mycorrhizal fungus in Drakaea elastica habitat (Chapter III and chapter IV). N and P sources are present in small quantities relative to C in soil. Different preferences for certain N and P sources is of ecological significance to minimize competition for nutrients that are scarce in soil for the coexistence of these sympatric orchid mycorrhizal fungi (mycorrhizal fungi of Drakaea elastica, Diuris corymbosa, Caladenia flava, and Pterostylis recurva) at the same habitat. Other studies also reported the different niche in the utilisation of N and P sources between mycorrhizal fungi from the same habitat for their coexistence (Anderson et al, 1999; Whittaker and Cairney, 2001; Adams et al; 2002; Chen et al; 1999). Presented data indicated that different niche for N and P sources between Drakaea elastica mycorrhizal fungus and other fungi coexisting at the same habitat may occur. Still much to learn about interaction between Drakaea elastica mycorrhizal fungus and other fungi as well as other soil microorganisms and how they coexist at the same habitat. Screening of a variety of C, N, P sources is required to investigate if they have partitioning resources at the same habitat.

6.5. Propagation of Drakaea elastica

Understanding the biology and ecological requirements of D. elastica’s mycorrhizal fungus will benefit the propagation of this orchid, especially seedling development and establishment. Presented data showed that the development of Drakaea elastica seedlings from seeds required temperature of 15°C-20°C, with the seed germination (the development of the seeds into protocorms) required dark condition and the development of protocorms into seedlings (seedlings development) needed light condition as the light induces the formation of the seedlings’ leaf. This is to be general required condition for the seedlings development from seeds of other orchids from Australia. This matches winter growing season’s conditions in Australia with the seeds will germinate in the winter with the average temperature between 15°C-20°C and the seeds will interact with the compatible mycorrhizal fungi underground in the dark.

When the seeds germinate and develop into protocorms, the leaves will develop from protocorms triggered by photosynthesis.

This study also showed that mycorrhizal fungi isolated from leafing, budding, flowering, and fruiting stages of Drakaea elastica from glass house and the natural habitat were able to germinate the seeds and most of them develop the seeds into seedlings. Selection of the best fungal isolates of all fungal isolates showed that few isolates had a high capacity to germinate the seeds to the “leaf stage” (seedlings development), which can be implemented in the future to generate a large number of D. elastica seedlings for reintroduction purposes. The result of selection of the best substrates for the establishment of seedlings in soil indicated that sandy soils and nutrient deficient substrates were suitable substrates for the establishment of Drakaea elastica seedlings in soil. Further investigation is required to follow up this experiment as well as optmisation propagation of Drakaea elastica in the longer terms for the translocation of Drakaea elastica seedlings and the establishment of seedlings in its natural habitat.

6.6. Conclusion

This study has elucidated some critical aspects of the biology and ecology of Drakaea elastica in terms of factors determining the growth and the survival of its slow growing mycorrhizal fungus that is a key determinant for the existence of Drakaea elastica. Data in present study can be incorporated into the fundamental knowledge of the biology and ecology of Drakaea elastica mycorrhizal fungus which is substantial in the conservation of Drakaea elastica. The growth and the survival of the slow growing mycorrhizal fungus related to its nutritional requirement for growth, factors limiting its growth and survival, its saprophytic competency in terms of its capacity to utilise a variety of nutrient sources relative to faster growing fungi have been elucidated in this study. Drakaea elastica mycorrhizal fungus required and utilised a wide range of nutrient sources including a variety of C, N, and P sources for its growth.

This study also showed that the growth and the survival of Drakaea elastica mycorrhizal fungus were limited by the availability of external organic compounds (thiamine and PABA). These exogenous organic compounds were required by Drakaea elastica mycorrhizal fungus to be able to utilise a wide range of nutrient sources (C, N, and P sources) for growth. Without these exogenous organic compounds, Drakaea

85 elastica mycorrhizal fungus grew poorly and was less capable to utilise a wide range of nutrient sources.

Although Drakaea elastica has a capacity to utilise a wide range of nutrient sources, its slow growing characteristic can be an intrinsic factor that impedes its competency to access a variety of nutrient sources in the competition for nutrient sources with many other fungi. This study also suggests that the optimisation of propagation of Drakaea elastica needs an understanding of the biological and ecological requirement for seedling development and establishment. Further study of biology and ecology requirement and the best conditions for seedlings development and establishment of Drakaea elastica to optmise propagation of Drakaea elastica is still required for the establishment of Drakaea elastica seedlings in the longer term in its natural habitat.

REFERENCES

Abuarghub, S. M. and Read, D. J. 1988. The biology of mycorrhiza in the Ericaceae .11. The distribution of nitrogen in soil of a typical upland callunetum with special reference to the free amino-acids. New Phytologist, 108, 425-431.

Abuarghub, S. M. and Read, D. J. 1988. The biology of mycorrhiza in the Ericaceae.12. Quantitative-analysis of individual free amino-acids in relation to time and depth in the soil- profile. New Phytologist, 108, 433-441.

Adams, M. A. 1992. Phosphatase activity and phosphorus fractions in Karri Eucalyptus diversicolor F. (Muell) Forest Soils. Biol. Fertil. Soil, 14, 200-204.

Adams, M. A., Bell, T. L. and Pate, J. S. 2002. Phosphorus sources and availability modify growth and distribution of root clusters and nodules of native Australian legumes. Plant, Cell and Environment, 25, 837-850. Alexander, C., Alexander, I. J. and Hadley, G. 1984. Phosphate uptake by Goodyera repens in relation to mycorrhizal infection. New Phytologist, 97, 401-411.

Alexander, C. and Hadley, G. 1985. Carbon movement between host and mycorrhizal endophyte during the development of the orchid Goodyera repens Br. New Phytologist, 101, 657-665.

Allen, M. F., Swenson, W., Querejeta, J. I., Egerton-Warburton, L. M. and K.K.Treseder 2003. Ecology of mycorrhizae:a conceptual framework for complex interactions among plants and fungi. Ann. Rev. Plant Phytopathol., 2003, 271-304.

Anderson, I. C., Chambers, S. M. and Cairney, J. W. G. 1999. Intra- and interspecific variation in patterns of organic and inorganic nitrogen utilization by three Australian Pisolithus species. Mycological Research, 103, 1579-1587.

Antibus, R. K., Bower, D. and Dighton, J. 1997. Root surface phosphatase activities and uptake of 32P-labelled inositol phosphate in field-collected gray birch and red maple roots. Mycorrhiza, 7, 39-46.

Antibus, R. K., Kroehler, C. J. and Linkins, A. E. 1986. The effects of external pH, temperature, and substrate concentration on acid phosphatase activity of ectomycorrhizal fungi. Can. J. Bot., 64, 2383-2387.

Antibus, R. K., Sinsabaugrh, R. L. and Linkins, A. E. 1992. Phosphatase activities and phosphorus uptake from inositol phosphate by ectomycorrhizal fungi. Can. J. Bot., 70, 794-801.

Arditti, J. 1992. Fundamentals of orchid biology, John Wiley and Sons, New York.

Arditti, J. 1967. Factors affecting the germination of orchid seeds. Botanical Review, 33, 1-197.

87 Arditti, J. and Ghani, A. K. A. 2000. Tansley Review No. 110 Numerical and physical properties of orchid seeds and their biological implications. New Phytologist, 145, 367-421.

Arnosti, C. 2003. Microbial extracellular enzymes and their role in dissolved organic matter cycling. Aquatic ecosystems: interactivity of dissolved organic matter. USA: Elsevier Science.

Attiwill, P. M. and Adams, M. A. 1993. Nutrient cycling in forests. New Phytologist, 124, 561-582.

Avila-Dı´az, I., Oyama, K., Go´Mez-Alonso, C. and Salgado-Garciglia, R. 2009. In vitro propagation of the endangered orchid Laelia speciosa. Plant Cell Tiss Organ Cult 99, Plant Cell Tiss Organ Cult (2009) 99:335–343.

Bajwa, R., Abuarghub, S. and Read, D. J. 1985. The biology of mycorrhiza in the Ericaceae .10. The utilization of proteins and the production of proteolytic-enzymes by the mycorrhizal endophyte and by mycorrhizal plants. New Phytologist, 101, 469-486.

Bartlett, E. M. and lewis, D. H. 1973. Surface phosphatase activity of mycorrhizal roots of beech. Soil Biol. Biochem, 5, 249-257.

Bassett, G. J. C., Quinlivan, E. P., Gregory, J. F. and Hanson, A. D. 2005. Folate synthesis and metabolism in plants and prospects for biofortification. Crop Science, 45, 449-453.

Bassett, G. J. C., Ravanel, S., Quinlivan, E. P., White, R., Giovannoni, J. J., Rebeille, F., Nichols, B. P., Shinozaki, K., Seki, M., Gregory, J. F. and Hanson, A. D. 2004. Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant Journal, 40, 453-461.

Batty, A. L., Brundrett, M. C., Dixon, K. W. and Sivasithamparam, K. 2006. In situ symbiotic seed germination and propagation of terrestrial orchid seedlings for establishment at field sites. Australian Journal of Botany, 54, 375-381.

Batty, A. L., Brundrett, M. C., Dixon, K. W. and Sivasithamparam, K. 2006. New methods to improve symbiotic propagation of temperate terrestrial orchid seedlings from axenic culture to soil. Australian Journal of Botany, 54, 367-374.

Batty, A. L., Dixon, K. W., Brundrett, M. C. and Sivasithamparam, K. 2002. Orchid conservation and mycorrhizal associations. In: Sivasithamparam, K., Dixon, K. W. and Barrett, R. L. (eds.) Microorganisms in plant conservation and biodiversity. Kluwer Academic Publishers, Dordrecht.

Batty, A. L., Dixon, K. W. and Sivasithamparam, K. 2000. Soil seed-bank dynamics of terrestrial orchids. Lindleyana, 15, 227-236.

88 Bell, T. L., Pate, J. S. and Dixon, K. W. 1994. Response of mycorrhizal seedlings of SW Australian sandplain Epacridaceae to added nitrogen and phosphorus. Journal of Experimental Botany, 45, 779-790.

Berg, B. 1984. Decomposition of root litter and some factors regulating the process:long term root litter decomposition in a Scots Pine forest. Soil Biology and Biochemistry, 16, 609-617.

Beyrle, H., Penningsfeld, F. and Hock, B. 1991. The role of nitrogen concentration in determining the outcome of the interaction between Dactylorhiza incarnata (L.) Soo and Rhizoctonia sp. New Phytologist, 117, 665-672.

Beyrle, H. F. and Smith, S. E. 1993. The effect of carbohydrate on the development of a cattleya hybrid in association with its mycorrhizal fungus. Mycorrhiza, 3, 57-62.

Bieleski, R. L. 1973. Phosphate pools, phosphate transport, and phosphate availability. Ann. Rev. Plant Physiol. , 24, 225-252.

Blair, J. M. and D. A. Crossley, J. 1988. Litter decomposition, nitrogen dynamics and litter microarthropods in a southern appalachian hardwood forest 8 years following clearcutting Journal of Applied Ecology, 25, 683-698.

Brown, A., Thomson-Dans, C. and Marchant, N. 1998. Western Australia’s threatened flora, Western Australia., Department of Conservation and Land Management.

Brundrett, M. C. 2007. Scientific approaches to Australian temperate terrestrial orchid conservation. In, 2007. CSIRO Publishing, 293-307.

Brundrett, M. C., Scade, A., Batty, A. L., Dixon, K. W. and Sivasithamparam, K. 2003. Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycological Research, 107, 1210-1220.

Bucking, H. 2004. Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations. Mycol. Res. 108, 599-609.

Bücking, H., Abubaker, J., Govindarajulu, M., Tala, M., Pfeffer, P. E., Nagahashi, G., Lammers, P. and Shachar-Hill, Y. 2008. Root exudates stimulate the uptake and metabolism of organic carbon in germinating spores of Glomus intraradices. New Phytologist 180, 684-695.

Burgeff, H. 1936. Samenkeimung der orchideen, Jena, Gustav Fischer cited in Midgley, D. J., Jordan, L. A., Saleeba, J. A. and Mcgee, P. A. 2006. Utilisation of carbon substrates by orchid and ericoid mycorrhizal fungi from Australian dry sclerophyll forests. Mycorrhiza, 16, 175-182.

89 Burke, R. M. and Cairney, J. W. G. 1997. Purification and characterization of a β-1,4-endoxylanase from the ericoid mycorrhizal fungus Hymenoscyphus ericae. New Phytologist, 135, 345- 352.

Cairney, J. W. G. and Burke, R. M. 1998. Extracellular enzyme activities of the ericoid mycorrhizal endophyte Hymenoscyphus ericae (Read) Korf and Kernan: their likely roles in decomposition of dead plant tissue in soil. Plant and Soil, 205, 181-192.

Cameron, D. D., Johnson, I., Leake, J. R. and Read, D. J. 2007. Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens. Annals of Botany, 99, 831-834.

Cameron, D. D., Leake, J. R. and Read, D. J. 2006. Mutualistic mycorrhiza in orchids: evidence from plant-fungus carbon and nitrogen transfers in the green-leaved terrestrial orchid Goodyera repens. New Phytologist, 171, 405-416.

Carstairs, S. and ., D. Coates. 1994. Conservation genetics and population ecology of five rare and threatened western australian orchids. Final Report to the Western Australian Department of Conservation and Land Management, and Australian Nature Conservation Agency.

Chaikelis, A. S. 1946. The thiamine content of herbs and medicinal plants. Journal of the American Pharmaceutical Association-Scientific Edition 35, 343-346.

Chalot, M. and Brun, A. 1998. Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. Fems Microbiology Reviews, 22, 21-44.

Chen, A., Chambers, S. M. and Cairney, J. W. G. 1999. Utilisation of organic nitrogen and phosphorus sources by mycorrhizal endophytes of Woollsia pungens (Cav.) F. Muell. (Epacridaceae). Mycorrhiza, 8, 181-187.

Clements, M. A. 1989. Catalogue of Australian Orchidaceae. Australian Orchid Research, 1, 1- 160.

Coates, D. J. and Atkins, K. A. 2001. Priority setting and the conservation of Western Australia's diverse and highly endemic flora. Biological Conservation, 97, 251-263.

Cochrane, V. W. 1958. Physiology of fungi, New York, John Wiley and Sons Inc.

Colpaert, J. V. and Tichelen, K. K. V. 1996. Decomposition, nitrogen and phosphorus mineralization from beech leaf litter colonized by ectomycorrhizal or litter-decomposing Basidiomycetes. New Phytologist, 134, 123-132.

Colpaert, J. V., Tichelen, K. K. V., Assche, J. A. V. and Laere, A. V. 1999. Short-term phosphorus uptake rates in mycorrhizal and non-mycorrhizal roots of intact Pinus sylvestris seedlings. New Phytologist, 143, 589-597.

90 Connell, M. J., Raison, R. J. R., and Khanna, P. K. 1995. Nitrogen mineralization in relation to site history and soil properties for a range of Australian forest soils. Biol Fertil Soils 20, 213- 220.

Cossins, E. 2000. The fascinating world of folate and one-carbon metabolism. Canadian Journal of Botany, 78, 691-708.

Cossins, E. A. and Chen, L. 1997. Folates and one-carbon metabolism in plants and fungi. Phytochemistry, 45, 431-452.

Couteaux, M., Bottner, P. and Berg, B. 1995. Litter decomposition,climate and litter quality. Tree10, 63-66.

Currah, R. S. & Zelmer, C. 1992. A key notes for the genera of fungi mycorrhizal with orchids and a new species in the genus Epulorhiza Reports of the Tottori Mycological Institute, 30, 43- 59 cited in Swarts, N. 2007. Integrated conservation of the rare and endangered terrestrial orchid Caladenia huegelii H. G. Reichb. . PhD Thesis. The University of Western Australia. Western Australia.

Daynes, C. M., Mcgee, P. A. and Midgley, D. J. 2008. Utilisation of plant cell-wall polysaccharides and organic phosphorus substrates by isolates of Aspergillus and Penicillium isolated from soil. Fungal Ecology, 1, 94-98.

Dearnaley, J. D. W. 2007. Further advances in orchid mycorrhizal research. Mycorrhiza 17, 475- 486.

Department of the Environment, Water, Heritage and Arts. 2007. Recommendation report commercial development on Jandakot airport (epbc 2007/3599). Available: http://www.environment.gov.au/ [Accessed August 29, 2008].

Díaz, M. S. S. and Álvarez, C. C. 2009. Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of Encyclia mariae (Orchidaceae), a threatened Mexican orchid. In Vitro Cell.Dev.Biol.Plant 45, 162-170.

Dickie, I. A., Xu, B. and Koide, R. T. 2002. Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytologist, 156, 527-535.

Dighton, J. 1983. Phosphatase production by mycorrhizal fungi. Plant and Soil, 71, 455-462.

Dixon, K. D. and Tremblay, R. L. 2009. Review biology and natural history of Caladenia. Australian Journal of Botany, 57, 247-258.

Dressler, R. 1993. Phylogeny and classification of the orchid family. Dioscorides Press, Portland.

Ezawa, T., Smith, S. E. and Smith, F. A. 2002. P metabolism and transport in AM fungi. Plant and Soil, 244, 221-230.

91 Fay, M. F. and Krauss, S. L. 2003. Orchid conservation genetics in the molecular age. In: Dixon, K. W., Kell, S. P., Barrett, R. L. and Cribb, P. J. (eds.) Orchid conservation. Borneo, Kota Kinabalu, Sabah, Malaysia: Natural history publications.

Finlay, R. D., Frostegard, A. and Sonnerfeldt, A. M. 1992. Utilization of organic and inorganic nitrogen-sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl ex Loud. New Phytologist, 120, 105-115.

Gambonnet, B., Jabrin, S., Ravanel, S., Karan, M., Douce, R. and Rebeille, F. 2001. Folate distribution during higher plant development. Journal of the Science of Food and Agriculture, 81, 835-841.

Goyer, A. 2010. Thiamine in plants: Aspects of its metabolism and functions. Phytochemistry

Gressel, N., Mccoll, J. G., Preston, C. M., Newman, R. H. and Powers, R. F. 1996. Linkages between phosphorus transformations and carbon decomposition in a forest soil. Biogeochemistry, 33, 97-123.

Griffin, D. H. 1994. Fungal Physiology, Wiley-liss. Inc, New York.

Gunawardena, U., Zhao, X. and Hawes, M. C. 2006. Roots: Contribution to the rhizosphere.

Guzman, S., Ramos, I., Moreno, E., Ruiz, B., Rodriguez-Sanoja, R., Escalante, L., Langley, E. and Sanchez, S. 2005. Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius. Applied Microbiology and Biotechnology, 69, 200-206.

Hadley, G. 1969. Cellulose as a carbon source for orchid mycorrhiza. New Phytologist, 68, 933- 939.

Hadley, G. 1970. Non-specificity of symbotic infection in orchid mycorrhiza. New Phytologist, 69, 1015-1023.

Hadley, G. 1984. Uptake of [C-14]-labeled glucose by asymbiotic and mycorrhizal orchid protocorms. New Phytologist, 96, 263-273.

Hadley, G. and Perombelon, M. 1963. Production of pectic enzymes by Rhizoctonia solani and orchid endophytes. Nature, 1337.

Hadley, G. and Williams.B 1971. Analysis of post-infection growth stimulus in orchid mycorrhiza. New Phytologist, 70, 445-455.

Hadley, G. and Ong, S. H. (1978) Nutritional requirements of orchid endophytes New Phytologist, 81, 561-569.

Hanson, A. D. and III, J. F. G. 2002. Synthesis and turnover of folates in plants. Current Opinion in Plant Biology 5, 244-24.

Harvais, G. and Hadley, G. 1966. Development of orchis purpurella in asymbiotic and inoculated cultures. New Phytologist, 66, 217-and.

Hegeman, C. E., Good, L. and Grabau, E. A. 2001. Expression of D-myo-inositol-3-phosphate synthase in soybean. Implications for phytic acid biosynthesis. Plant Physiology, 125, 1941- 1948.

Hijner, J. A. and Arditti, J. 1973. Orchid mycorrhiza - vitamin production and requirements by symbionts. American Journal of Botany, 60, 829-835.

Hilger, A. B. and Krauseh, H. 1989. Growth characteristics of Laccaria laccata and Parillus involutus in liquid culture media with inorganic and organic phosphorus sources. Can. J. Bot., 67, 1782-1789.

Hinsinger, P. 2001. Bioavailability of soil inorganic P in the rhizosphere as affected by root- induced chemical changes: a review. Plant and Soil 237, 173-195.

Hinsinger, P., Plassard, C. and Jaillard, B. 2006. Rhizosphere: A new frontier for soil biogeochemistry. Journal of Geochemical Exploration, 88, 210-213.

Ho, I. and Zak, B. 1979. Acid phosphataseactivity of six ectomycorrhizal fungi. Can. J. Bot., 57, 1203-1205.

Hodge, A. 2005. Plant Uptake. Nitrogen in Soils.

Hoffman, N. and Brown, A. 1998. Orchids of South-West Australia. University of Western Australia Press, Nedlands, Western Australia.

Holländer, S. 1932. Ernährungsphysiologische untersuchungen an wurzelpilzen saprophytisch lebender orchideen, Julius-Maximilian-Universität, Würtzberg cited in Midgley, D. J., Jordan, L. A., Saleeba, J. A. and Mcgee, P. A. 2006. Utilisation of carbon substrates by orchid and ericoid mycorrhizal fungi from Australian dry sclerophyll forests. Mycorrhiza, 16, 175-182..

Hollick, P. S. 2004. Mycorrhizal specificity in endemic Western Australia terrestrial orchids (tribe Diuridae): Implications for conservation. PhD thesis, Murdoch University, Western Australia.

Hopper, S. D. and Brown, A. P. 2007. A revision of Australia's hammer orchids (Drakaea: Orchidaceae), with some field data on species-specific sexually deceived wasp pollinators. Australian Systematic Botany, 20, 252-285.

Huynh, T. T., Mclean, C. B., Coates, F. and Lawrie, A. C. 2004. Effect of developmental stage and peloton morphology on success in isolation of mycorrhizal fungi in Caladenia formosa (Orchidaceae). Australian Journal of Botany, 52, 231-241.

Huynh, T. T., Thomson, R., Mclean, C. B. and Lawrie, A. C. 2009. Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae). Annals of Botany, 1-9.

IGEM 2008. In vivo folate production. California Institute of Technology.

Jabrin, S., Ravanel, S., Gambonnet, B., Douce, R. and Rebeille, F. 2003. One-carbon metabolism in plants. Regulation of tetrahydrofolate synthesis during germination and seedling development. Plant Physiology, 131, 1431-1439.

Jackson, L. E., Burger, M. and Cavagnaro, T. R. 2008. Roots, nitrogen transformations, and ecosystem services. Annu. Rev. Plant Biol. 59, 341-363.

Jafra, S., Figura, I., Hugouvieux-Cotte-Pattat, N. and Lojkowska, E. 1999. Expression of Erwinia chrysanthemi pectinase genes pelI, pelL, and pelZ during infection of potato tubers. Molecular Plant-Microbe Interactions, 12, 845-851.

Jayachandran, K., Schwab, P. and Hetrick, B. A. D. 1992. Mineralization of organic phosphorus by vesicular-arbuscular mycorrhizal fungi. Soil Biol. Biochem, 24.897-903.

Jennings, D. H. 1995. The physiology of fungal nutrition, Cambridge University Press, Cambridge,UK.

Joner, E. J., Ravnskov, S. and Jakobsen, I. 2000. Arbuscular mycorrhizal phosphate transport under monoxenic conditions using radio-labelled inorganic and organic phosphate. Biotechnology Letters, 22, 1705-1708.

Jones, D. L., Kielland, K., Sinclair, F. L., Dahlgren, R. A., Newsham, K. K., Farrar, J. F. and Murphy, D. V. 2009. Soil organic nitrogen mineralization across a global latitudinal gradient. Global Biogeochemical Cycles, 23.

Jones, D. L., Nguyen, C. and Finlay, R. D. 2009. Carbon flow in the rhizosphere: carbon trading at the soil–root interface. Plant Soil, 321, 5-33.

Julou, T., Burghardt, B., Gebauer, G., Berveiller, D., Damesin, C. and Selosse, M. A. 2005. Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium. New Phytologist, 166, 639- 653.

Karandashov, V. and Bucher, M. 2005. Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science 10, 22-29.

Kemmitt, S. J., Wright, D., Murphy, D. V. and Jones, D. L. 2008. Regulation of amino acid biodegradation in soil as affected by depth. Biol Fertil Soils, 44, 933-941.

94 Kieliszewska-Rokicka, B. 1992. Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizai fungus Paxillus involutus cultured in vitro. Plant and Soil 139, 229-238.

Knox, B., Ladiges, P. and Evans, B. 1995. Biology, McGraw-Hill Book Company, Sydney.

Kononova, M. M., Nowakowski, T. Z. and Greenwood, G. A. 1961. Soil organic matter its nature, its role in soil formation and in soil fertility, Pergamon press, Oxford.

Kuperman, R. G. 1999. Litter decomposition and nutrient dynamics in oak hickory forests along a historic gradient of nitrogen and sulfur deposition. Soil Biology and Biochemistry 31, 237- 244.

Leake, J. R. and Miles, W. 1996. Phosphodiesters as mycorrhizal P sources .1. Phosphodiesterase production and the utilization of DNA as a phosphorus source by the ericoid mycorrhizal fungus Hymenoscyphus ericae. New Phytologist, 132, 435-443.

Leake, J. R. and Read, D. J. 1990. Proteinase activity in mycorrhizal fungi. II. The effects of mineral and organic nitrogen sources on induction of extracellular proteinase in Hymenoscyphus ericae (Read) Korf and Kernan. New Phytologist, 116, 123-128.

Leake, J. R. and Read, D. J. (eds.) 1997. Mycorrhizal fungi in terrestrial habitats, Springer-Verlag, Berlin.

Lin, J., Dickinson, D. B. and Ho, T. D. 1987. Phytic acid metabolism in lily (Lilium longiflorum Thunb.) pollen. Plant Physiology, 83, 408-413.

Lindahl, B. O., Taylor, A. F. S. and Finlay, R. D. 2002. Defining nutritional constraints on carbon cycling in boreal forests-towards a less 'phytocentric' perspective. Plant and Soil, 242, 123- 135.

Littke, W. R., Bledsoe, C. S. and Edmonds, R. L. 1984. Nitrogen uptake and growth-in vitro by Hebeloma crustuliniforme and other Pacific Northwest mycorrhizal fungi. Canadian Journal of Botany. 62, 647-652.

Maccheroni, W. and Azevedo, J. L. 1998. Synthesis and secretion of phosphatases by endophytic isolates of Colletotrichum musae grown under conditions of nutritional starvation. J. Gen. Appl. Microbiol., 44, 381-387.

Margesin, R. and Schinner, F. 1994. Phosphomonoesterase, phosphodiesterase, phosphotriesterase and inorganic pyrophosphatase activities in forest soils in an alpine area: effect of pH on enzyme activity and extractability. Biol. Fertil. Soil, 18.

Marschner, B., Brodowski, S., Dreves, A., Gleixner, G., Gude, A., Grootes, P. M., Hamer, U., Heim, A., Jandl, G., Ji, R., Kaiser, K., Kalbitz, K., Kramer, C., Leinweber, P., Rethemeyer, J., Schaffer, A., Schmidt, M. W. I., Schwark, L. and Wiesenberg, G. L. B. 2008. How

95 relevant is recalcitrance for the stabilization of organic matter in soils? J. Plant Nutr. Soil, 171, 91-110.

McDowell, R. W., Condron, L. M., Stewart, I. and Cave1, V. 2005. Chemical nature and diversity of phosphorus in New Zealand pasture soils using 31P nuclear magnetic resonance spectroscopy and sequential fractionation. Nutrient Cycling in Agroecosystems 72, 241-254.

Midgley, D. J., Chambers, S. M. and Cairney, J. W. G. 2004. Inorganic and organic substrates as sources of nitrogen and phosphorus for multiple genotypes of two ericoid mycorrhizal fungal taxa from Woollsia pungens and Leucopogon parviflorus (Ericaceae). Australian Journal of Botany, 52, 63-71.

Midgley, D. J., Chambers, S. M. and Cairney, J. W. G. 2004. Utilisation of carbon substrates by multiple genotypes of ericoid mycorrhizal fungal endophytes from eastern Australian Ericaceae. Mycorrhiza 14, 245-251.

Midgley, D. J., Jordan, L. A., Saleeba, J. A. and Mcgee, P. A. 2006. Utilisation of carbon substrates by orchid and ericoid mycorrhizal fungi from Australian dry sclerophyll forests. Mycorrhiza, 16, 175-182.

Mitchel, D., Williams, K. and Desmond, A. 2002. Swan Coastal Plain 2 (SWA2 – Swan Coastal Plain subregion). Available: http://www.dec.wa.gov.au/.

Murphy, A. M., Otto, B., Brearley, C. A., Carr, J. P. and Hanke, D. E. 2008. A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens. The Plant Journal 56, 638-652.

Nardi, S., Concheri, G., Pizzeghello, D., Sturaro, A., Rella, R. and Parvoli, G. 2000. Soil organic matter mobilization by root exudates. Chemosphere 41, 653 -658.

Nehls, U. and Hampp, R. D. 2000. Carbon allocation in ectomycorrhizas. Physiological and Molecular Plant Pathology 57, 95-100.

Norkrans, B. 1963. Degradation Of Cellulose. Annu. Rev. Phytopathol, 1, 325-350. Paul, E. A. and Clark, F. E. 1996. Soil microbiology and biochemistry, Academic press, California.

Otero, J. T., Ackerman, J. D., and Bayman, P. (2002) Diversity and host specificity of endophytic rhizoctonia-like fungi from tropical orchids. American Journal of Botany, 89, 1852-1858.

Peakall, R. 1990. Responses of male Zaspilothynnus trilobatus Turner wasps to females and the sexually deceptive orchid it pollinates. Functional Ecology, 4, 159-167.

Perotto, S., Coisson, J. D., Perugini, I., Cometti, V. and Bonfante, P. 1997. Production of pectin- degrading enzymes by ericoid mycorrhizal fungi. New Phytologist, 135, 151-162.

96 Peterson, R., Y, U. and C, Z. 1998. Fungal symbioses with orchid protocorms Symbiosis, 25, 29-55.

Quastel, J. H. 1965. Soil metabolism. Ann. Rev. Plant Physiol. 16, 217-240.

Raghothama, K. G. and Karthikeyan, A. S. 2005. Phosphate acquisition. Plant and Soil 274, 37-49.

Ramsay, R. R., Dixon, K. W. and Sivasithamparam, K. 1986. Patterns of infection and endophytes associated with Western Australian Orchids. Lindleyana, 1, 203-214.

Rasmussen, H. N. 2002. Recent developments in the study of orchid mycorrhiza. Plant and Soil, 244, 149-163.

Read, D. J. and Perez-Moreno, J. 2003. Mycorrhizas and nutrient cycling in ecosystems - a journey towards relevance? New Phytologist, 157, 475-492.

Rosling, A., Landeweert, R., Lindahl, B. D., Larsson, K. H., Kuyper, T. W., Taylor, A. F. S. and Finlay, R. D. 2003. Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytologist, 159, 775-783.

Rouifed, S., Handa, I. T., David, J. F. and Hattenschwiler, S. 2010. The importance of biotic factors in predicting global change effects on decomposition of temperate forest leaf litter. Oecologia, 163, 247-256.

Sawyer, N. A., Chambers, S. M. and Cairney, J. W. G. 2003. Utilisation of inorganic and organic nitrogen sources by Amanita species native to temperate eastern Australia. Mycological Research, 107, 413-420.

Sawyer, N. A., Chambers, S. M. and Cairney, J. W. G. 2003. Utilisation of inorganic and organic phosphorus sources by isolates of Amanita muscaria and Amanita species native to temperate eastern Australia. Australian Journal of Botany, 51, 151-158.

Sawyer, N. A., Chambers, S. M. and Cairney, J. W. G. 2003. Variation in nitrogen source utilisation by nine Amanita muscaria genotypes from Australian Pinus radiata plantations. Mycorrhiza, 13, 217-221.

Scott, J., Rebeille, F. and Fletcher, J. 2000. Folic acid and folates: the feasibility for nutritional enhancement in plant foods. Journal of the Science of Food and Agriculture, 80, 795-824.

Sharples, J. M. and Cairney, J. W. G. 1997. Organic nitrogen utilization by an unidentified mycobiont isolated from mycorrhizas of Pisonia grandis N utilization by the Pisonia mycobiont. Mycol. Res. 101, 315-318.

Smith, S. E. 1966. Physiology and ecology of orchid mycorrhizal fungi with reference to seedling nutrition. New Phytologist 65 (4) 488-499.

97 Smith, S. E. 1967. Carbohydrtae translocation in orchid mycorrhizal fungi. New Phytologist, 66, 371-378.

Smith, S. E. and Read, D. J. 1997. Mycorrhizal symbiosis, London, Academic Press.

Sparks, D. L. 1999. Soil physical chemistry. 2nd edition, CRC Press, Boca Raton.

Steffen, K. T., Cajthaml, T., Snajdr, J. and Baldrian, P. 2007. Differential degradation of oak (Quercus petraea) leaf litter by litter-decomposing basidiomycetes. Research in Microbiology 158, 447-455.

Stephen, R. C. and Fung, K. K. 1971. Nitrogen requirements of the fungal endophytes of Arundina chinensis. Canadian Journal of Botany, 49, 407-410.

Stephen, R. C. and Fung, K. K. 1971. Vitamin requirements of the fungal endophytes of Arundina chinensis. Canadian Journal of Botany, 49, 411-415.

Swarts, N. 2007. Integrated conservation of the rare and endangered terrestrial orchid Caladenia huegelii H. G. Reichb. . PhD. Thesis. The University of Western Australia. Western Australia.

Swarts, N. D. and Dixon, K. W. 2009. Terrestrial orchid conservation in the age of extinction. Annals of Botany, 104, 543-556.

Taylor, D. L., Leake, J. R., Quirk, J., Hardy, K., Banwart, S. A. and Beerling, D. J. 2009. Biological weathering and the long-term carbon cycle: integrating mycorrhizal evolution and function into the current paradigm. Geobiology, 7, 171-191.

Tedersoo, L., Pellet, P., Ljalg, U. K. and Selosse, M. 2007. Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae. Oecologia, 151, 206-217.

Tibbett, M., Sanders, F. E., Minto, S. J., Dowell, M. and Cairney, J. W. G. 1998. Utilization of organic nitrogen by ectomycorrhizal fungi (Hebeloma spp.) of arctic and temperate origin. Mycological Research, 102, 1525-1532.

Todar, K. 2008. Todar's Online Textbook of Bacteriology. http://www.textbookofbacteriology.net/.

IUCN. 2001. IUCN red list categories:version 3.1., Gland, Switzerland, IUCN.

Valaskova, V. and Baldrian, P. 2006. Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases. Microbiology 152, 3613-3622.

Valaskova, V., Snajdr, J., Bittner, B., Cajthaml, T., Merhautova, V., Hofrichter, M. and Baldrian, P. 2007. Production of lignocellulose-degrading enzymes and degradation of leaf litter by

98 saprotrophic basidiomycetes isolated from a Quercus petraea forest. Soil Biol. Biochem, 39, 2651-2660. Warcup, J. H. & Talbot, P. H. B. (1967) Perfect states of rhizoctonias associated with orchids. New Phytologist, 66, 631-&.

Warren, R. A. J. 1996. Microbial hudrolysis of polysaccharides. Annu. Rev. Microbiol., 50, 183- 212.

Whittaker, S. P. and Cairney, J. W. G. 2001. Influence of amino acids on biomass production by ericoid mycorrhizal endophytes from Woollsia pungens (Epacridaceae). Mycological Research, 105, 105-111.

Wright, M., Cross, R., Dixon, K., Huynh, T., Lawrie, A., Nesbitt, L., Pritchard, A., Swarts, N. and Thomson, R. 2009. Propagation and reintroduction of Caladenia. Australian Journal of Botany, 57, 373-387.

99 Appendix I

Formulae for media (Swarts, 2007) 1. Oatmeal Agar (OMA) For 2 L: 5 g crushed oats 2 L reverse osmosis water 16 g agar The crushed oats and agar were put into the reverse osmosis water to make up 2 L media. The solution were mixed well and pH was adjusted to 5.5 by adding hydrochloric acid (HCl) or potassium hydroxide (NaOH). The solution was microwaved for 15 minutes to dissolve the agar, stirred every 5 min. The media were poured into four 500 mL glass stock bottles and autoclaved for 20 min at 121º C at 1.05 kg/cm2 15-20 psi. The bottles were placed in a hot water bath until cool enough to handle then media were pourud into Petri dishes under sterile conditions. 2. Soil Solution Equivalent (SSE) media with streptomycin sulfate For 2 L: 0.4 g MES buffer 1400 mL reverse osmosis water 200 mL of stock of solution A, B, and C 4 g sucrose 16 g agar The MES buffer was added to the reverse osmosis water. Each stock solution was measured and poured separately into the water with the MES buffer. Sugar and agar were added to the media and pH was adjusted to 5.5 by adding HCl or KOH. The media were microwaved to dissolve the agar, stirred every 15 min then the media were poured into 500 mL glass bottles, autoclaved for 20 min at 121º C at 1.05 kg/cm2 15-20 psi. Following autoclaving, bottles were placed in hot water bath until cool enough to handle. 5 mL of streptomycin sulfate was added per 500 mL bottle using a syringe and sterile 0.22 μm Millipore filter attachment, mix well and pour media into Petri dishes under sterile conditions.

Stock solutions Three stock solutions were prepared by weighing the quantities of salts in the table and placing them into stock bottles (table 1). 1 L reverse osmosis water was added and

100 mixed well. Separate stocks are required to avoid reactions. Stocks will make 10 L of media and should be stored in a refrigerator between users.

Table 1 Stock solutions for SSE media Common name g/L g/500 mL STOCK A

NH4NO3 Ammonium nitrate 0.4 0.2

KH2PO4 Potassium dihydrogen orthophosphate 0.0136 0.0068

MgCl.6H2O Magnesium chloride 0.61 0.305 NaCl Sodium chloride 0.058 0.029

STOCK B

CaSO4.2H2O Calcium sulphate 0.861 0.4305

STOCK C FeEDTA (Na) Iron 0.073 0.0365

3. Potato Dextrose Agar (PDA) For 2 L: 13.6 g PDA powder 12 g agar 2000 mL reverse osmosis water PDA powder and agar was added into 2000 mL reverse osmosis water in a microwave proof jug. pH was adjusted to 6.8. The solution was microwaved until the agar dissolve and poured into 500 mL media bottles before autoclaving 20 min at 121º C at 1.05 kg/cm2 15-20 psi. The bottles were placed in a hot water bath until cool enough to handle then pour into Petri dishes under sterile conditions.

101 Appendix II Calculation of carbon, nitrogen, and phosphorus sources to be added into CN MMN liquid media

Carbon calculation Hidrogen carbon sources to be added No Carbon sources Carbon C (H) Oxigen (O) FW % of C total C into liquid media (g) in liquid no of FW no of FW no FW media C (C) Total H (H) Total of O (O) Total (g/l) g/1000ml g/500 ml 1 Glucose (C6H12O6) 6 12 72 12 1 12 6 16 96 180 0.4 2 5 2.5 2 arabinose (C5H10O5) 5 12 60 10 1 10 5 16 80 150 0.4 2 5 2.5 3 tannic acid (C76H52O46) 76 12 912 52 1 52 46 16 736 1700 0.53647 2 3.72807 1.864035 4 cellulose (C6H10O5)n 6 12 72 10 1 10 5 16 80 162 0.44444 2 4.5 2.25 5 galactose (C6H12O6) 6 12 72 12 1 12 6 16 96 180 0.4 2 5 2.5 6 cellobiose (C12H22O11) 12 12 144 22 1 22 11 16 176 342 0.42105 2 4.75 2.375 7 pectin (C6H10)7) 6 12 72 10 1 10 7 16 112 194 0.37113 2 5.388889 2.694444 8 xylan (C5H10O5)n 5 12 60 10 1 10 5 16 80 150 0.4 2 5 2.5 9 mannose (C6H12O6) 6 12 72 12 1 12 6 16 96 180 0.4 2 5 2.5 rhamnose 10 (C6H12O5.H2O) 6 12 72 14 1 14 6 16 96 182 0.3956 2 5.055556 2.527778 11 starch(C6H10O5)n 6 12 72 10 1 10 5 16 80 162 0.44444 2 4.5 2.25 12 CMC(C6H12O6)n 6 12 72 12 1 12 6 16 96 180 0.4 2 5 2.5

FW= formula weight

102

Nitrogen calculation

total N in Nitrogen sources to be added Amount of liquid into liquid media glucose media Total C (g/1000ml) No Nitrogen sources FW N % N (g/l) g/1000 ml g/500 ml C %C total C glucose 1 (NH4)2HPO4 132.07 28 0.212009 0.053002 0.25 0.125 0.4 2 5 2 (NH4)2SO4 132.14 28 0.211896 0.053002 0.2501316 0.12506579 2 5 3 NaNO3 84.9947 14 0.164716 0.053002 0.3217778 0.1608889 2 5 4 Ca(NO3)2 164.088 28 0.17064 0.053002 0.3106069 0.15530343 2 5 organic nitrogen 4.659 1 Alanine (C3H7NO2) 89.09 14 0.157144 0.053002 0.337282 0.16864101 36 0.404086 0.136291 1.863709143 4.716 2 glutamine (C5H10N2O3) 146.14 28 0.191597 0.053002 0.2766326 0.13831629 60 0.410565 0.113576 1.886424286 4.773 3 histidine (C6H9N3O2) 155.15 42 0.270706 0.053002 0.1957919 0.09789596 72 0.464067 0.090861 1.909139429 4.773 4 Glycine (C2H5NO2) 75.07 14 0.186493 0.053002 0.2842043 0.14210215 24 0.319702 0.090861 1.909139429 aspartic acid 4.546 5 (C4H7NO4) 133.1 14 0.105184 0.053002 0.5038976 0.25194879 48 0.360631 0.181721 1.818278857 glutamic acid 4.432 6 (C5H9NO4) 147.13 14 0.095154 0.053002 0.5570132 0.27850658 60 0.407803 0.227151 1.772848571 4.83 7 arginine (C6H14N4O2) 174.2 56 0.32147 0.053002 0.1648741 0.08243704 72 0.413318 0.068145 1.931854571 BSA (Leake and Miles, 4.3666 8 1996) 0.331 4.432 10 Proline (C5H9NO2) 115.13 14 0.121602 0.053002 0.4358657 0.21793287 60 0.52115 0.227151 1.772848571 4.773 11 Asparagine (C4H8N2O3) 132.12 28 0.211929 0.053002 0.2500937 0.12504686 48 0.363306 0.090861 1.909139429 FW= formula weight

Amino acids also contain carbon (C), and the total of glucose as the carbon source in N treatments was calculated based on the proportion of C in each amino acid.

103

Phosphorus calculation

N Na or Hidrogen Phosphorus o Phosphorus sources C (H) Oxigen (O) (P) FW % of P total P F amount of P No of W in liquid sources to be added Na or F Tot no FW Tot no of (O Tot no FW media in liquid media (g) C W al of H (H) al O ) al of P (P) Total (g/l) g/1000ml

1 NaH2HPO4.2H2O Na=1 22 22 7 1 7 6 16 96 1 31 31 156 0.198718 0.06 0.3

2 phytic acid (C6H18O24P6) C=6 12 72 18 1 18 24 16 384 6 31 186 660 0.281818 0.06 0.212903 DNA (calculation adapted 3 from Midgley, 2004) 0.06 0.6605

Na=sodium C= Carbon FW= formula weight

104