Chiang Mai J. Sci. 2014; 41(2) 275

Chiang Mai J. Sci. 2014; 41(2) : 275-285 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Culture Conditions and Some Properties of Pure Culture of Ectomycorrhizal , Scleroderma sinnamariense Jariwat Siri-in, Jaturong Kumla, Nakarin Suwannarach and Saisamorn Lumyong* Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 14 September 2012 Accepted: 22 April 2013

ABSTRACT Scleroderma are ectomycorrhizal fungi which have a worldwide distribution. In 2010, basidiomes of Scleroderma sp. CMUS01 were collected from northern Thailand and pure cultures were obtained. Samples were identified based on morphology and molecular characteristics as Scleroderma sinnamariense. In this paper we report the optimum in vitro culture conditions of S. sinnamariense. Among ten culture media tested, fungal-host agar was the best medium for the mycelial growth and biomass yield. The fungus was able to grow at temperatures ranging from 20-35°C, with an optimal growth temperature of 30°C. The optimal pH for mycelial growth was 5.0. In addition, this strain produced indole-3-acetic acid and siderophore in pure culture. This report provides valuable information for S. sinnamariense mycelial cultivation in Thailand.

Keywords: cultivation, puffball mushroom, indole-3-acetic acid, siderophore

1. INTRODUCTION It is estimated that there are between Rhizopogon, Scleroderma and Suillus [3, 5]. It is 7,000 and 10,000 ectomycorrhizal fungi difficult to cultivate ectomycorrhizal fungi species in the world [1]. Ectomycorrhizal fungi because of a lack of understanding of can enhance water and nutrient uptake and the general ecological and physiological produce metal chelating compounds, such as requirements. The physiological factors such siderophore and organic acid. These promote as nutrition, pH, temperature and water stress plant growth by producing phytohormones on pure cultures of ectomycorrhizal fungi such as indole-3-acetic acid (IAA), cytokinins, can be controlled in the laboratory [5-8]. gibberirin-like substance, increase tolerance to The fungal Scleroderma is an environmental stresses and protect against ectomycorrhizal fungus belonging to pathogens [2-4]. Many ectomycorrhizal fungi the , order , family have been cultivated into pure culture such as [9]. It exists worldwide in Amanita, , Cortinarius, Hebeloma, Laccaria, temperate and tropical regions with host plants Lactarius, Laccinum, Phlebopus, Pisolithus, in the families Caesapinioideae, Dipterocarpaceae, 276 Chiang Mai J. Sci. 2014; 41(2)

Fagaceae, Myrtaceae, Phyllanthaceae and Pinaceae from dried basidiomes extracted according [10, 11]. Scleroderma species have been used to to a CTAB method [8]. The internal increase the growth of tree seedlings, both in transcribed spacer (ITS) regions of the RNA nurseries and the field [12]. This study gene were amplified by polymerase chain evaluated the culture conditions for mycelial reaction (PCR) with ITS4 and ITS5 primers growth in pure culture of S. sinnamariense. under the flowing thermal condition: 94°C In addition, fungal identification was based for 2 min; 35 cycles of 95°C for 30 s, 50°C on morphological and physiological for 30 s, 72°C for 1 min and 72°C for characteristics. Moreover, some properties 10 min. The PCR products were then checked such as IAA and siderophore production of on 1% agarose gels stained with ethidium pure culture of this fungus were investigated. bromide and visualized under UV light. The results of this study can help select suitable PCR products were purified using the PCR conditions to support mycelial growth and clean up gel extraction NucleoSpin® mycelial inocula production of selected Extract II Purification Kit (Macherey-Nagel, fungi for application and management of Germany Catalog no. 740 609.50) following the plant inoculation programs in Thailand. the manufacturer’s protocol. The purified PCR products were directly sequenced. 2. MATERIALS AND METHODS Sequencing reactions were preformed and 2.1 Fungal Strain the sequences were automatically determined Basidiomes of wild ectomycorrhizal in the genetic analyzer (1ST Base, Malaysia) fungus, Scleroderma sp. CMUS01 was collected using the PCR primers mentioned above. from the Medicinal Plant Garden, Doi Suthep- Sequences were used to query GenBank via Pui National Park, Chiang Mai Province, BLAST (http://blast.ddbj.nig.ac.jp/top- Thailand in July, 2010 and kept at the Research e.html). For the phylogenetic analysis, a Laboratory for Excellence in Sustainable multiple aliment subroutine in Clustal X [15] Development of Biological Resource. A small and a maximum parsimony analysis using the piece of mycelium was aseptically removed PAUP beta 10 software version 4.0 [16] were from inside the basidiome and transferred conducted. on to modified Melin Norkrans (MMN) medium. The mycelial were isolated from 2.3 Fungal Cultivation basidiomes and cultivated in MMN medium. Twenty five milliliters of culture media The plates were incubated at 30°C in the dark. for each experiment were poured into Petri The mycelial emerging from tissue were dishes after autoclaving for 15 min at 121°C. picked and transferred to MMN medium. A cellophane disc (9.0 cm diam) was The pure cultures were kept on MMN weighted, sterilized and placed on the medium slants for further use. Duplicate surfaces of the tested media. Mycelial plugs cultures were kept in both sterile distilled with 5 mm diameter were obtained from water at 4°C and 20% glycerol at -20°C for 21-day-old cultures at 30°C on MMN long-term preservation. medium and transferred to the tested media. The inoculated plates were sealed with 2.2 Fungal Identification Parafilm after inoculation and incubated at Basidiomes were identified based on 30°C for 21 days. Colony diameter at three morphology (macroscopic and microscopic weeks after inoculation was measured. characteristics) [13, 14] and genomic DNA Mycelial dry weight was obtained after drying Chiang Mai J. Sci. 2014; 41(2) 277

at 60°C overnight and maintained in CMUS01 was determined according to the desiccators for 20 min. The final net weights method of Niemi et al. [24]. Mycelial plugs, of fungal biomass were calculated. Three each 5 mm in diameter and cut from the replications were made for each treatment. margin of the fungal colony, were transferred to 5 mL MMN liquid medium containing 2.4 Effect of Cultivation Media on 2 mg/mL of L-tryptophan. The culture was Mycelial Growth incubated at 30°C with shaking at 125 rpm The following ten different media for 30 days and then harvested by were used in this experiment: Ammonium centrifugation at 11,000 rpm for 15 min. choloride glucose agar [17], Fungal-host agar One milliliter of the supernatant was mixed [18], Heli medium [17], Hagem’s medium with 2 mL of Salkowski ’s reagent; the [19], Melin-Norkans medium [20], G-MMN appearance of a pink color indicated IAA medium [21], L-MMN medium [22], production. Optical density (OD) was read modified Murashige and Skoog medium at 530 nm. The level of IAA produced was [23], malt extract agar [8], and modified SH estimated against the IAA standard. medium [22], All tested media were adjusted to pH 6.0 with 1N HCl or 1N NaOH. The 2.8 Determination of Siderophores inoculated plates were incubated at 30°C in Production darkness for three weeks. The screening siderophore production used the CAS assay [25]. The culture was 2.5 Effect of Temperature on Mycelial inoculated on CAS agar and incubated in Growth the dark at 30°C for 30 days. The colonies In this experiment, cultures of producing a yellow, orange, purple or S. sinnamariense on fungal-host agar, pH 5.6 red zone were considered siderophore (the best medium for mycelial growth) [8] production. Consequently, to provide a were incubated in the dark at 20, 25, 30, 35, better interpretation, the relative index of 40 and 45°C. After 21 days of inoculation, produced activity was calculated as the ratio colony diameters and dry weight were of diameter (mm) of the clear zone to that measured. of the ectomycorrhizal fungi colony and was used for this comparative analysis. 2.6 Effect of pH on Mycelial Growth Pure culture of S. sinnamariense was 2.9 Statistical Analysis inoculated on fungal-host agar with pH Analysis of variance was used to ranging from 4.0-9.0. The pH was adjusted determine the significant differences among with various buffers before autoclaving [5], mean values between treatments, and Tukey’s and the inoculated plates were incubated in test (P<0.05) was used for significance the dark at 30°C (the best temperature for between treatments. The software of SPSS mycelial growth). Colony diameters and dry (version 16.0) was applied. weight were measured after 21 days of incubation. 3. RESULTS AND DISCUSSION 3.1 Fungal Identification 2.7 Determination of Indole-3-Acetic The morphological features of Acid (IAA) Production the basidiomes suggested that the fungal The production of IAA by S.sinnamariense strain CMUS01 was S. sinnamariense, an 278 Chiang Mai J. Sci. 2014; 41(2)

ectomycorrhizal fungi which found chrome-yellow. Gleba white when young in Southeast Asia [14, 26]. Basidiomes globose become dark brown to back at maturity to subglobose 21.0-64.0 mm diameter, 10.0- and pulverulent. Basidiospores globose 20.0 mm height with basal rhizomorph. to subglobose with short spines, (Figure 1, A and B). Peridium 0.8-1.5 mm 4.18-6.15 μm diameter (n = 50) (Figure 1, thick when fresh, leathery, surface partially C and D), yellowish brown in water smooth with scattered, small and thin dark or KOH and not changing in Melzer’s brown to back scales, lemon-yellow to reagent.

Figure 1 Scleroderma sinnamariense CMUS01 A, B. basidiomes. C. basidiospores under compound light microscrope. D. basidiosopre under scanning electron microscope. Bar. A, B = 20 mm, C = 10 μm and D = 2 μm.

The ITS sequence of S. sinnamariense Scleroderma bovista, S. citrinum, S. containing 555 bp, was deposited in meridionale, S. michiganense, S. patagonicum GenBank as HQ687222 and analyzed and S. septentrionale. Clade 2 comprised phylogenetically with 18 sequences of with S. areolatum, S. bermudense, S. cepa, Scleroderma species and the outgroup S. laeve, S. polyrhizum and S. verrucosum. (Pisolithus albus and P. microcarpus) Scleroderma sinnamariense was placed obtained from the GenBank database. within clade 3 with 100% bootstrap Heuristic searches resulted in a tree length support. Our analysis showed that the of 703 steps, CI = 0.723, RI = 0.827, RC separation into clades of recticulate spore = 0.598 and HI = 0.277. One of the species and spiny or subrecticulate spore maximum-parismony trees is shown in species had 67% bootstrap support. Figure 2. A phylogenetic dendrogram This result was similar to previous separated Scleroderma fungi into 3 clades. molecular phylogenetic studies in that Clade 1 constituted species with recticulate Scleroderma fungi were morphologicalls spores, while the other clades included separated into two groups which were the species with spiny or subrecticulate moderately to well supported by molecular spores. Clade 1 included six species, analysis [26, 27]. Chiang Mai J. Sci. 2014; 41(2) 279

Figure 2 A maximum parsimonious tree inferred from a heuristic search of the internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 sequence alignments of 21 sequences. Pisolithus albus and P. microcarpus were used to root the tree. Branches with bootstrap values ≥ 50% are shown an earch branch and bar respresents 10 showed substitutions per nucleotide position.

3.2 Effect of Cultivation Media for found on G-MMN medium (15.10±0.04 Mycelial Growth mm) and Modified SH medium (2.60±0.95 Mycelial growth and biomass yield of mg), respectively. Generally, growth of S. sinnamariense on ten different growth media ectomycorrhizal fungi in pure culture were is presented in Figure 3, A and B. After three greatly influenced by changes to the weeks of incubation, the fungus grew best cultivation media. For example, Kalm and on fungal-host agar (popular medium for Kalyoncu [23] reported that potato dextrose tissue culture of conifers), followed by agar and malt extract agar were the most modified Murashige and Skoog medium and suitable media for mycelial growth of MMN medium. These three media were Morchella spp. Pure culture of Phlebopus modified for cultivation the ectomycorrhizal portentosus grows best on fungal-host agar fungi and they were not need the plant and modified Murashige and Skoog medium material for preparation [18, 23]. For all [5, 8]. In addition, Xu et al., [17] reported tested media the fungal colonies exhibited that L. insulsus and Boletus edulis grew a yellow color and clamp connections best on potato juice iron-magnesium agar, were observed under a light microscope. while Lactarius deliciosus grew best on pine juice The statistical analysis showed that the iron-magnesium agar. In this study, mycelia highest colony diameter (43.5±0.36 mm) and of S. sinnamariense grew best in fungal-host biomass yield (14.27±0.86 mg) were observed agar which has a high nutritional content on fungal-host agar. While, the smallest and vitamins. The experimental results colony diameter and biomass yield was were similar with previous reports in that 280 Chiang Mai J. Sci. 2014; 41(2)

ectomycorrhizal fungi need high nutrition better in medium which contains natural for mycelial growth and they can grow components and vitamins [8, 17].

Figure 3 Growth of Scleroderma sinnamariense on different culture media. A. colony diameter and B. biomass yield. HI = Heli medium, ACG = amonium choloride glucose agar, MMN = Melin-Norkans medium, FH = fungal-host agar, MEA = malt extract agar, modified MS = Murashige and Skoog medium, HG = Modifide Hagem′s medium, G-MMN = G-MMN medium, L-MMN = L-MMN medium and SH = modified SH medium.

3.3 Effect of Temperature on Mycelial et al. [8], who reported the maximum biomass Growth yield of P. portentosus was observed on Temperature had a significant effect fungal-host medium at 30°C. However, on the growth of S. sinnamariense. The fungus the optimum temperature for the pure was able to grow in temperatures ranging culture growth of ectomycorrhizal fungi from 25-35°C (Figures 4, A and B). depended on fungal species. For example, The statistical analysis indicated that 30°C Sanchez et al. [6] reported that most of was the best temperature for mycelial the ectomycorrhizal fungi species from growth which showed the largest colony Mediterranean forest showed the greatest diameter (56.6±0.04 mm) and biomass mycelial growth at 23°C. While, Xu et al. [17] yield (41.33±0.98 mg). After three weeks of reported that 25°C was the optimum incubation, the fungal mycelia did not temperature for growth of L. deliciosus develope at 40 and 45°C and failed to L. insulsus, whereas B. edulis grew best at resume growth after an additional week 28°C. Furthermore, pure culture of Amanita incubation at 30°C. These results were caesarea has an optimum growth temperature according to Sanmee et al. [5] and Kumla at 24-28°C [7]. Chiang Mai J. Sci. 2014; 41(2) 281

Figure 4 Growth of Scleroderma sinnamariense CMUS01 on fungal-host agar pH 5.6, at different temperature for 30 days. Colony diameter (A) and biomass yield (B).

3.4 Effect of pH on Mycelial Growth example, B. edulis and Hebeloma sp. showed The mycelial growth in different the largest colony diameters at pH 5.0 pH values is shown in Figure 5, A and [17, 29]. However, the optimum pH value B. Scleroderma sinnamariense mycelia had the for ectomycorrhizal mycelial growth is not ability to grow at a pH range of 2.0-9.0. homologous for the ectomycorrhizal fungi The pH 5.0 was the optimum pH value and species [6]. For example, optimal pH of P. produced the largest colony diameter (40.37± portentosus was 4.0 [5, 8]. Hebeloma vinosophyllum, 1.00 mm) and biomass yield (49.70±0.05 mg). Laccaria bicolor, and L. deliciosus grow best at The smallest colony diameter was observed pH 6.0 [17, 28]. Amanita caesarea had the largest at pH 2.0 (21.33±0.58 mm) and biomass radial growth at pH 6.0-7.0 [7]. However, yield (19.20±0.00 mg). The result was similar some species favor neutral to slightly alkaline to several studies that ectomycorrhizal conditions such as H. edurum, L. insulsus and fungi favor acidic conditions [28, 29]. For Suillus collinitus [17, 30]. 282 Chiang Mai J. Sci. 2014; 41(2)

Figure 5 Colony diameter (A) and biomass (B) of Scleroderma sinnamariense on fungal-host agar with different pH value at 30°C for 30 days.

3.5 Determination of Indole-3-Acetic 3.6 Determination of Siderophore Acid (IAA) Production Production After three weeks of incubation, the In this study, S. sinnamariense CMU S01, supernatant of S. sinnamariense produced IAA was grown on CAS agar. Under such yielding 8.13±0.06 μM/L. This result is conditions, the fungus produced a light similar to several ECM fungi [24, 31], for yellow zone around the colony which example, P. tinctorius or Paxillus involutus indicated siderophore production. This produce IAA in vitro and IAA from both result is similar to Machuca [26] who fungi increased rooting and subsequent root reported that pine ectomycorrhizal growth on Scots pine hypocotyl cuttings fungi, S. luteus, Rhizopogon luteolus and S. in vitro [32, 33]. verrucosum produced iron-chelating compounds including siderophore in culture. In addition, iron concentration Chiang Mai J. Sci. 2014; 41(2) 283

was reported as the key factor regulating [3] Brundrett M., Bougher N., Dell B., siderophores production by ectomycorrhizal Grove T. and Malajczuk N., Working fungi [33-36]. Further, Haselwandter with in Forestry and and Winkelmann [37] reported that Agriculture, ACIAR Monograph, ectomycorrhizal leads to greater Camberra, 1996. Fe uptake rates by a range of different host [4] Smith S.E. and Read D.J., Mycorrhizal plants. Symbiosis, 2nd ed. Academic Press London, 1997. 3. CONCLUSIONS [5] Sanmee R., Lumyong P., Dell B. and Scleroderma sinnamariense grew best Lumyong S., In Vitro cultivation and on fungal-host agar which contained a high fruit body formation of black bolete, nutritional and vitamin content. The Phlebopus portentosus, a popular edible fungus was able to produce IAA and ectomycorrhizal fungus in Thailand, siderophore in pure culture. The optimum Mycoscience, 2010; 51: 15-22. temperature and pH values for the mycelia [6] Sanchez F., Honrubia M. and Torres were 30°C and pH 5.0 these factors well P., Effects of pH, water stress and supported mycelial growth, respectively. temperature on in vitro cultures These results can be used for mycelial of ectomycorrhizal fungi from inocula production of useful , Mediterrannean forests, Cryptogamie and eventually for the management of Mycol., 2001; 22: 243-258. eucalypthus seedlings inoculated with [7] Daza A., Manjon J.L., Camacho M., ectomycorrhizal fungi for greenhouse and Romero de la Osa, L., Aguilar A. and field inoculation programs in Thailand. Santamaria, C., Effect of carbon and nitrogen sources, pH and temperature ACKNOWLEDGEMENTS on in vitro culture of several isolates This project was supported by grants of Amanita caesarea (Scop.:Fr.) Pers., from the Graduate School and Center of Mycorrhiza, 2006; 16: 133-136. Excellent for Renewable Energy, Chiang [8] Kumla J., Danell E., Bussaban B. and Mai University, the Thailand Research Lumyong S., Suitable growth conditions Fund (IUG5380003) and Tree Tech Co., and nutrition factors on in vitro culture Ltd. We are grateful to Keegan Kennedy of Phlebopus portentosus (Boletales), for improving the English text. Chiang Mai J. Sci., 2011; 38: 156-159. [9] Binder H. and Hibbet D.S., Molecular REFERENCES systematic and biological diversifica- tion of boletales, Mycologia, 2006; 98: [1] Taylor A.F.S. and Alexander I., The 971-981. ectomycorrhizal symbiosis: Life in the real world, Mycologist, 2005; 19: 102- [10] Munyanziza E. and Kuyper T.W., 111. Ectomycorrhizal synthesis on seedings of Afzelia quanzensis Welw. using [2] Shaw L.M., Messier J.M. and Mercurio various types of inoculums, Mycorrhiza, A.M., The activation dependent 1995; 5: 283-287. adhesion of macrophages to laminin involves cytoskeletal anchoring and [11] Sanon K.B., Ba A.M., Delaruelle C., phosphorylation of the a6β1 integrin, Duponnois R. and Matin F., Cell Biol., 1990; 110: 2167-74. Morphological and molecular analyses 284 Chiang Mai J. Sci. 2014; 41(2)

in Scleroderma species associated [19] McLaughlin D.J., Environmental with some Caesalpinioid legumes, control of fruitbody development in Diptrocapaceae and Phyyllanthaceae Boletus rubinellus in axenic culture, tree in southern Burkina Faso, Mycologia, 1970; 62: 307-331. Mycorrhiza, 2009; 19: 571-584. [20] Langer I., Krpata D., Peintner U., [12] Chen Y., Kang L., Malajczuk N. and Wenzel, W.W. and Schweiger P., Dell B., Selecting ectomycorrhizal Media formulation influences in vitro fungi for inoculating plantations in ectomycorrhizal synthesis on the south China: Effect of Scleroderma on European aspen Populus tremula, colonization and growth of exotic Mycorrhiza, 2008; 18: 297-307. Eucalyptus globulus, E. urophylla, Pinus [21] Gafur A., Schutzendubel A., elliottii and P. radiata, Mycorrhiza, Langenfeld-Heyser R., Fritz E. and 2006; 16: 251-259. Polle A., Compatible and incompetent [13] Sims K.P., Watling R. and Jeffries P., Paxillus involutus isolates for A revised key to the genus Scleroderma. ectomycorrhiza formation in vitro Mycotaxon, 1995; LVI: 403-420. with poplar (Populus canescens) differ in H O production, Plant Biol., 2004; [14] Guzman G., Monografia del genero 2 2 6: 91-99. Scleroderma, Darwiniana, 1970; 16: 233-407. [22] Guerin-Laguette A., Vaario L.M., Gill W.M., Lapeyrie F., Matsushita N. [15] Tompsom J.D., Gibson T.J., and Suzuki K., Rapid in vitro Plewniak F., Jeanmougin F. and ectomycorrhizal infection on Pinus Higgins D.G., The Clustal X densiflora roots by Tricholoma windows interface: Flexible strategies matsutake, Mycoscience, 2000; 41: 389- for multiple sequence alignment aided 393. by quality analysis tools, Nucleic. Acids Res., 1997; 24: 4876-4882. [23] Kalm E. and Fatih Kalyoncu F., Mycelial growth rate of some morels [16] Swofford D.L., Phylogenetic Analysis (Morchella spp.) in four different Using Parsimony (* and Other Methods) microbiological media, American- Assosiates, Sunderland, Massachusetts, Eurasian J., 2008; 3: 861-864. 2002. [24] Niemi K.H., Haggman T. and [17] Xu M., Zhu J., Kang H., Xu A., Zhang Sarjala T., Effects of diamines on the J. and Li F., Optimum conditions for interaction between ectomycorrhizal pure culture of major ectomycorrhizal fungi and adventitious root formation fungi obtained from Pinus sylvestris on scots pine in vitro, Tree Physiol., var. mongolica plantations in 2002; 22: 373-381. southeastern Keerqin sandy lands, China, J. Forest. Res., 2008; 19: 113- [25] Machuca A., Pereira G., Aguiar A. 118. and Milagres A.M., Metal-chelating compounds produced by ectomy- [18] Vaario L., Gill W., Tanaka M., Ide Y. and corrhizal fungi collected from pine Suzuki K., Aseptic ectomycorrhizal plantations, Lett. Appl. Microbiol., synthesis between Abies firma and 2007; 44: 7-12. Cenococcum geophilum in artificial culture, Mycoscience, 2000; 41: 395-399. Chiang Mai J. Sci. 2014; 41(2) 285

[26] Phosri C., Martin M.P., Watling R., [32] Sztein A.E., Cohen J.D. and Cooke Jeppson M. and Sihanonth P., T.J., Evolutionary patterns in the Molecular phylogeny and re-assessment auxin metabolism of green plants, Int. of some Scleroderma spp. J. Plant Sci., 2000; 161: 849-859 (Gasteromycetes), Mycol. Res., 2007; [33] Niemi K.M., Salonen A., Ernstsen H., 166: 83-91. Heinonen-Tanski H. and Haggman [27] Nouhra E.R., Caffot M.L.H., Pastor H., Application of ectomycorrhizal N. and Crespo E.R., The species of fungi in rooting of Scots pine fascicular Scleroderma from Argentina, including shoots, Can. J. For. Res, 2000; 30: 1221- a new species from the Nothofagus 1230. forest, Mycologia, 2012; 104: 488-495. [34] Haselwandter K. and Winkelmann G., [28] Dix N.j. and Webster J., Fungal Ferricrocin - an ectomycorrhizal Ecology, Chapman and Hall, London, siderophore of Cenococcum geophilum, 1995. BioMetals, 2002; 25: 73-77. [29] Yamanaka T., The effect of pH on [35] Heymann P., Ernst J.F. and the growth of saprotrophic and Winkelmann G., Identification and ectomycorrhizal ammonia fungi in substrate specificity of a ferichrome- vitro, Mycologia, 2003; 95: 584-589. type siderophore transporter (Arn l p) in Saccharomyces cerevisiae, FEMS. [30] Hung L.L. and Trappe J.M., Growth Microbiol. Lett., 2000; 186: 221-227. variation between and within species of ectomycorrhizal fungi in response [36] Renshaw G.M.C., Kerrisk C.B. and to pH in vitro, Mycologia, 1983; 75: Nilsson G.E., The role of adenosine 234-241. in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum, Comp. [31] Rudawska M.B. and Kieliszewska- Biochem. Physiol., 2002; 131: 133-141. Rokicka B., Mycorrhizal formation by Paxillus involutus in relation to [37] Haselwandter K. and Winkelmann G., their IAA-synthesizing activity, New Siderophores of symbiotic fungi: In Phytologist, 1997; 137: 509-517 Soil Biology- Microbial Siderophores, Berlin Heidelberg, Springer-Verlag, 2007.