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Studies of interactions among cultured ericoid mycorrhizal fungi of salal (gaultheria shallon pursh) Nafar, Firoozeh 1998

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STUDIES OF INTERACTIONS A M O N G CULTURED ERICOID MYCORRHIZAL FUNGI OF S A L A L (GAULTHERIA SHALLONPURSH) by Firoozeh Nafar B.Sc, The University of Tehran, 1989 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A April 1998 © Firoozeh Nafar, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of T^^TCA n ^ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT This thesis compares molecular and cultural methods for the detection and identification of the fungal partners of mixed in vitro mycorrhizae of salal. Mixed in vitro mycorrhizae were synthesized by inoculating salal with pairs of three different fungi in all possible combinations. The fungi were sterile isolates S246 and S9, and Oidiodendron griseum. Mycorrhizae formed after three to four months and the fungi in the mycorrhizal roots were detected by (1) re-isolation and morphological identification of the cultured fungi and (2) direct polymerase chain reaction (PCR) amplification of the ribosomal internal transcribed spacer regions using primers ITS1-F and ITS4, followed by restriction fragment length polymorphism (RFLP) identification. The detection results from the synthesized mycorrhizae were compared with PCR/RFLP detection from mixtures of fungal DNAs at known concentrations. The O. griseum. D N A could not be detected at any concentration when it was combined with D N A from either S9 or S246, indicating a PCR bias against O. griseum. This bias explained why O. griseum never appeared in the RFLP patterns from amplified D N A from synthesized mycorrhizae despite being re-isolated from 71% of the root segments. The RFLPs proved useful in clearly indicating mixtures of the two sterile isolates, especially because mixtures of sterile isolates were difficult to recognize based on morphology. This thesis also compared culture and PCR/RFLP methods to identify ericoid mycorrhizal fungi colonizing mapped roots. Two mycorrhizal fungi often occupied the same 5 mm segment of a salal root. From the roots located under both inocula with the same chance for colonization, 70% of the cultures recovered were mixed cultures when a sporulating and sterile fungi were used as inoculum. The three ericoid mycorrhizal fungi mentioned above were paired in culture to look at their competitive abilities. A l l pairings showed deadlock, an inability of the paired species to invade the territory occupied by the other one. For better observation of the colonized roots within the agar medium, phytagel was substituted by granulated agar. Phytagel improved the clarity of the gel and significantly improved the shoot growth of salal; the percent root colonization did not show any significant difference. T A B L E OF CONTENTS A B S T R A C T ii T A B L E O F C O N T E N T S iii L I S T O F T A B L E S v L I S T O F F I G U R E S vi A C K N O W L E D G E M E N T S viii CHAPTER 1 LITERATURE 1 1.1 LITERATURE REVIEW 1 1.1.1 General mycorrhizae 1 1.1.2 The history of isolation and identification of ericoid mycorrhizae 2 1.1.3 Physiology of ericoid mycorrhizal fungi 6 1.1.4 Culture conditions used for testing the mycorrhizal ability of isolated endophytes 7 1.1.5 Rationale for the present study 8 1.2 O V E R A L L OBJECTIVES 11 1.3 SPECIFIC OBJECTIVES 12 CHAPTER 2: MOLECULAR AND CULTURAL IDENTIFICATION OF ERICOID MYCORRHIZAL FUNGI OF GAULTHERIA SHALLON 13 2.1 INTRODUCTION 13 2.1.1 Isolation techniques and identification of ericoid mycorrhizal fungi 13 2.1.2 Identification by electron microscopy 14 2.1.3 Identification by molecular methods 15 2.1.4 Identification of ectomycorrhizal fungi by direct DNA amplification from roots 15 2.1.5 Identification of vesicular arbuscular mycorrhizae by direct DNA amplification from soil 16 2.1.6 Molecular identification of cultured ericoid mycorrhizae 17 2.2 OBJECTIVES 18 2.3 M A T E R I A L S A N D METHODS 19 2.3.1 General preparation of synthetic soldi mycorrhizae 19 2.3.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae 20 2.3.3 Molecular detection of the fungi in the mixed, synthetic mycorrhizae 21 2.3.4 Competitive PCR between pairedfungal DNA extracts of known concentration 23 2.3.4.1 D N A preparation 23 2.3.4.2 Tests of P C R amplification with artificial mixtures of D N A 23 2.4 RESULTS 24 2.4.1 Observations on ericoid mycorrhiza formation in vitro 24 2.4.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae 24 2.4.3 DNA extraction 26 2.4.4 Results from competitive PCR between paired fungal DNA extracts of known concentration 27 iii 2.5 DISCUSSION 2 7 2.5.1 Recovery of fungal mixtures from 5 or 10 mm root segments 27 2.5.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae 28 2.5.3 Culturing bias against sterile isolates 29 2.5.4 DNA extraction 30 2.5.5 PCR amplification bias against Oidiodendron griseum 30 2.6 CONCLUSION 31 CHAPTER 3: IN VITRO COMPETITIVE STUDIES AMONG THREE ERICOID MYCORRHIZAL FUNGI OF GAULTHERIA SHALLON 55 3.1 INTRODUCTION 55 3. 2 M A T E R I A L S A N D M E T H O D S 57 3.2.1 Fungal isolates 57 3.2.2 Dual cultures 57 3.2.3 Observations and measurements 58 RESULTS 59 3.4 DISCUSSION 61 3.4.1 Deadlock 61 3.4.2 My eel ial expansion 61 CHAPTER 4: OPTIMIZATION OF GELLING AGENT CONDITIONS 68 4.1 INTRODUCTION 68 4.2 M A T E R I A L S A N D METHODS 70 4.2.1 Media and Random sampling 70 4.2.2 Percent root colonization 70 4.2.3 Shoot dry weight. 71 4.3 RESULTS 71 4.4 DISCUSSION 72 CHAPTER 5: GENERAL CONCLUSIONS AND DISCUSSION 75 REFERENCES 79 APPENDICES 86 APPENDIX A. l Measurements of mycelial growth Oidiodendron griseum (O.g) away from interacting colonies.. 86 APPENDIX A.2 Measurements of mycelial growth Oidiodendron griseum (O.g) towards interacting colonies 87 APPENDIX A.3 Tukey test for Mycelial growth of'Oidiodendron griseum (O.g) towards interacting colonies... 88 APPENDIX B. 1 Measurements of mycelial growth of S246 away from interacting colonies 89 APPENDIX B.2 Measurements of mycelial growth of S246 towards interacting colonies 90 APPENDIX B.3 Tukey test for mycelial growth of S246 towards interacting colonies 91 APPENDIX C. 1 Measurements of mycelial growth of S9 away from interacting colonies 92 APPENDIX C.2 Measurements of mycelial growth of S9 away from interacting colonies 93 APPENDIX C.3 Tukey test of mycelial growth of S9 towards interacting colonies 94 APPENDIX C.4 Tukey test of mycelial growth of S9 away from interacting colonies 95 APPENDIX D. 1 Measurements of shoot dry weight of salal seedlings in culture 96 APPENDIX D.2 Measurements of perecent root colonization of salal seedlings in culture 97 LIST OF TABLES Table 2.1 Fungal isolates used in this study, source of isolate, mycorrhizal 33 status and isolation data. Table 2.2 Various concentrations of O. griseum D N A in pure and mixed solutions with S9 DNA. 33 Table 2.3 Various concentrations of O. griseum D N A in pure and mixed solutions with S246 DNA. 34 Table 2.4 Various concentrations of S9 D N A in pure and mixed solutions with S246 D N A . 34 Table 2.5 Identified ericoid mycorrhizal fungi from colonized salal roots using traditional cultural isolation techniques and molecular methods. 35 Table 2.6 Summary of the ericoid mycorrhizal fungi recovered from 5-mm root segments of salal under one inoculum or both inocula. 36 Table 3.1 Measurements of mycelial expansion of Oidiodendron griseum in dual culture. 64 Table 3.2 Measurements of mycelial expansion of S246 in dual culture. 64 Table 3.3 Measurements of mycelial expansion of S9 in dual culture. 65 Table 4.1 Measurements of shoot dry weight of salal inoculated with two 74 known ericoid mycorrhizal fungi. Table 4.2 Measurements of percent root colonization of salal inoculated with two known ericoid mycorrhizal fungi. 74 V LIST OF FIGURES Figure. 2.1 Figure. 2.2 Figure. 2.3 Figure. 2.4 Figure. 2.5 Figure. 2.6 Figure. 2.7 Figure. 2.8 Figure. 2.9 Figure. 2.10 Figure. 2.11 A mapped root of salal. Roots segments to be used for either 37 isolation by culturing or D N A amplification were chosen from different parts, according to the color scheme shown. A mapped root of salal. Roots segments to be used for either 39 isolation by culturing or D N A amplification were chosen from different parts, according to the color scheme shown. Results of isolation from culture (salal Oidiodendron griseum). + S246 and 41 Mixed cultures of S9 and Oidiodendron griseum recovered 42 from root segments of in vitro mycorrhizae. Results of cultural isolation (salal + S9 and Oidiodendron 44 griseum) Mixed cultures of S246 and S9 recovered from root segments of 45 in vitro mycorrhizae. Results of cultural isolation (salal + S9 and S246) 47 RFLP results from mixed cultures of S9 and Oidiodendron 48 griseum. RFLP pattern of PCR products of fungal and ericoid mycorrhizal D N A , from salal seedlings inoculated with S9 and O. griseum, using the restriction enzyme Msp I. RFLP results from mixed cultures of S246 and Oidiodendron 49 griseum. RFLP pattern of PCR products of fungal and ericoid mycorrhizal D N A , from salal seedlings inoculated with S246 and O. griseum, using the restriction enzyme Msp I. RFLP results from mixed cultures of S9 and S246. RFLP 50 pattern of PCR products of fungal and ericoid mycorrhizal D N A , from salal seedlings inoculated with S9 and S246, using the restriction enzyme Msp I. RFLP results from D N A mixture of S9 and Oidiodendron 51 griseum. RFLP pattern of PCR products obtained using fungal D N A mixture of S9 and O. griseum D N A at various concentrations, using the restriction enzyme Msp I. Figure. 2.12 RFLP results from D N A mixture of S246 and Oidiodendron 52 griseum. RFLP pattern of PCR products obtained using fungal D N A mixture of S246 and 0. griseum D N A at various concentrations, using the restriction enzyme Msp I. Figure. 2.13 RFLP results from D N A mixture of S9 and S246. RFLP 53 pattern of PCR products obtained using fungal D N A mixture of S9 and S246 D N A at various concentrations, using the restriction enzyme Msp I. Figure. 2.14 Summary of the ericoid mycorrhizal fungi detected by PCR- 54 RFLP techniques from 10-mm root segments of salal under one or both inocula. Figure. 3.1 Deadlock interaction on M M N (phytagel), shown between two 66 colonies of S9 four months after inoculation. Figure. 3.2 Deadlock interaction on M M N (phytagel), shown between an 66 S9 colony and an S246 colony four months after inoculation. Figure. 3.3 Deadlock interaction on M M N (phytagel), shown between 67 colonies of Oidiodendron griseum and S9 four months after inoculation. Figure. 3.4 Deadlock interaction on M M N (phytagel), shown between colonies of Oidiodendron griseum and S246 four months after inoculation. 67 vii ACKNOWLEDGMENTS The author wishes to thank God and gratefully acknowledge the help and guidance of the following persons during the course of this thesis. Dr. Mary Berbee whose deep knowledge of the research area and kind and intelligent supervision paved the way for the successful completion of this thesis. Dr. Shannon Berch whose insightful comments and continuous guidance revealed the complexities of the work. Dr. Rob DeWreede for his advise and insightful comments on statistical approaches. My friend Dr. Barbara Mable for her advise on statistics, experimental design, and constant support during completion on this work. My friends Tammara Allen and Andrea Sussmann for their advice, encouragement and caring support. My husband Majid for his love and support, my daughter Sara whose sweet behavior has always helped me through the most difficult situations and my parents for their patience, devotion and encouragement. viii CHAPTER 1 LITERATURE 1.1 Literature Review /././ General mycorrhizae The rhizosphere and the surfaces of plants are surrounded by many different microorganisms. Among the organisms that can be present on root surfaces, or in the tissues or root cells, are filamentous fungi. Root tissue or root cells may be colonized by fungal pathogens or mycorrhizal symbionts. A n estimated 90% of plant families form the symbiotic associations between plant roots and fungi termed mycorrhizae (Peterson et al. 1984; Trappe 1987). Mycorrhizae were first described in 1885 by A. B. Frank, a German pathologist. From the mycorrhizal association, the plant receives additional nutrients, enhanced water uptake and, occasionally protection from root pathogens (Peterson and Farquhar 1991), while the fungus absorbs and utilizes photosynthate produced by the host as an energy source. In this way, both organisms benefit from the association and therefore the mycorrhizal association is most often referred to as a mutualistic relationship. The mycorrhizal association can potentially occur anywhere that plants and fungi are present. Mycorrhizae have been observed in Arctic regions (Allen et al. 1987), temperate rain forests (Christy et al. 1982), grasslands (Stahl and Christensen 1983), tropical rain forests (Janos 1987), serpentine soils (Koide and Mooney 1987), and aquatic ecosystems (Bagyaraj et al. 1979). Mycorrhizae occur in pteridophytes, all groups of gymnosperms, and almost all families of angiosperms. The fungi involved in mycorrhizal formation include members of the Zygomycotina, Basidiomycotina, Ascomycotina and Deuteromycotina (Fungi Imperfecti). Harley and Smith (1983) described seven different mycorrhizal types based on the taxa of fungi and the morphological alterations that occur in both fungus and plant during the 1 development of mycorrhizae: vesicular-arbuscular mycorrhizae, ectomycorrhizae, ericoid mycorrhizae, orchid mycorrhizae, arbutoid mycorrhizae, monotroid mycorrhizae, and ectendo-mycorrhizae. This literature review will focus on ericoid mycorrhizae. 1.1.2 The history of isolation and identification of ericoid mycorrhizae . Fungi were first isolated from roots of ericaceous plants by Ternez in 1907 (Ternez, cited by Harley, 1969). She isolated five species of Phoma from roots of Vaccinium, Andromeda and Erica species which formed pycnidia in culture and were called Phoma radicis. Because of the difficulty in obtaining sterile seedlings, she was unable to test the fungi for mycorrhiza formation by back inoculation and she assumed that the seeds must be carrying the mycorrhizal fungi. Rayner (1913) was unable to isolate the mycorrhizal fungus from roots of Calluna and assumed that Calluna was systemically infected. Later, however, Rayner isolated a species of Phoma from seeds of Calluna, and suggested it was the same root-infecting fungus isolated by Ternez (Rayner 1913, 1915). Later on, researchers failed to observe the colonization of young seedlings by back inoculation with Phoma and so the role of Phoma radicis remained unclear. Doak (1928) isolated a slow-growing sterile mycelium from Vaccinium corymbosum L . , which produced typical mycorrhizae by back inoculation. Friesleben (cited by Bain 1937) isolated endophytic fungi from roots of Vaccinium uliginosum L . , which could form the same structure in root cells of Calluna vulgaris (L.) Hull and Vaccinium spp. under control conditions. Despite the early suggestions of systemic infection (Rayner 1913, 1915), researchers agreed that ericaceous plants possess typically root inhabiting mycorrhizal fungi. The fungi formed a typical structure in root cells referred to as a "hyphal complex", "knot", or "hyphenknauel" (Bain 1937). 2 In 1937, Bain isolated mycorrhizal fungi from "hyphal complex" cells in roots of Vaccinium macrocarpon Ait., Vaccinium canadense Kalm (blueberry), Chamadaphne calyculata (L.) Moench and Ledum groenlandium Oeder that did not form fruiting bodies in culture and were tested positive for mycorrhiza formation by back inoculation in cranberry seedlings (Bain, 1937). MacNabb (1961) isolated similar sterile, dark, slow-growing endophytes that were mycorrhiza-formers on ericaceous plants. Harley (1969) first used the term "ericoid mycorrhizae" for the association observed in Ericales and he suggested that there was a structural distinction between the ericoid mycorrhizae in most Ericaceae, Epacridaceae, and Empetraceae, and the arbutoid mycorrhizae in Arbutus and Pyrolaceae. Pearson and Read (1973a) used a similar method to that of Bain's for isolating endophytes. The cortical cells from washed roots containing mycorrhizal hyphae were first plated on a layer of nutrient agar on a cover glass. The hyphae that grew out from the roots were then subcultured under aseptic conditions and used for further studies. One of Read's isolates from Calluna vulgaris was mycorrhizal with various species of Ericaceae such as C. vulgaris and Erica spp. This fungus was initially named Pezizella ericae Read (Read 1974) but was renamed Hymenoscyphus ericae (Read) Korf and Kernan due to the lack of gelatinized excipular cells (Kernan and Finocchio 1983). It has subsequently been shown that H. ericae can form ericoid mycorrhizae with many ericaceous species including C. vulgaris, Vaccinium myrtillus L., Erica cinerea L . , Rhododendron ponticum L., and Gaultheria procumbens L . (Pearson and Read 1973a; Bonfante-Fasalo et al. 1981). The ascomycete H. ericae is one of the most studied ericoid mycorrhizal fungi. Burgeff (1961) reported the isolation of Oidiodendron spp. from mycorrhizal hair roots of ericaceous species. Couture (1983) described Oidiodendron griseum Roback as an endophyte 3 of ericoid mycorrhizae. O. griseum isolated from surface sterilized roots of Vaccinium corymbosum was used as an inoculum for seedlings of Vaccinium angustifolium Ait and it successfully colonized their roots (Couture 1983). Dalpe (1986) tested five different species of Oidiodendron for their mycorrhizal ability on axenic seedlings of Vaccinium angustifolium. Three of the five species (O. griseum, Oidiodendron rhodogenum Roback, and Oidiodendron truncatum Barron) were proven to form mycorrhizae on Vaccinium seedlings (Dalpe 1986). In 1989, Dalpe found that Myxotrichum setosum (Eidam) Orr and Kuehn, Gymnascella dankalienses (Castellani) Currah, and Pseudogymnoascus roseus Raillo were able to form ericoid mycorrhizae with V. angustifolium. These fungi are ascomycetes in the order Onygenales. However, in Italy the occurrence of a basidiomycete was also detected in cortical cells of C. vulgaris and the presence of dolipore type septa confirmed the identification (Bonfante-Fasolo 1980). The mycorrhizal fungi of the Ericaceae have been studied in North America, Europe, and South Africa, but mycorrhizal fungi of Epacridaceae were examined only recently. Studies carried out in Australia (Hutton et al. 1996; Steinke et al. 1996) have now added to our knowledge. Roots of Leucopogan parviflorus (Andr.) Lindl, a species in Epacridaceae, have been examined for ericoid mycorrhizae. The structure of the hair roots in L. parviflorus is similar to other members of Epacridaceae and Ericaceae. They have a small stele, a cortex that is two cells thick, and an epidermal layer. The fungal endophyte penetrates into individual cells without entering the adjacent cells. Steinke et al. (1996) found two isolates with dark, sterile, slow-growing hyphae, one of which was similar to the ericoid endophyte Hymenoscyhus ericae, and the other was a thick walled, brown-pigmented fungal isolate which formed coils and large vesicles within the cells. In another study in Australia, several epacrid species were collected from three sites in Victoria (Mclean and Lawrie 1996). The ericoid mycorrhizal fungi found in the root and 4 epidermal cells of plants at two of these sites were similar in size and formed typical ericoid mycorrhizae. At the third site, however, most root epidermal cells in the plants contained fungal hyphae different from those found at the other sites. The fungal hyphae of this endophyte were coiled and thicker compared to the fungal hyphae of the endophytes recovered from the other two sites, and unlike the other sites, vesicle structures were seen in all root samples. This study also reported, for the first time, the direct cell to cell invasion of adjacent epidermal cells (Mclean and Lawrie 1996). This finding contrasts with observations (Harley and Smith 1983) that the ericoid endophyte is first attracted to the roots and then penetrates into individual cells, with single entry points from the outside and without entering adjacent cells. The lack of photographs of fungal penetration into adjacent cells in Mclean and Lawrie's work makes this conclusion unconvincing and more careful developmental studies should be done before drawing definite conclusions. Differences in structure, such as the formation of the vesicles, suggest that these fungi are different from typical ericoid mycorrhizae. Further investigation using polymerase chain reaction (PCR) is also needed in order to compare these fungi at the molecular level. At present, more than 130 species of vesicular-arbuscular mycorrhizal fungi (Morton 1990) and 5000 species of ectomycorrhizal fungi (Molina et al. 1992) have been identified, while the number of known ericoid mycorrhizal fungi is still relatively small. Straker (1996) concluded that the exact number of fungal taxa involved in ericoid mycorrhizal formation is not clear. Despite the extensive studies done on Hymenoscyphus ericae, only a small number of fungi have been identified as typically ericoid mycorrhiza-forming, and they are taxonomically diverse. To date, there are about 18 known fungal species that form ericoid mycorrhizae with ericaceous plants in axenic culture. Further research is needed to look at the diversity of ericoid mycorrhizal fungi. However, before a complete and comprehensive study on the diversity and distribution of 5 ericoid mycorrhizal fungi can be conducted, the possible advantages and disadvantages (limitations) of using different methods needs to be assessed. 1.1.3 Physiology of ericoid mycorrhizal fungi One of the advantages of working with ericoid mycorrhizal fungi is that they can be grown in culture. Experiments have shown that ericoid mycorrhizal fungi can grow as saprophytes independently of their hosts (Read 1996). In contrast, the vesicular-arbuscular mycorrhizal fungi are obligately associated with their plants (Trappe 1987) and they cannot be grown in pure culture (Simon et al. 1993). Many ectomycorrhizal fungi are obligately mycorrhizal as well (Molina et al. 1992). This obligate relationship between the fungi and the plant makes it difficult to conduct biological studies. In part because the ericoid fungus Hymenoscyphus ericae does grow well in culture, its physiology has been extensively studied by Read and colleagues. One of the first studies on the physiology of the mycorrhizal endophyte H. ericae reported the optimal temperature for growth and pH of the growth medium (Pearson and Read 1975). In the same study, nitrogen and phosphorus utilization were briefly explored. More recent studies have shown that H. ericae can utilize amino acids, proteins, peptides and chitin as nitrogen sources (Stribley and Read 1980; Bajwaand Read 1985; Langdale and Read 1989; Leake and Read 1989; Kerely and Read 1995). The ericaceous plants with ericoid mycorrhizal associations are dominant in mor-humus soils where available nitrogen is low. The results of nitrogen utilization studies raise the possibility that H. ericae can access nitrogen that would otherwise be unavailable to plants, and then possibly share this nitrogen with its plant partners. 6 Phosphorus is the second major limiting nutrient for plant growth in environments dominated by ericaceous plants. Leake and Miles (1995) designed an experiment in which Hymenoschypus ericae was grown with DNA, orthophosphate, or without a phosphorus source. Phosphodiesters such as nucleic acids are high in soils with slow rates of decomposition and the results showed that the fungus grew best on D N A indicating that H. ericae can produce an extra-cellular enzyme which hydrolyses D N A to use as a phosphorus source (Leake and Miles 1995). 1.1.4 Culture conditions usedfor testing the mycorrhizal ability of isolated endophytes The mycorrhizal ability of field isolated fungi should be tested by back inoculation onto a sterile plant. In this way the possibility of mistaking one of the rhizosphere fungi for a mycorrhizal fungus is eliminated. In all the methods used for back inoculation, sterile seeds and pure cultures of isolated fungi were used, however, there were slight differences in the methods employed by various researchers. Bain (1937) planted sterile cranberry seeds (Vaccinum macrocarpon) in tubes on 1% agar to which no nutrients of any kind were added. Seed germination was followed by root penetration into the agar. Bain suggested that the seedling cultures may be inoculated with the mycorrhizal fungi, by spreading fungal hyphae from pure cultures, at the time the seed is planted or after seed germination. Observation of the colonization was done under the light microscope. Couture and Dalpe (1983) used similar methods to those of Pearson and Read (1974), for preparation of Vaccinium angustifolium seedlings. Sterile seeds were germinated on fine silica sand, and 3 weeks later the germinated seedlings were transferred to agar in glass test tubes. The agar was dusted with just enough soil to cover the agar surface and then the seedlings were inoculated with different fungal isolates. The inoculum consisted of 6 mm fungal plugs taken 7 from the edge of a growing colony. The seedlings were then placed in a growth chamber under specific conditions and after 2 months the entire root system was pulled out of agar, washed, stained and, examined under the light microscope. Stoyke and Currah (1990) used a similar method for testing the mycorrhizal ability of the isolated fungi from alpine plant communities dominated by ericaceous plants in the Rocky Mountains of Alberta. The sterile seeds of Menziesia ferruginea J. E. Smith (Ericaceae) were germinated on water agar then transferred to cereal agar and, apparently 7 days after inoculation with mycorrhizal fungi, the whole root system was excised, mounted and observed under the compound microscope. Xiao and Berch (1995) introduced a new synthesis chamber where the colonized roots could be observed without pulling the roots out of agar. They germinated salal seeds on 8% water agar and, after leaf emergence, one seedling of salal was placed at the center of a petri dish with half of the media disc cut out. The medium used in the synthesis chamber was modified Melin Norkran Agar (MMN). The advantage of using this closed system is that the colonization of salal roots can be observed by mounting the whole plate on a light microscope without pulling the roots out. In this thesis, the Xiao and Berch (1995) synthesis chambers were used and in Chapter 4 phytagel was replaced by granulated agar in an attempt to improve the visibility of agar and hence allow better observation of the roots. 1.1.5 Rationale for the present study In Europe and North America, important forest lands have been overtaken by ericaceous plants after logging and burning (Weetman et al. 1989). For example, the ericaceous shrub Kalmia angustifolia L. is found in eastern North America and is commonly found as an understory of 8 black spruce forests. K. angustifolia can grow rapidly after stand disturbances such as harvesting and reduce the growth of naturally or artificially regenerated seedlings (Titus 1995). On Northern Vancouver Island, old growth forests of western red cedar {Thuja plicata Donn) and western hemlock (Tsuga heterophylla (Raf) Sarge) have salal {Gaultheria shallon Pursh) as a dominant understory. Salal is an ericaceous shrub with an extensive system of fine roots that are colonized by ericoid mycorrhizal fungi in the field (Xiao and Berch 1995). These cedar-hemlock sites are characterized by low pH, low available nutrients, and high organic matter content, while the major growth-limiting factor is nitrogen. After clear-cutting and slash-burning, salal comes back quickly and covers the cutblock. Once salal is established, it spreads very quickly by sprouting from rhizomes. The SCHIRP (Salal Cedar Hemlock Integrated Research Program) project (Prescott 1996) determined that the presence of salal causes an extreme reduction in growth of coniferous seedlings. According to Messier and Kimmins (1991), the dominance of salal on sites disturbed by clearcutting and burning is due to: (1) salal's ability to survive these treatments; (2) salal's rhizomes, which allow it to occupy both below and above ground environments, rapidly preempting resources and therefore resisting invasion by other species. The dominance of salal may also be attributed in part to its mycorrhizal association. Recently, ericoid mycorrhizae have received more attention because of their suggested role in dominance of their ericaceous hosts. This possibility prompted several research attempts to examine the fungi involved in the mycorrhizal association of salal from the C H (cedar-hemlock) site using both isolation in culture (Xiao, 1992; Monreal, 1996) and molecular methods (Monreal, 1996). Xiao (1992) concluded that the recovery rate of the endophytes from highly colonized field roots via traditional culture isolation methods was around 15% and of the fungi detected, Oidiodendron griseum was recovered most frequently. In culture, Xiao (1992) isolated two 9 sporulating fungi, O. griseum and Acremonium strictum, and two nonsporulating fungi, unknown 1 (isolates U B C S9, U B C S222, and U B C S245) and unknown 2 (isolates U B C S246, U B C S203, and U B C S226) from field-colonized roots of salal. Back inoculation of salal seedlings showed that A. strictum formed pseudomycorrhizae while O. griseum and the nonsporulating fungi formed typical ericoid mycorrhizae in culture. Based on restriction fragment analysis of ribosomal D N A , Monreal (1996) recognized two new ericoid mycorrhizal fungi isolated from salal field roots that remained sterile in culture: unknown 3 (isolate U B C M8) and unknown 4 (isolate U B C M20). Monreal generated a synoptic key for identification of known ericoid mycorrhizal fungi. She detected a fifth group, unknown 5 (isolate U B C M5) based on sequences of the ribosomal ITS regions. Phylogenetic analysis based on the sequence analysis of the ITS 2 region showed that all the ericoid mycorrhizal fungi can be classified into two main groups: (1) the Hymenoscyphus group and (2) the Oidiodendron group (Monreal 1996). A l l five sterile, unknown species clustered loosely with European ericoid mycorrhizal isolates of the species, Hymenoscyphus ericae (Monreal 1996). The Hymenoscyphus group segregated into three subgroups. One branch included H. ericae and isolates unknown 3 (UBC M8), and unknown 4 (UBC M20). The second branch included Phialophora flnlandia and sterile isolates unknown 1 (UBC S9) and unknown 2 (UBC S246) while the third branch led to unknown 5 (UBC M5). Molecular tools have also been used in attempts to identify the fungal endophytes directly from the field roots (Monreal 1996). However, the multiple bands generated in direct PCR reactions from salals field roots were difficult to interpret. 1.2 Overall objectives Traditional isolation by culturing is one of the most common practices used for identifying endophytes from colonized roots, although recent studies using molecular techniques reported a higher diversity of ericoid mycorrhizal fungi compared to that determined by culture isolation methods (Monreal 1996). The use of molecular tools has now changed the way we conduct studies on the abundance and distribution of ectomycorrhizal fungi. Researchers used to assume that structure of the fruit body could be used accurately to reflect the distribution and abundance of ectomycorrhizal fungi, but studies of this nature can only represent the distribution of reproductive structures, not vegetative structures (Bradbury 1996). It is known that both isolation in culture and molecular methods can be used to identify ericoid mycorrhizal fungi, but the capabilities of these two methods have not been compared in the same study. This thesis compares the results of these two methods with each other. The question here is whether, by using traditional methods, we are missing out on some fungi that do not grow well in culture but are easily detected with molecular tools or vice versa. Could molecular tools detect the presence of more than one fungus in salal's colonized roots? Would traditional isolation by culturing detect the presence of more than one fungus in salal's colonized roots and would it give the same results as molecular methods? It has been reported that there can be high diversity of ericoid mycorrhizae on a small scale (Perotto et al. 1996; Monreal 1996). This thesis tests this finding in culture and looks at the colonization patterns in salal's roots and addresses these questions: (1) If salal seedlings are in close contact with two different ericoid mycorrhizal fungi, can both mycorrhizal fungi colonize the same root system or will one fungus out-compete the other? (2) What is the colonization 11 pattern in plant roots? Wil l we be able to observe high diversity (i.e., more than one fungus) in a single root system? 1.3 Specific objectives (1) To compare traditional isolation techniques with molecular methods for the identification of ericoid mycorrhizal fungi from inoculated salal roots. (2) To map roots and look at the colonization patterns. (3) To compare the competitive interactions of various ericoid mycorrhizal fungi in pure culture and when inoculated onto salal roots in culture. (4) To compare the growth of ericoid mycorrhizal fungi and mycorrhizal salal on media gelled with agar vs. phytagel. 12 CHAPTER 2: MOLECULAR AND CULTURAL IDENTIFICATION OF ERICOID MYCORRHIZAL FUNGI OF GAULTHERIA SHALLON 2.1 Introduction Ericoid mycorrhizae are formed by plants in the order Ericales, in families such as Ericaceae, Empetraceae, and Epacridaceae. Salal (Gaultheria shallon Pursh) is an ericaceous shrub with fine roots that are colonized by ericoid mycorrhizal fungi in the field. In western Canada, on some reforestation sites, salal has become the dominant understory. On these sites, about 8 years after clearcutting conifers grew poorly but salal was thriving (Weetman et al. 1990). Xiao (1992) suggested that the success of salal on these sites might be due to its mycorrhizal association. Studies on mycorrhizal associations of salal and on the effect of mycorrhizal fungi on each other might help us understand what is happening in the field. The fungal symbionts associated with ericoid mycorrhizae are mostly ascomycetes and a few basidiomycetes, but their exact number and diversity remain uncertain (Starker 1996). It is impossible to identify an ericoid mycorrhizal fungus just by microscopic examination of the fungus in a plant root. As a result researchers have used a variety of different methods to identify the ericoid mycorrhizal fungi. 2.1.1 Isolation techniques and identification of ericoid mycorrhizal fungi Isolation techniques followed by microscopic study were the earliest methods used for identification of ericoid mycorrhizal fungi. For the first time in 1907, Ternez isolated fungi from roots of ericaceous plants (cited by Harley 1996). Later Bain isolated mycorrhizal fungi from root cells of Vaccinium uliginosum L. (Bain 1937). Pearson and Read (1973a) used a similar method to that of Bain's for isolating the mycorrhizal endophyte from heather, Calluna vulgaris 13 (L.) Hull., roots. Read's isolate, originally called Pezizella ericae Read (Read 1974) and later transferred to Hymenoscyphus ericae (Read) Korf & Kerman, is a soil saprophyte that has been isolated from different substrates including forest soils, paper samples, wood pulp, tree leaves, humus, and rhizosphere soil (Dalpe 1986; Stoyke and Currah 1991). Sporulating fungi isolated from ericoid mycorrhizae include Oidiodendron griseum Roback isolated from Vaccinium corymbosum L. (Couture et al. 1983), Phialocephala fortinii Wang & Wilcox from Cassiope mertensiana (Bong.) D. Don and Arctostaphylos uva-ursi (L.) Spreng (Ericaceae) as well as from Luetkea pectinata (Pursh) Kuntze (Rosaceae) (Stoyke and Currah 1990), Scytalidium vaccina (Dalpe) Litten & Siglerfrom Vaccinium angustifolium Ait. (Dalpe et al. 1989), and Acremonium strictum W. Gams from salal (Xiao and Berch 1996). Several sterile mycorrhizal fungi [Unknown 1 (UBC S9), Unknown 2 (UBC S246), Unknown 3 (UBC M8), and Unknown 4 (UBC M20)] have been isolated from salal roots (Xiao and Berch 1996 and Monreal 1996). Until recently the identity of fungi forming ericoid mycorrhizae in Epacridaceae has not been addressed (Read 1996). In a recent study, roots of Leucopogan parviflorus (Andr.) Lindl, an epacrid species, have been examined for ericoid mycorrhizae. The structure of the epacrid root was similar to ericaceous plants and a sterile slow-growing isolate similar to the ericoid endophyte Hymenoscyphus ericae was isolated from the plant roots (Steinke et al. 1996). Except for P. fortinii, all these sporulating and sterile ericoid mycorrhizal fungi listed above have been shown to form mycorrhizae in vitro. 2.1.2 Identification by electron microscopy Another method used for identification of ericoid mycorrhizal fungi was based on ultrastructural features using electron microscopy. Under the light microscope, all the colonized 14 cortical cells will show similar ericoid mycorrhiza formation and the fungal species cannot be differentiated in this way. However, Bonfante-Fasolo (1980) used electron microscopy to differentiate between ascomycetes and basidiomycetes in the cortical cells. The basidiomycetes have a single electron dense layer in their hyphal walls and dolipore type septa whereas the hyphal wall of the ascomycetes have an outer and an inner layer as well as simple septa with Woronin bodies (Bonfante-Fasolo 1980). The ultrastructural features observed in field collected mycorrhizal hair roots of Calluna vulgaris suggested the presence of a basidiomycete. In another study using ultrastructural features, the presence of dolipore septa indicated that a basidiomycete was present in roots of Rhododendron (Peterson et al. 1980). 2.1.3 Identification by molecular methods Immunocytochemical techniques were used to detect the presence of the fungus Clavaria spp. in thin sections of Rhododendron roots (Muller et al. 1986). Pectic zymograms provide another broad screening technique for grouping ericoid mycorrhizal fungi (Hutton et al. 1996). In this technique, fungi are grown in a liquid induction medium for pectic enzymes. After 7 days, the pectic enzymes are detected by gel electrophoresis. In a survey of pectic zymograms of cultured endophytes from Australia, fungi from wetland sites showed homogeneity of banding patterns, while those from dry land habitat were more heterogeneous (Hutton et al. 1996). 2.1.4 Identification of ectomycorrhizal fungi by direct DNA amplification from roots Molecular techniques based on D N A are now commonly used to study ectomycorrhizae and vesicular arbuscular mycorrhizae, whereas ericoid mycorrhizae have received little attention. 15 In most ecological studies, the diversity of ectomycorrhizal basidiomycetes has been inferred from fruiting records. However, studies showed that fruiting-body appearance may not be a good indication of the true fungal diversity in the ectomycorrhizae (Gardes and Bruns 1993; Bradbury 1996). In order to assess the diversity of ectomycorrhizal fungi and to overcome the assumption that fruit body distribution accurately represent the fungal diversity, molecular tools have been employed. In 1993, Gardes and Bruns concluded that the diversity of ectomycorrhizal fungi involved in an ecosystem as determined by counting fruiting bodies was half of what they found using molecular techniques. In the same study, Gardes and Bruns designed two selective primers for the internal transcribed spacer (ITS) regions in the ribosomal repeat unit, one to amplify all fungi and the other to amplify only basidiomycetes. The primers successfully discriminated between fungal and plant D N A of mycorrhizae and rust infected tissue (Gardes and Bruns 1993) and therefore the presence of the plant D N A was no longer a problem. Using these primers, the unknown fungal partner in a mycorrhizal root could be selectively amplified and sequenced, then identified through comparisons with sequences from known species. This approach is permitting surveys of root fungi that are improving our understanding of mycorrhizal ecology. 2.1.5 Identification of vesicular arbuscular mycorrhizae by direct DNA amplification from soil Molecular identification has also been applied to vesicular arbuscular mycorrhizae. Determining which vesicular arbuscular mycorrhizal fungi are present on a site currently requires that host plants be used to trap inoculum and eventually produce spores for identification. Drawbacks of the current culture and green house methods are: (1) they require several months of growth under greenhouse conditions, and (2) fungal strains with low density or low tolerance for 16 green house conditions cannot be observed. Because of these two problems the population diversity cannot be detected accurately. As an alternative, direct D N A extraction from the soil followed by PCR amplification using specific primers from the nuclear 17S R N A sequence has proved an effective way for direct identification (Claassen et al 1996). 2.1.6 Molecular identification of cultured ericoid mycorrhizae The molecular identification of ericoid mycorrhizal fungi, unlike the identification of ectomycorrhizal fungi, has required pure fungal cultures as starting material. Egger and Sigler (1993) used PCR amplification, RFLPs, and D N A sequencing to show a low degree of variation in ribosomal D N A between isolates of Hymenoscyphus ericae and Scytalidium vaccinii, indicating a possible anamorph-teleomorph relationship between the two species. Perotto et al. (1996) investigated the molecular diversity of fungal endophytes cultured from ericoid mycorrhizal roots of Calluna vulgaris. PCR-RFLP techniques revealed that several groups of ericoid mycorrhizal fungi were present in a single plant root system. In addition, highly sensitive PCR-RAPD techniques showed high genetic polymorphism among different fungal mycelia of the same RFLP group (Perotto et al. 1996). Identification of fungi in ericoid mycorrhizae by direct amplification from the mycorrhizal roots has failed, probably because the root segments contained more than one fungus. Monreal (1996) generated a synoptic key for identification of 34 fungal isolates representing a total of 18 known ericoid mycorrhizal fungi using RFLP patterns from the PCR amplified ITS regions. Monreal then tried to identify ericoid mycorrhizal fungi associated with salal through direct D N A amplification of the ITS region from field sampled salal mycorrhizae using analysis of RFLPs. The numerous bands generated by the restriction enzymes indicated the presence of D N A from 17 more than one species, revealing the complexity of the ecosystem. Identification of the individual fungal species was not possible from the mixed banding patterns. My long term objective is to find out which ericoid mycorrhizal fungi are the most important and most abundant in nature. To achieve this objective, diversity studies should be conducted, but before that, we need to know which identification methods will give the most accurate results. As a simple system for testing identification methods, I grew salal plants in petri dishes, inoculated them with two different mycorrhizal fungi to simulate a simple mixed fungal population present in nature and allowed mycorrhizae to form. The present study compares the two most common approaches for the detection of ericoid mycorrhizal fungi, (1) D N A amplification followed by RFLP analysis and (2) traditional isolation followed by morphological classification,. With the two methods, I reconstructed and compared the pattern of fungal colonization within synthesized mycorrhizal root systems. 2.2 Objectives The objectives of this chapter were: (1) To compare traditional isolation techniques with molecular methods for identification of ericoid mycorrhizal fungi from inoculated salal roots. (2) To map roots and select colonized roots under each inoculum and under both inocula to observe the colonization patterns. (3) To observe the results of competitive PCR between paired fungal D N A extracts of known concentrations. 18 2.3 Materials and methods The three fungal isolates used in this study were obtained from the Departments of Botany and Soil Science of the University of British Columbia (Table 2.1). The three isolates were chosen because they represent common mycorrhizal fungi from British Columbia, can also be distinguished morphologically, and their RFLP patterns from the enzyme Msp I are clearly different. Two of the fungi, isolate S246 and isolate S9 are sterile. The third, Oidiodendron griseum, sporulates readily. On potato dextrose agar (PDA) S246 colonies were shiny, moist and funiculose with ropes radiating out (Xiao and Berch 1996) while S9 colonies had a powdery look and as they grew they changed the media color to brown. O. griseum colonies on P D A were whitish gray, cottony, and sporulated with long-stalked conidiophores arising from the hyphae at the agar surface (Xiao and Berch 1996). Fungal isolates originally maintained on modified Melin Norkran agar (MMN) (CBS List of Cultures, Fungi and Yeasts, 32nd edition, 1990; Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) were used as the inoculum for these experiments. The medium used for the mycorrhizal synthesis was M M N with the exclusion of mineral nitrogen, malt extract, and glucose. Glutamine was added as a nitrogen source to the medium. Deletion of mineral nitrogen, malt extract, and glucose slows the growth of mycorrhizal fungi and ensure the colonization of the roots of salal (Xiao, 1992). 2.3.1 General preparation of synthetic salal mycorrhizae Salal seeds were surface sterilized in 30% hydrogen peroxide, rinsed in distilled water, and then placed on water agar to germinate. When true leaves emerged, each salal seedling was transferred to a petri dish containing M M N with half of the media disc cut out. After two weeks 19 the seedlings were inoculated with fungal plugs 3 mm diameter that were cut from the edge of a pure fungal colony. The fungal plugs were then placed on the cut edge of the medium on either side of the shoot of the salal plant. Each seedling was inoculated with two different fungi at a time and three different treatments were used (S9 and S246 + salal, S9 and O. griseum + salal, and S246 and O. griseum + salal) (Fig. 2.2, 2.4, 2.6). A total of 32 plates were inoculated with combination of two fungi. The plates were then sealed with Parafilm and placed in growth chamber at 21° C, 13 hr light at 128 umol m~2 sec~l and 16° C, 11 h dark. The petri dishes were kept in the growth chamber for four to five months and the whole plates were examined under the compound microscope for colonization. A total of 24 healthy salal cultures with similar root systems, eight for each combination of fungi, were selected for isolation by culturing and D N A amplification. 2.3.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae The unopened petri dish containing an inoculated colonized salal seedling was placed under a compound microscope and observed for mycorrhiza formation. Colonized roots under each inoculum as well as under both inocula were marked and mapped (Fig. 2.1). Roots of salal were harvested by gently pulling them out of the agar; the roots were then rinsed in distilled water and examined under the light microscope to confirm the presence of ericoid mycorrhizae. Five mm root segments were placed on 1/3 strength potato dextrose agar (PDA), and the position of each 5 mm segment was recorded on the root map. Four plates were used as replicates for each fungal treatment and a total of 12 plates was used for the experiment. Fifteen root segments were taken from each original plate, so that for each treatment, sixty 5-mm root segments were cut and plated. The new plates were observed very carefully every day under the dissecting 20 microscope to watch for fungal growth. At the first sign of fungal growth, each root segment with a fungus growing out of it was individually transferred to a new 1/3 P D A plate to keep the fungi from different segments from growing together. These plates were then observed periodically for identification of the fungus or fungi according to morphology. 2.3.3 Molecular detection of the fungi in the mixed, synthetic mycorrhizae The same mapping technique as described above was used prior to D N A extraction. D N A was extracted from 10 mm root segments using a phenol-chloroform method (Lee and Taylor 1990). D N A pellets were suspended in 40 ul of TE (low EDTA) and used as stock solutions. Twenty ul of the stock solution were further diluted with 10 ul of sterile distilled water. Diluted D N A was amplified with PCR using Ready-To-Go beads (Pharmacia Biotech Inc. P.O. Box 2027, 1250 University Ave, Station "B", Montreal, PQ H3B 4H4). To preferentially amplify the fungal D N A from the mixture of plant and fungal D N A , the fungal-specific primer ITS1-F (Gardes and Bruns 1993) and the universal primer ITS4 (White et al. 1990) were used. PCR temperature cycling began with an initial denaturation step at 95° C for 5 min, continued by 30 cycles of denaturation at 95° C for 1 min, annealing at 56° C for 1 min, and extension at 72° for 45 sec with initial dwell time of 5 min at 95° C. Cycling was followed by a final dwell time of 7 min at 72° C. To test for D N A contamination, one negative control with sterile distilled water substituted for the D N A template was used for each PCR reaction. Positive controls used for the PCR experiment were: (1) total genomic D N A from pure fungal cultures amplified with ITS4 and ITS 1-F, and (2) D N A and primers provided with the PCR beads. PCR products were digested with the restriction enzyme Msp I. D N A fragments were then electrophoresed and identified based on banding patterns. 21 A control experiment was set up to test the possibility of amplification of the fungal hyphae that were present around the root but not colonizing the roots. In these preliminary experiments, as a control, 10 mm uncolonized salal roots were chosen from an inoculated petri dish with fungi growing around the roots. D N A extraction was followed by PCR amplification and RFLP identification. Preliminary experiments were set up to amplify fungal D N A from salal's colonized roots by direct PCR amplification using primers ITS4 and ITS 1 -F followed by RFLP identification. These experiments included all three fungal treatments and the results showed that 10 mm root segments are the ideal size for consistently successful direct D N A extraction. Five mm root segments sometimes provided enough D N A , but the results were less consistent. In the main experiment, D N A was extracted from mycorrhizal roots from the four petri plates that constituted four replicates of the S9 and S246 + salal treatment. From each petri plate, twelve root segments were sampled, four 10-mm root segments from under each inoculum and four 10-mm root segments from under both inocula. From the four replicates a total of forty-eight 10-mm root fragments were used for D N A extraction. In the plates containing S9 and O. griseum and S246 and O. griseum treatments, only one plate was used for D N A extraction for each combination because of time constraints. However, previous D N A extractions from preliminary experiments from plates containing S9 and O. griseum and S246 and O. griseum brought the total up to 36 for each treatment. 22 2.3.4 Competitive PCR between pairedfungal DNA extracts of known concentration 2.3.4.1 DNA preparation For this study, D N A was extracted from each of the three ericoid mycorrhizal fungi, S246, S9 and O. griseum, after they had been growing for five weeks at room temperature on full strength PDA. Mycelium from the edge of the fungal colony was used for D N A extraction (Lee and Taylor 1990). To verify initial D N A quality, the ITS regions were PCR amplified using primers ITS 4 and ITS 1-F (Gardes and Bruns 1993) and the PCR conditions explained in Section 2.3.3. To test for contamination, a negative control replacing the D N A template with sterile distilled water was used in one PCR reaction. PCR products were then run on an agarose gel and the fungal bands were observed. Extracted total genomic D N A [stock solution] from these fungi was used for the next part. 2.3.4.2 Tests of PCR amplification with artificial mixtures of DNA Spectrophotometer readings were used to measure the D N A concentration in the three different total genomic fungal D N A solutions and appropriate dilutions with similar D N A concentrations were prepared. The diluted DNAs from two different fungal isolates were then mixed (Tables 2.2, 2.3, 2.4). The total D N A concentration was held constant at 91 ng/|j,l, but the proportions of each of the constituent DNAs was varied. Four artificial mixtures were examined: 1) Both constituents present at the same D N A concentration (50:50), 2) the concentration of one isolate present at twice the concentration of the other isolate (33:66), 3) the concentration of one isolate present at half the concentration of the other isolate (66:33), 4) the concentration of one isolate present at 1/4 the concentration of the other isolate (75:25). The positive controls contained D N A from one fungus at the same four concentrations at which it appeared in the 23 mixture (Tables 2.2, 2.3, 2.4). To test for contamination, negative controls containing sterile distilled water were used for each PCR reaction. PCR amplification of the D N A was carried out as described above. The RFLP banding patterns generated were used for identification. 2.4 Results 2.4.1 Observations on ericoid mycorrhiza formation in vitro A l l three ericoid mycorrhizal fungi S9, S246 and Oidiodendron griseum formed typical ericoid mycorrhizae with dense coils inside the cortical cells. Microscopic observation of the mycorrhizal formation showed that the fungal hyphae started growth from the inocula. Sometimes the fungal hyphae would initially pass over or under salal roots, apparently without detecting the root. However, after the fungal mycelium had grown past the root, some of the hyphae appeared to notice the presence of the root and grew towards it. In early stages I observed more colonization at the root tips while given time and proximity of fungal hyphae to the root eventually resulted in colonization along the root. Hyphae of the sporulating fungus O. griseum and the sterile fungi S9 and S246 all behaved similarly in the presence of salal roots. Oidiodendron griseum spores usually scatter around the plate and the result might be a root segment that is colonized near the inoculum (near the stem of the salal plant) and at the root tips but not in the middle. Fungal species could not be distinguished in colonized cells by microscopic observation. 2.4.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae In cultures where salal seedlings were inoculated with S246 and Oidiodendron griseum, both mixed cultures and pure cultures were obtained (Table 2.5, Figs. 2.2, 2.3). Mixed cultures of 24 S246 and O. griseum accounted for 70% (42 out of 60) of the isolates (Table 2.5, Fig. 2.3). Twenty-eight percent (17 out of 60) of the cultures were pure S9 and only 2% (1 out of 60) of cultures were pure O. griseum. Similarly, from cultures where salal seedlings had been inoculated with S9 and O. griseum, 69% (29 out of 42) of the cultures were mixed (Fig. 2.4, 2.5) and 31% (13 out of 42) were pure S91 (Table 2.5). From salal seedlings inoculated with S9 and S246, 88% (53 out of 60) of the isolates were pure S9 and 12% (7 out of 60) of the cultures were mixed cultures of S9/ S246 (Table 2.5, Figs. 2.6, 2.7). When salal was inoculated with either S9 and Oidiodendron griseum or S246 and Oidiodendron griseum, over 80% of the isolates from roots equally close to both inocula were mixed cultures. Even from roots that were closer to the point of inoculation of one fungal species than the other, 50% or more of the root segments gave rise to mixed cultures (Table 2.6). In salal seedlings inoculated with S246 and Oidiodendron griseum, about 50% of the isolates recovered from the roots located under the S246 inocula were pure S246 and 50% of the isolates were mixed cultures of S246 and O. griseum. Ninety-five percent of the fungi recovered from the roots located under both inocula were mixed cultures of S246 and O. griseum while only 5% were pure O. griseum. Of the isolates recovered from the roots located under O. griseum inoculum, 65% were mixed cultures of both fungi and 35% were pure S246 (Table 2.6). 1 One of the replicates was not counted because the S9 inoculum died and only O. griseum was re-isolated from the roots also three individual plates were contaminated and were not counted, which decreased the total number of plates from 60 to 42. 25 In salal seedlings inoculated with S9 and S246, 80% of the isolates recovered from under the S9 inoculum were pure cultures of S9 and 20% were mixed cultures of S9 and S246. Over 90% of the isolates from the roots located under S246 inoculum or both S9/S246 inocula were pure S9. Pure cultures of S246 were not isolated even in the colonized roots located under S246 inoculum (Table 2.6). In salal seedlings inoculated with S9 and Oidiodendron griseum, about 46% of the isolates recovered from under S9 inoculum were pure S9 and 54% of the isolates were mixed cultures of S9 and O. griseum. Eighty percent of the fungi recovered from the roots located under both inocula were mixed cultures of S9 and O. griseum while only 20% were pure S9. Seventy-one percent of the isolates recovered from the roots located under O. griseum inoculum were mixed cultures of both fungi and 29% of the isolates were pure S9 (Table 2.6). However pure cultures of O. griseum were not isolated. 2.4.3 DNA extraction PCR amplification and Msp I RFLP pattern revealed only one fungus in D N A extracts from mycorrhizae where salal seedlings had been inoculated with either Oidiodendron griseum and S9 (Fig. 2.8) or O. griseum and S246 (Fig. 2.9). Either S246 or S9 could be detected but O. griseum was not detected in any one of the 71 D N A extracts from root segments (Fig. 2.14). In cultures where salal seedlings were inoculated with S9 and S246, PCR amplification and RFLP analysis revealed both of the fungi (Table 2.5, Fig. 2.10). Of 48 D N A extractions from 10 mm colonized salal roots, fungal D N A was amplified from 42 of them. RFLP banding patterns 26 showed that 16 extractions (38%) contained mixed D N A and 26 (62%) were pure S9 (Table 2.5, Fig. 2.10). In cultures where salal seedlings were inoculated with S9 and S246, PCR amplification and RFLP analysis showed that in the roots located under S246 inoculum 7 or 50% of the extracts were pure S9 and the other 50% contained both fungi. In the roots located under the S9 inoculum 9 extracts (64%) were pure S9 and 5 (36%) were mixed D N A . In the roots located under both inocula 10 extracts, or 71% were still pure S9 (Fig. 2.14). 2.4.4 Results from competitive PCR between paired fungal DNA extracts of known concentration When Oidiodendron griseum D N A was mixed with D N A from S246 or S9 (Tables 2.2, 2.3), it no longer amplified even though pure O. griseum D N A did amplify at equivalent concentrations (Fig. 2.11, 2.12). In contrast, regardless of the concentration, when S9 or S246 DNAs were mixed (Table 2.4), both DNAs amplified (Fig. 2.13). 2.5 Discussion 2.5.1 Recovery offungal mixtures from 5 or 10 mm root segments None of the three fungi I studied consistently excluded either of the two other competitors to take sole possession of a root. In this thesis, the method of isolation by cultoing showed that in axenically grown salal seedlings that were inoculated with Oidiodendron griseum and S9 or O. griseum and S246, almost 70% of the root segments gave rise to mixed cultures (Table 2.5, Figs. 2.2, 2.3, 2.4, 2.5). This means that two or more different ericoid mycorrhizae can colonize a single 5 mm salal root segment. These results are consistent with the possibility 27 that salal roots in nature posses a high diversity of ericoid mycorrhizal fungi on a small scale. The information presented in this thesis is also consistent with the research done by Bonfante-Fasolo and Gianinazzi- Pearson (1982) showing that external hyphae infect individual cells but that hyphae within cells do not colonize neighboring cells. If a new colonization is needed for each cell, then neighboring cells are perhaps likely to be colonized by different fungi in an environment where a fungal mixture is present. I cannot rule out that the fungi recovered were not colonizing the roots but were merely in close proximity to the root. However, the results from PCR-RFLP identification clearly showed the presence of both sterile fungi from 10 mm root segments. The D N A amplification from the control experiment where uncolonized salal roots were chosen from an inoculated petri dish with fungal hyphae growing around the roots, showed that the fungal D N A was not detected in this case. I think it is most reasonable to assume that the recovered fungi were in fact the fungi colonizing the roots and not the fungal hyphae growing around the root. In nature, in addition to ericoid mycorrhizal fungi, other nonmycorrhizal fungi are present forming a complex rhizosphere community. The existence of other unculturable and nonmycorrhizal fungi might explain the presence of numerous bands generated in PCR reactions and the unsuccessful attempt to identify ericoid mycorrhizal fungi via direct amplification from sampled salal field roots (Monreal 1996). 2.5.2 Re-isolation of the fungi from the mixed, synthetic mycorrhizae In cultures where salal seedlings were inoculated with Oidiodendron griseum and either S9 or S246, the root segments located under both inocula in over 87% of the isolates gave rise to mixed cultures (Figs. 2.3, 2.5, Table 2.6), showing that given the same opportunity both fungi 28 jointly colonize the root segments. The isolated fungi recovered from the roots located under either inoculum (S9, S246, or O. griseum) were mixed in over 50% of the cultures. These roots were not preferentially colonized by the inoculum located above them. In cultures where salal seedlings were inoculated with S9 and S246, over 80% of the isolates that were recovered from under each inoculum or both inocula appeared to be pure cultures of S9 (Fig. 2.7, Table 2.6). The reasons for the difference observed in the results from inoculation with Oidiodendron griseum and either sterile fungi compared to both sterile fungi are explained in part 2.5.3. 2.5.3 Culturing bias against sterile isolates Isolation in culture favors the recovery of the sporulating fungus Oidiodendron griseum. This finding is consistent with the results of previous researchers who attempted to isolate ericoid mycorrhizal fungi associated with salal. Xiao used 560 salal root pieces from a site on Vancouver Island, British Columbia, Canada. Oidiodendron griseum was isolated 28 times out of 87 (32%>) and it was the most abundant fungal isolate from the site (Xiao 1992). However the fungal cultures recovered from salal seedlings that were inoculated with S9 and S246, the two sterile fungi, all at first appeared to be pure cultures of S9. On closer inspection, I detected small shiny patches on the agar. Subsequent subculturing from the shiny patches revealed mixed cultures of S9 and S246 (Fig. 2.6). To the untrained eye, the pure and mixed cultures will look similar. It would be almost impossible to recognize mixed cultures of sterile fungi from field collected roots, especially with undescribed, nonsporulating isolates. However, molecular techniques using PCR-RFLP analysis can clearly distinguish the two sterile fungi from one another and will be extremely useful for field isolation. This is also consistent with findings from 29 previous researchers. Monreal (1996) collected salal roots near Xiao's (1992) collecting site. Monreal's approach for identification of ericoid mycorrhizal fungi of salal using PCR- RFLP techniques on cultured fungi from twenty 5 mm root segments resulted in the detection of three different sterile mycorrhizal fungi and only one sporulating O. griseum culture. Xiao (1992) had recognized two types of sterile fungal isolates in his previous study of ericoid mycorrhizae from this site. With minimum effort, Monreal had more than doubled the known number of types of sterile ercoid mycorrhizal fungi from the area. Possibly, using PCR/RFLP identification even more sterile mycorrhizal fungi will be found. D N A amplification is a very useful and satisfactory tool for identification of sterile ericoid mycorrhizal fungi. 2.5.4 DNA extraction In cultures where salal seedlings were inoculated with S9 and S246, over 70% of the extracts from roots located under both inocula were pure S9. A possible explanation is that S9 grew faster compared with S246, resulting in more colonization by fungal hyphae of S9. In cultures where salal seedlings were inoculated with Oidiodendron griseum and either S9 or S246, regardless of the location of the roots O. griseum D N A was never detected (Table 2.5). Some possible explanations are covered in part 2.5.5. 2.5.5 PCR amplification bias against Oidiodendron griseum Isolation by culturing showed that 70% of the root segments gave rise to mixed cultures of Oidiodendron griseum with either S9 or S246 but the PCR-RFLP combination never detected O. griseum (Table 2.5) in these mixtures. One possible explanation for the lack of PCR-RFLP detection is that DNAs from S9 and S246 outcompete D N A from O. griseum during PCR 30 amplification. A second possible explanation is that the concentration of O. griseum D N A in the mycorrhizae was (much) lower than that of S9 or S246. Judging from the variation in the amount of each fungus that grew out of each root, the relative proportion of O. griseum to either S9 or S246 within different roots varied but was perhaps too low for DNA-based detection. To test these possibilities artificial D N A mixture experiments were designed. The results from these experiment clearly indicated that Oidiodendron griseum D N A , at various concentrations, will amplify i f it is alone but will not amplify in mixtures with either S9 or S246 (Figs. 2.11, 2.12) using the primer combination and PCR conditions employed here. Oidiodendron griseum D N A may not amplify in mixed cultures for a number of reasons. (1)0. griseum may have one or two mismatches with ITS 1-F or ITS 4 while S9 and S246 match perfectly. This difference in binding regions might be sufficient to favor S9 and S246 amplification over O. griseum amplification but would permit O. griseum to amplify in the absence of competing fungi. To test whether a priming mismatch is the problem would require that the ITS 1 -F and ITS 4 priming sites in O. griseum and the other two fungi be sequenced, using primers that bracket the primer sites. (2) Alternatively, the D N A of O. griseum may fold in such a way that the priming site is less well exposed in O. griseum than in S9 or S246, again resulting in preferential amplification of the sterile isolates. Experimenting with different temperatures to change the specificity of the primers or with different sets of primers might give interesting results. 2.6 Conclusion The results obtained from this research showed that both cultural isolation and PCR amplification, two commonly used methods for identification of ericoid mycorrhizal fungi, have 31 their own advantages and disadvantages. Using only isolation by culturing for identification of mycorrhizal fungi from field collected roots will probably overlook the number and diversity of sterile fungi while favoring the identification of sporulating fungi (i.e. Oidiodendron griseum). PCR-RFLP identification directly from field collected roots will fail to detect occasional fungi that have had substitutions in their PCR binding sites, but will generally work better than morphology-based methods for the detection of sterile fungi. The bias observed in both methods should influence the future design of field studies. The ecosystem surrounding the salal roots contains numerous mycorrhizal and nonmycorrhizal fungi which have the potential to colonize the root cells. Since the hyphae infect individual cells without colonizing neighboring cells, the roots possibly have "vacant cells" available for colonization by a second fungus. We should not assume that the roots have a single mycorrhizal fungus. According to the information obtained from this study, both molecular and cultural methods should be used to minimize the bias in the final results. One possible approach is to first isolate the mycorrhizal fungi from field collected roots using culture methods and then use these cultures for PCR-RFLP identification using the synoptic key generated by Monreal (1996). 32 Table 2.1 Fungal isolates used in this study, source of isolate, mycorrhizal status and isolation data. Fungal species Source Mycorrhizal status Isolation data Oidiodendron Xiao, + Roots of Gaultheria shallon, griseum U B C Vancouver Island, B.C., Canada Unknown 1 Xiao, + Roots of Gaultheria shallon, U B C S9 Vancouver Island, B.C., Canada Unknown 2 Xiao, + Roots of Gaultheria shallon, U B C S246 Vancouver Island, B.C., Canada Table 2.2 Various concentrations of Oidiodendron griseum D N A in pure and mixed solutions with S9 DNA. Lane No. Fungal species Total S 9 D N A 0. griseum Relative D N A ng/ul D N A ng/ul proportions of S9 ng/ul to O. griseum 1 S9, O. griseum 91 45.5 45.5 50: 50 3 S9, O. griseum 91 31 60 33: 66 5 S9, 0. griseum 91 61 30 66: 33 7 S9, 0. griseum 91 73 18 75:25 2 O. griseum 45.5 - 45.5 0: 50 4 O. griseum 60 - 60 0: 66 6 0. griseum 30 - 30 0: 33 8 O. griseum 18 - 18 0:25 Table 2.3 Various concentrations of Oidiodendron griseum D N A in pure and mixed solutions with S246 D N A . Lane No. Fungal species Total D N A ng/ul S246 D N A ng/ul O. griseum D N A ng/ul Relative proportions of S246 to O. griseum 1 S246, O. griseum 91 45.5 45.5 50: 50 3 S246, 0. griseum 91 31 60 33: 66 5 S246, 0. griseum 91 61 30 66: 33 7 S246, 0. griseum 91 73 18 75:25 2 O. griseum 45.5 - 45.5 0: 50 4 0. griseum 60 - 60 0: 66 6 O. griseum 30 - 30 0: 33 8 O. griseum 18 - 18 0: 25 Table 2.4 Various concentrations of S9 D N A in pure and mixed solutions with S246 D N A Lane No. Fungal species Total D N A ng/ul S 9 D N A ng/ul S246 D N A ng/ul Relative proportion of S9 to S246 1 S9, S246 91 45.5 45.5 50: 50 3 S9, S246 91 31 60 33: 66 5 S9, S246 91 61 30 66: 33 7 S9, S246 91 73 18 75:25 2 S246 45.5 - 45.5 0: 50 4 S246 60 - 60 0: 66 6 S246 30 - 30 0: 33 8 S246 18 - 18 0: 25 Table 2.5 Identified ericoid mycorrhizal fungi from colonized salal roots using traditional cultural isolation techniques and molecular methods. The percentage recovery of the fungi using both cultural techniques and mo ecular methods are included. Inocula Cultural isolation Molecular technique S246, 0. griseum 1 (2%) pure 0. griseum 17(28%) pureS246 42 (70%) mixed cultures of S246 and 0. griseum 36 (100%) S246 S9, 0. griseum 13 (31%) pureS9 29 (69%) mixed cultures of S9 and O. griseum 35 (100%) S9 S9, S246 53 (88%) pure S9 7 (12%)2 mixed cultures of S9 and S246 26 (62%) pure S9 16(38%) both S9 and S246 2 Initially all cultures were similar and they looked as if they were all pure cultures of S9, however, on further observation colonies of S246 could be recognized. 35 c O 0s-L/1 O 0s-0s* 00 O a-o 0s-O -U> 5 T/3 00 o 0s-o 0s-•IS. 0s-O to 0s-o ST n C/3 35 ON « 3 ON ON ON *9 •9 1/3 (to (to O 1/3 NO ON Ore & 3 O ore C/3 NO a-s 3 H as CD 03 o n Fig. 2.1 A mapped root of salal. Roots segments to be used for either isolation by culturing or D N A amplification were chosen from different parts, according to the color scheme shown. Roots from below S246 inoculum are shown in black, roots from below S9 inoculum are shown in blue, and roots from below both inocula are shown in red. 37 Fig. 2.2 Mixed cultures of S246 and Oidiodendron griseum recovered from root segments from in vitro mycorrhizae. Ericoid mycorrhizal fungi were isolated from 5 mm root segments of salal seedlings that had been inoculated with S246 and O. griseum. A - D . The relative proportions of S246 to O. griseum differed in different root segments. The square patch on top was the initial inoculum. The gray patches are O. griseum. 39 3 era" It* a oo C O >-t> 00° O o 13 O 3 o C I + N> ON o TO s o a TO CD 00 00 i-i •a CD a T3 o n « ' oo 00 o oo' 02. <' as lett n> a 13 o on o i-l o> CD he ri; d fro er to (JQ cr 3 i a 5 - ^ oo T3 n> o <• 5' o o 00 >1 ST ^ 00 1 00 |—I CfQ -3 .fif o S g 3 &«Q O M 3 . (I m P 3, » » 3 & S, Fig. 2.4 Mixed cultures of S9 and Oidiodendron griseum recovered from root segments of in vitro mycorrhizae. Ericoid mycorrhizal fungi were isolated from 5 mm root segments of salal seedlings that had been inoculated with S9 and O. griseum. A-D. The relative proportions of S9 to O. griseum differed in different root segments. The square patch on top was the initial inoculum. The gray patches are O. griseum. 42 43 op Ifl ft TO C/3 c/3 O •+> O E? P O O 13 P~ GO O' TO s o s TO s 3 03 H i i — °a M TO ST re fa 1 00 TO TO TO C/3 N O 1' O C O N O 3 cr TO o S-o' TO 3 © a to TO s o ^ 1. * H £L e? SL TO O p 2. a 2* o £ 3 r " + ' o 13 o on TO CTQ 3 TO 13 C/3 TO 1-1 TO C/3 T3 TO O <' TO P -TO i-t cr o 8 o 5* 2 3 3 & & o o & p TO l-P o TO >-! TO C/3 TO 13 C/3 tt» O O era H 2 TO i-i o K c/3 X3 (3 . . P TO TO » o 2, 5 P TO TO* <*> M P EL P § & B > Z S9 »-t TO i-i C L TO TO |3 TO hj, i t £• 3 o S. o era N ' TO SL Fig. 2.6 Mixed cultures of S246 and S9 recovered from root segments of in vitro mycorrhizae. Ericoid mycorrhizal fungi were isolated from 5 mm root segments of salal seedlings that had been inoculated with S246 andS9. The square patch on top was the initial inoculum. The S9 mycelium grew fastest and was usually obvious while the S246 mycelium was often small and difficult to find. The small shinny patch on the agar is S246 45 46 3 era* CD o o C B <-t fa CO O O 3 + S O p. G O K ) O N 03 & "a 0 1 ST* 00 CD CD CD Replicates III Unde Root r S246 No. W * * II -• •. tft • ON Unde Root llll • 1^ 2 •» o cz> • SO 00 SO 1—' o 1 i mi - Unde Root 1 h-1 ts> r S246 and S9 No. | w r S246 and S9 No. r S246 and S9 No. llll 11-' 1 'Ji 4i> 1 2 3 4 5 6 7 8 9 10 11 12 13 M Fig. 2.8 RFLP results from mixed cultures of S9 and Oidiodendron griseum. RFLP pattern of PCR products of fungal and ericoid mycorrhizal DNA, from salal seedlings inoculated with S9 and O. griseum, using the restriction enzyme Msp I. M = molecular fragment size marker; lanes 1-12 = digested mycorrhizal D N A ; lane 13 = digested fungal D N A of S9. 48 Fig. 2.9 RFLP results from mixed cultures of S246 and Oidiodendron griseum. RFLP pattern of PCR products of fungal and ericoid mycorrhizal D N A , from salal seedlings inoculated with S246 and O. griseum, using the restriction enzyme Msp I. M = molecular fragment size marker; lanes 1-7 = digested mycorrhizal D N A ; lane 8 = digested fungal D N A of S9; lane 9 = digested fungal D N A of S246; lane 10 = digested fungal D N A of O. griseum. Fig. 2.10 RFLP results from mixed cultures of S9 and S246. RFLP pattern of PCR products of fungal and ericoid mycorrhizal DNA, from salal seedlings inoculated with S9 and S246, using the restriction enzyme Msp I. M = molecular fragment size marker; lanes 1-9 = digested mycorrhizal D N A ; lane 10 = digested fungal D N A of S246. Lanes 7, 8, 9 show RFLP patterns indicating the presence of both S9 and S246. The arrows indicate the bands generated by S9. 1 2 3 4 5 6 7 8 M Fig. 2.11 RFLP results from a mixture of S9 and Oidiodendron griseum DNA. RFLP pattern of PCR products obtained using fungal D N A mixture of S9 and O. griseum D N A at various concentrations, using the restriction enzyme Msp I. M = molecular fragment size marker; lane 1 = O. griseum at the same concentration as S9 (50:50); lane 2 = 0. griseum alone at the same concentration as in lane 1; lane 3 = O. griseum at twice the concentration of S9 (66:33); lane 4 = 0. griseum alone at the same concentration as in lane 3; lane 5 = O. griseum at half the concentration of S9 (33:66); lane 6 = O. griseum alone at the same concentration as in lane 5; lane 7 = O. griseum at a quarter of the concentration of S9 (25:75); lane 8 = 0. griseum alone at the same concentration as in lane 7. 51 1 2 3 4 5 6 7 8 M Fig. 2.12 RFLP results from D N A mixture of S246 and Oidiodendron griseum. RFLP pattern of PCR products obtained using fungal D N A mixture of S246 and 0. griseum D N A at various concentrations, using the restriction enzyme Msp I. M = molecular fragment size marker; lane 1 = O. griseum at the same concentration as S246 (50:50); lane 2 = 0. griseum alone at the same concentration as in lane 1; lane 3 = O. griseum at twice the concentration of S246 (66:33); lane 4 = 0. griseum alone at the same concentration as in lane 3; lane 5 = O. griseum at half the concentration of S246 (33:66); lane 6 = 0. griseum alone at the same concentration as in lane 5; lane 7 = O. griseum at a quarter of the concentration of S246 (25:75); lane 8 = 0. griseum alone at the same concentration as in lane 7. 52 1 2 3 4 5 6 8 M Fig. 2.13 RFLP results from D N A mixture of S9 and S246. RFLP pattern of PCR products obtained using fungal D N A mixture of S9 and S246 D N A at various concentrations, using the restriction enzyme Msp I. M = molecular fragment size marker; lane 1 = S246 at the same concentration as S9 (50:50); lane 2 = S246 alone at the same concentration as in lane 1; lane 3 = S246 at twice the concentration of S9 (66:33); lane 4 = S246 alone at the same concentration as in lane 3; lane 5 = S246 at half the concentration of S9 (33:66); lane 6 S246 alone at the same concentration as in lane 5; lane 7 = S246 at a quarter of the concentration of S9 (25:75); lane 8 = S246 alone at the same concentration as in lane 7. 53 p O M B B-CD P S P x <*» ce a C/5 4*. O N SO O N to so o 2! > 2 > 2! > o o o o o o o o o O O O 0s* 2 2 > 2 > O N 01 O 2 > 2 > 21 > v o O o o o o ST a so tZ! so 4^. O N 4A. O N O N CZ2 * » . O N s (to p in SO CZ> SO (to P (to C/3 SO 4*. O N (Z! K * 4A. ON r s \ TO a P (to !Z3 SO a I a 1-TO & 3 P Orci CHAPTER 3: IN VITRO COMPETITIVE STUDIES AMONG THREE ERICOID MYCORRHIZAL FUNGI OF GAULTHERIA SHALLON 3.1 Introduction A l l organisms in their natural habitats come into proximity or contact with a diverse group of other organisms and those interactions between species influence the organization of communities. In natural environments, both interactions among fungi and between fungi and other organisms play an important role in the recycling process that will eventually affect the dynamics of all organisms in the community (White and Boddy 1992). This chapter examines intra- and interspecific interactions between ericoid mycorrhizal fungi colonizing roots of salal plants (Gaultheria shallon Pursh). If the presence of one organism in some way affects the performance of another organism the two organisms are said to be interacting with each other. When mycelia of different species (interspecific interaction) or the same species (intraspecific interaction) come into proximity or contact with each other, there may be different responses (Cooke and Rayner 1984). Three possible interactions are as follows: (1) neutral intermingling of hyphae; (2) deadlock, in which neither mycelium can enter the other's domain; and (3) partial or complete replacement of one mycelium by the other. Such interactions among fungi may have implications for biological control. For example, to control decay in freshly-felled pine, instead of using chemical treatments, sixteen isolates of microfungi, obtained from the same environment, were screened for inhibitory activities on some wood decay fungi (Schoeman et al. 1994) and the results showed that at least four of the biological treatments, including Trichoderma harzianum Rifai, Cryptosporiopsis tarraconensis Gene and Guarro, Ascocoryne sarcoides (Jacq. ex Gray) Groves and Wilson, and Potebniamyces 55 coniferarum (Hahn) Di Cosmo, showed successful establishment in the field-test logs and were able to significantly inhibit the growth of wood decay fungi (Schoeman et al. 1994). In an attempt to find biological control agents for the blue stain fungus, Ceratocystis coerulescens (Munch) Bakeshi (which attacks felled logs) other fungal species were screened to see if their metabolic products would inhibit the growth of the pest species (Croan and Highley, 1991). The results showed that four of the fungal species tested, Antrodia (= Poria) carbonica (Overh.) Ry v. et Gilbn., Bjerkandera adusta (Wild.: Fr.) Karst, Gloeophyllum trabeum (Pers. Ex. Fr.) Murr., and Neolentinus (=Lentinus) lepideus (Fr.: Fr.) Readhead & Ginns, not only killed but also decolorized the blue stain (Croan and Highley 1991). Interactions between fungi and their symbiotic host plants may affect competitive abilities with other plant species. In mycorrhizal association, the close proximity of root cells and fungal hyphae enables the fungus to absorb and transport nutrients to the host. Mycorrhizal association plays an important role in plant survival, not only by providing enhanced nutrient uptake, but also by providing access to organic nutrients, protection from diseases, and detoxification of heavy metals (Read 1991). Mycorrhizae have been shown to be influential in plant-plant competition especially in exploitation competition where available resources are limited. The presence and activities of mycorrhizal fungi may improve the competitiveness of the host plant. In vitro study of interactions among ericoid mycorrhizal fungi of salal and the ectomycorrhizal fungi of western hemlock (Tsuga heterophylla (Raf.) Sarge.) showed that all the ectomycorrhizal fungi tested were inhibited to some degree by one or other of the ericoid mycorrhizae of salal, whereas none of the ericoid mycorrhizal roots of salal were inhibited by the ectomycorrhizal fungi of western hemlock. This negative influence of ericoid mycorrhizal fungi 56 of salal on ectomycorrhizal fungi of western hemlock may be a part of a possible explanation for the success of salal in cutblocks compared to the poor growth of western hemlock (Xiao 1992). The objective of this study was to examine the in vitro interactions among ericoid mycorrhizal fungi of salal. Three species of fungi were used in the study: Oidiodendron griseum, an identified sporulating species, and two unidentified sterile isolates that were found to colonize salal roots by Xiao 1992. I recognize that laboratory tests do have drawbacks because artificial situations are used to predict performance in the field; nevertheless, laboratory tests are easy to perform and provide the first indication of the possible interactions between the species examined. Studying the effects these fungi have on each other's growth in vitro may improve our understanding of the interactions of these fungi in nature. 3. 2 Materials and Methods 3.2.1 Fungal isolates The three ericoid mycorrhizal fungi of salal used in this study were Unknown 1 (isolate S9), unknown 2 (isolate S246), and Oidiodendron griseum. The first two are the unidentified, non-sporulating isolates, and will be referred to by their strain numbers. A l l isolates were maintained on petri dishes on modified Melin Norkran agar (MMN). 3.2.2 Dual cultures To examine the interactions among different ericoid mycorrhizal fungi, two growth media, normal M M N and buffered M M N containing 20 m M 2-[N-morpholino]ethanesulphonic acid (MES), were used. As fungi grow, they change the pH of their media. To test the effect of pH, 57 normal and buffered M M N were used. The pH of both media was adjusted to 6 before autoclaving. Media were solidified using phytagel (Sigma Chemical Co. P.O. Box 14508, St. Louis, M O 63178, USA). Each ericoid mycorrhizal fungus of salal was grown opposite to another ericoid mycorrhizal fungus of salal and each fungus was grown opposite to itself on both media ( M M N , MES). Fungal plugs of about 3 mm in diameter were cut from the edge of a fungal colony with a cork borer and placed 3 cm away from the competing colony (Figs. 3.1, 3.2). Three replicates were used for each treatment. A l l of the cultures were incubated at about 25 C in the dark. The cultures were observed periodically for up to 4 months, until the hyphae of the paired fungi met. 3.2.3 Observations and measurements To determine colony expansion, mycelial growth towards and away from the interacting colony was measured. Colony expansion was statistically compared for interstrain competitions versus self-paired controls to determine if fungal growth was affected by competition among fungal strains. The data were analyzed using a two way analysis of variance (ANOVA) with species combination (e.g., S9 vs. S246) and gel type (e.g., M M N or MES = buffered M M N ) as factors. Growth towards and away from the interacting colony were analyzed separately. To pinpoint the source of significant differences, Tukey's tests (Zar 1984) were performed to determine which means were different. When the A N O V A showed a significant difference among species combinations but not among gel types, the data were pooled across gel types prior to performing Tukey's tests on species combinations (n=6). When the A N O V A showed significant differences both among species combinations and among gel types, Tukey's tests were performed on each gel and species combination mean separately (n=3). In addition to the statistical 58 analysis, the cultures were observed for signs of mycelial mtermingling or overlapping, colony restriction, or any other kind of visible interactions on the surface of the agar. 3.3 Results Measurements (the mean of three replicates) of mycelial expansion of fungal colonies towards and away from competing colonies in dual cultures are presented in Tables 3.1-3.3. Results from the statistical analysis used in this chapter were mainly clustered in the two categories explained below. The growth of Oidiodendron griseum either towards or away from the other potentially interacting colony was not significantly different from when O. griseum was paired with itself, when paired with either S9 or with S246 (Table 3.1). However, the Tukey test showed that mycelial growth of O. griseum was significantly less towards S246 than towards S9. Whether the growth medium was buffered or not did not make a significant difference in the growth of O. griseum. Mycelial growth of S246 in interstrain dual cultures was not significantly different from the self-paired control except that mycelial growth towards Oidiodendron griseum was significantly increased compared to both S9 and the self-paired control. Although there was no significant difference in growth observed between the normal and buffered M M N (MES), there was a significant interaction term so the data were analyzed unpooled for the Tukey test. Mycelial growth of S9 both towards and away from the interacting colony was influenced by the presence of an interspecific competitor. There was, however, a significant difference between gel types for growth towards the interacting colony. The data were therefore pooled across gel types for growth away from the interacting colony but were unpooled for growth 59 towards the interacting colony in Tukey's tests. The results are presented in Table 3.3. There was a significant increase in growth of S9 away from S246 compared to both Oidiodendron griseum and the self-paired control (pooled across gel types). The significance of this result, however, might be questioned because there is a significant interaction term in the A N O V A for this comparison. On M M N , but not on MES, there was a significant increase in growth of S9 towards O. griseum compared to both S246 and the self-paired control (unpooled data). The growth of Oidiodendron griseum either towards or away from the other potentially interacting colony was not significantly different from when O. griseum was paired with itself, when paired with either S9 or with S246 (Table 3.1). However, the Tukey test showed that mycelial growth of O. griseum was significantly less towards S246 than towards S9. Whether the growth medium was buffered or not did not make a significant difference in the growth of O. griseum. Mycelial growth of S246 in interstrain dual cultures was not significantly different from the self-paired control except that mycelial growth towards Oidiodendron griseum was significantly increased compared to both S9 and the self-paired control. Again, no significant difference in growth was observed between the normal and buffered M M N (MES). Mycelial growth of S9 both towards and away from the interacting colony was influenced by the presence of an interspecific competitor. There was, however, a significant difference between gel types for growth towards the interacting colony. The data were therefore pooled across gel types for growth away from the interacting colony but were unpooled for growth towards the interacting colony in Tukey's tests. The results are presented in Table 3.3. There was a significant increase in growth of S9 away from S246 compared to both Oidiodendron griseum and the self-paired control (pooled across gel types). The significance of this result, 60 however, might again be questioned because there is a significant interaction term in the A N O V A for this comparison. On M M N , but not on MES, there was a significant increase in growth of S9 towards O. griseum compared to both S246 and the self-paired control (unpooled data). 3.4 Discussion 3.4.1 Deadlock When mycelia of different or the same species are paired in the laboratory, different responses, such as growth inhibition, replacement, or deadlock may occur. The interactions observed in this study all fall into one category, deadlock. The term deadlock, described by Cooke and Rayner (1984) was used in this study. Deadlock occurs when neither mycelium can invade the territory occupied by the other one (Figs. 3.1, 3.2, 3.3, 3.4). Deadlock may be due to competition for nutrients, release of fungistatic (antagonistic) compounds, or extracellular enzyme localization (Cooke and Rayner 1984; White and Boddy 1992). Deadlock has been reported among fungi in previous studies (Shearer and Zare-Maivan 1988; White and Boddy 1992; Xiao 1992). White and Boddy (1992) reported deadlock after one month of incubation of Phlebia radiata (Fr.) vs. Stereum hirsutum (Wild: Fr. Gray) on malt extract agar. They observed the production of aerial mycelia at the meeting point of the colonies, which was associated with high enzyme activity. During confrontation, the production of laccase-a-naphthol and peroxidase was detected. 3.4.2 Mycelial expansion The results of the experiments on growth of interacting colonies, combined with the results from paired fungi growing in synthetic mycorrhizae, are consistent with the hypothesis 61 that deadlock resulted from nutrient limitations or metabolic waste products. If fungi released species specific antagonistic substances when confronted with a mycelium from a different species, then the inhibited species should grow better when paired with itself than when paired with the antagonistic fungus. However, none of the pairings showed the growth pattern expected from species specific antagonism. None of the three fungi grew better towards another colony of the same species than towards another colony of a different species (Tables 3.1, 3.2, 3.3). Both S9 and S246 in fact grew better towards mycelia of Oidiodendron griseum than towards their own mycelia (Tables 3.2, 3.3). This may be because the O. griseum mycelium grew slowly (Figs. 3.3, 3.4) and either produced fewer metabolic waste products or left more available nutrients than the faster growing mycelia of either S9 or S246. Generally speaking, mycelial growth away from the interacting colony was not significantly different in all the pairings, except in one case where the mycelial expansion of S9 was favored when paired with S246, possibly because of the production or release of secondary metabolites or compounds that favored the growth of S9. Consistent with this fast growth of S9 in the presence of S246 in dual cultures the results from PCR-RFLP identification from Chapter 2 detected the presence of S9 D N A in every single extraction from mycorrhizal seedlings inoculated with S9 and S246 (Table 2.5). Growth responses of the majority of fungi on the two media were similar, which means that the effect observed is not from changes in pH alone. Only in one case was there a statistically significant difference between the two gels, when mycelial expansion of S9 towards the interacting colonies was measured. The significant difference observed between normal M M N and buffered M M N might be biologically insignificant or it could also be due to the small number of replicates. 62 Although the assumptions of A N O V A were not met, the analysis of variance was still useful in showing which data were not significantly different and could be pooled together. Because the sample size was only three, chance variation among the samples may have resulted in the lack of homogeneity in sample variance. The violation of the assumptions of A N O V A may have been responsible for the significant difference between gels (normal M M N and buffered M M N ) in Appendix C.2 as well as the significant difference observed in "Interaction" column in Appendix C . l . A n "interaction" tests whether the combination of factors together (different gels and species) have a greater effect compared to the effect of each of them individually. Although the competition experiments showed that the ericoid mycorrhizal fungi can limit one another's growth, the results from Ch. 2 show that they can also cohabit in the same 5 mm root segment in synthesis chambers. Seventy percent of the isolated fungi from 5mm colonized salal roots were mixed cultures of Oidiodendron griseum and S9 or O. griseum and S246. If deadlock was the result of limited nutrients, then in the synthesis chambers salal roots could be providing extra food which results in cohabitation of both fungi without showing the deadlock interaction. 63 Table 3.1 Measurements of mycelial expansion of Oidiodendron griseum in dual culture Mycelial growth away from interacting colony (mm) Mycelial growth toward interacting colony (mm) Opposing Fungi x (S.E.) x (S.E.) 10.1 (0.5) a 8.5 (0.55) ab Oidiodendron griseum 9.5 (0.5) a 10 (0.3) a S9 9.7 (0.45)a 7.3 (0.5) b S246 A two way analysis of variance showed no significant difference between growth media, so all the data from M M N and MES were pooled together; Tukey test performed on the pooled data showed that the means (pooled data) within the column with the same letters are not significantly different at a = 0.05. Table 3.2 Measurements of mycelial expansion of S246 in dual culture4 Mycelial growth away from interacting colony (mm) Mycelial growth toward interacting colony (mm) Opposing Fungi x (S.E.) x (S.E.) 21.8 (1.7) a 12.2 (0.3) a S246 21.8 (0.65) a 12.8 (0.25) a S9 22.3 (0.4) a 14.1 (0.6) b Oidiodendron griseum A two way analysis of variance showed no significant difference between growth media, so the data from M M N and MES were pooled together; Tukey test performed on the pooled data showed that the means (pooled data) within the column with the same letters are not significantly different at a = 0.05. 3 Typical calculations are presented in Appendix A 4 Typical calculations are presented in Appendix B Table 3.3: Measurements of mycelial expansion of S9 in dual culture5 M M N (phytagel) MES (phytagel) Mycelial growth away from interacting colony (mm) Mycelial growth toward interacting colony (mm) Mycelial growth away from interacting colony (mm) Mycelial growth toward interacting colony (mm) Opposing Fungi x (S.E.) x (S.E.) x (S.E.) x (S.E.) 20.3 (0.3) 1 11.7 (0.3) 3 18.3 (0.9) 5 11.0(0) 7 S9 20.7 (0.7) 2 12.7 (0.7) 22.7 (0.3) 6 11.3 (0.9) 7 S246 20.3 (0.9) 1 14.0 (0.6) 4 18.7 (0.7) 5 11.7 (0.3) 7 Oidiodendron griseum A two way analysis of variance showed a significant difference between gels for mycelial growth towards the interacting colony. Tukey's tests were performed on the individual (unpooled) data. The following pairs of means are significantly different at a=0.05, mycelial growth of S9 away from S9 and away from O. griseum (') is significantly different than mycelial growth of S9 away from S246 (6); mycelial growth of S9 away from S246 (6) is significantly different than mycelial growth of S9 away from S9 (5) and away from O. griseum (5); mycelial growth of S9 away from S246 (2) is significantly different than mycelial growth of S9 away from S9 (5). n=3. The following pairs of means are significantly different at a=0.05; mycelial growth of S9 towards O. griseum (4) is significantly different than mycelial growth of S9 towards S9 (3); mycelial growth of S9 towards O. griseum (4) is significantly different than mycelial growth of S9 towards S9 (7), S246 (7) and, towards O. griseum (7). 5 Typical calculations are presented in appendix C 65 Fig. 3.2 Deadlock interaction on M M N (phytagel), shown between an S9 colony and an S246 colony four months after inoculation. The colony on the right side is S246. Fig. 3.3 Deadlock interaction on M M N (phytagel), shown between colonies of Oidiodendron griseum and S9 four months after inoculation. The colony on the right side is O. griseum. Fig. 3.4 Deadlock interaction on M M N (phytagel), shown between colonies of Oidiodendron griseum and S246 four months after inoculation. The colony on the right side is O. griseum. CHAPTER 4: OPTIMIZATION OF GELLING AGENT CONDITIONS 4.1 Introduction From the early days, mycorrhizal studies have been associated with experiments in laboratory culture systems. In order to confirm mycorrhizal ability of an isolated fungus, the fungus in question was inoculated onto a sterile plant. The ability of the isolated fungus to form mycorrhizae on other plants was tested, especially in studies regarding fungal specificity. These studies began as early as 1937, when Bain planted sterile cranberry seeds in test tubes on 1% agar. After seed germination and root penetration into the agar, the seedlings were inoculated with mycorrhizal fungi and checked for entry of fungal hyphae into the cortical root cells and for the presence of hyphal complex structures. Mycorrhizal research continues to employ the culture media approach to confirm mycorrhizal abilities of field isolated fungi through back inoculation (Pearson and Read 1973; Couture and Dalpe 1983; Stribley and Read 1976; Dalpe 1986; Xiao 1992; Chabot et al 1994; Monreal 1996). Synthesis chambers designed by Xiao (1992) provide a closed and sterile environment where one can observe the interactions between plants and mycorrhizal fungi (mainly colonization), without opening the plate. Mycorrhizal colonization can be observed in roots in the growth medium inside the closed synthesis chamber using a compound microscope at 100X. One of the advantages of using this closed system is that chances of contamination are reduced. Another advantage is that the nutrient content of the medium can be manipulated and the effects of different nutrients on the studied mycorrhizal fungus can be observed. One limitation of this system is that, using the standard medium, visibility is limited. Improving the visibility of the agar to the extent that most of the root systems could be more clearly observed would help us confirm ericoid mycorrhizal formation. 68 In this study gellan gum, or phytagel, was used as a gelling agent to improve the optical properties of the medium as suggested by Kang et al. (1982) and Shungu et al. (1983). Kang et al. (1982) reported that anew species of Pseudomonas, isolated from plant {Elodea spp.) tissue collected in Pennsylvania, produced a polysaccharide (PS-60) after incubation for 3 days at 30 C in media containing 3% glucose as a carbon source. This polysaccharide displayed several interesting properties, including self-gelling in the presence of a cation, thermal stability, and optical clarity. Shungu et al. (1983) looked at the possible applications of gellan gum (formerly known as PS-60 and S-60) in bacteriology. They used fifty different bacterial species representing a total of 26 genera for testing routine medium applications and concluded that gellan gum can be substituted for agar. The successful application of gellan gum in bacteriology led to studies where gellan gum was used as a gelling agent for other microorganisms. In another report where bacto agar was replaced by phytagel (gellan gum), the entire life cycle of Glomus intraradix, a vesicular-arbuscular mycorrhizal fungus, was studied in culture. The growth of the initial germ tube, formation of the mycelial network and, finally spore formation were observed successfully (Chabot et al, 1994). In this study phytagel was used at a concentration of 0.4% for the establishment of vesicular arbuscular mycorrhizal association with carrot roots (Chabot et al. 1992). In the present study phytagel was used at 0.8% for the establishment of ericoid mycorrhizal association. A preliminary experiment showed that when phytagel was used at 0.4% or 0.6% the media was not solid enough. The objective of this chapter was to assess whether phytagel could substitute for granulated agar in synthesis chambers described in Chapter Two. To test for the possible effects 69 of phytagel vs. granulated agar, percent root colonization and shoot dry weight of salal seedlings were measured. 4.2 Materials and methods 4.2.1 Media and Random sampling The basal medium used in this study was M M N with the exclusion of mineral nitrogen, malt extract, and glucose and the addition of glutarnine as a nitrogen source. The M M N containing basic salts was prepared with either phytagel or granulated agar. Salal seedlings were then transferred to both media. For this experiment, the same synthesis chamber as described in Chapter 1 was used. After leaf emergence, plates were inoculated with about 3 mm diameter fungal plugs from the edge of a fungal colony. Each seedling was inoculated with two different fungi at the same time. The plates were then sealed with Parafilm and placed in a growth chamber at 21° C, 13 hr light at 128 umol m"2 sec"l and 16° C, 11 h dark. During the next five to six months, the plates were examined under the light microscope for colonization. To eliminate any bias in favor of either granulated agar or phytagel, the plates were randomly assigned alphabet letters. The randomly marked plates were then used for the percent root colonization and shoot dry weight experiments. 4.2.2 Percent root colonization To test for differences in percent root colonization, the roots were mounted on slides. A l l the roots were mounted in lactic acid on the same day [at the same time] and kept for further observation. Slides prepared with lactic acid can be kept for at least 2-3 weeks, which provides sufficient time to determine the percentage of root colonization. In each plate the whole root 70 system was harvested by gently pulling the root out of the agar, rinsing it in distilled water, cutting it into 1 to 1 1/2 cm segments, and finally mounting the roots in lactic acid. Percent colonization can be assessed in two ways: root colonization and cell colonization. For percent cell colonization, 100 contiguous cells within each colonized root can be counted as colonized or not colonized. For percentage root colonization, root pieces per sample can be counted as colonized or noncolonized (Xiao 1992). In this experiment, root pieces were examined under a compound microscope and percent root colonization was assessed by counting at least 100 cortical cells as colonized or noncolonized. The data were then analyzed by two factor analysis of variance (ANOVA) with species combination and gel types as factors. 4.2.3 Shoot dry weight To test for the possible effect of phytagel versus granulated agar on the general growth pattern of salal seedlings shoot dry weights were measured. To test for the differences in growth of seedlings in culture, the shoot dry weight of each salal plant was measured. Colonized salal seedlings were cut at the root collar, transferred to separate paper bags, and dried in an oven at 56 C for 24 hours. The shoot dry weight was then measured immediately using a sensitive balance. The data were then recorded with respect to the original plates where the shoots came from and analyzed by a two way A N O V A . 4.3 Results Visual observation of salal seedlings in phytagel showed improved visibility through the agar as well as increased shoot and root growth. The results from a two way A N O V A comparing the effect of different gel types on shoot growth, as well as differences between species, showed 71 that there was a significant increase in growth using phytagel. Statistical analysis thus supported the visual observation that the shoot growth rate of salal seedlings grown on M M N prepared with phytagel was increased (Table 4.1). The results from a two way A N O V A comparing the effect of the two gels on percent root colonization, as well as the difference between species, showed no significant difference (Table 4.2). 4.4 Discussion The results from this experiment showed that granulated agar can be replaced by phytagel at the same concentration in the media. Furthermore, statistical analysis showed that shoot dry weight increased significantly in the media prepared with phytagel (appendix D. l ) . At the present time, it takes several weeks to grow salal plants in culture. The increase in growth rate of salal plants may expedite experiments to test the effects of other parameters on salal in culture. For example, the effects of light on salals' root or shoot system or the influence of different nutrients on the growth of salal can be tested. Statistically there was no significant difference in percent root colonization when two different types of agar were used. Considering that the number of replicates included in this study was limited and that the variances between the data shown in appendix D.2 were high, further work will be required to verify the effect of phytagel on percent root colonization. The improved optical properties of the medium made it easier to observe the colonized cells in the mycorrhizal roots, and it is encouraging that, to the extent that it had an effect, phytagel generally increased root colonization. However, phytagel currently costs twice that of granulated agar. 72 There was no specific experiment designed to assess the effect of phytagel on fungal growth but visual observation clearly showed that fungal growth was faster and denser on media containing phytagel. The normally slow growth rate of mycorrhizal fungi in culture can be improved and facilitated by keeping the fungi on M M N prepared with phytagel. This will help me prepare cultures with good mycelial growth in a shorter period of time for further experiments. 73 Table 4.1 Measurements of shoot dry weight of salal inoculated with two known ericoid mycorrhizal fungi M M N (granulated agar) x (S.E.) M M N (phytagel) x (S.E.) Fungal treatment 4.6 (0.5) 8.5 (2.4) S9-S246 4.3 (0.3) 10.6(2.8) Oidiodendron griseum - S9 4.5 (0.2) 10.2 (4.3) Oidiodendron griseum - S246 Two way analysis of variance showed a significant difference between the two gel types ( M M N prepared with phytagel or granulated agar) at a=0.05. n=3. Table 4.2 Measurements of percent root colonization of salal inoculated with two known ericoid mycorrhizal fungi. M M N (granulated agar) x (S.E.) M M N (phytagel) x (S.E.) Fungal treatment 71 (8.1) 74.3 (3.8) S9- S246 57.3 (7.5) 65.7 (3.18) O. griseum - S9 65 (6.0) 76.3 (4.4) O. griseum - S246 Two way analysis of variance showed a significant difference between the two gel types ( M M N prepared with phytagel or granulated agar) at a=0.05. n=3. CHAPTER 5: GENERAL CONCLUSIONS AND DISCUSSION Monreal (1996) used molecular methods to identify mycorrhizal fungi directly from colonized field roots, and she found that more than one fungal species was present in a small segment of the root. Similarly, Perotto et al. (1996) found high diversity of mycorrhizal fungi on a small scale within the roots of the ericaceous shrub, Calluna vulgaris. The present research also showed that i f a salal seedling is in close proximity with two different fungi, both fungal endophytes can colonize a very small root segment. In fact, isolation by culturing showed that almost 70% of the isolates were mixed cultures when one of the fungal inoculum used in the experiment was Oidiodendron griseum. A possible explanation is that, since O. griseum has the ability to sporulate in culture, the spores dispersed all over the plate can act as an inoculum source and the fungal hyphae grown from these spores can enter salal roots and colonize them. In this way, the slow growth rate of O. griseum compared to that of S9 and S246 would be compensated for by the fact that O. griseum sporulates in culture while the fungal hyphae belonging to S9 and S246 must grow from the original inoculum till they meet other parts of a salal root system. This experiment once again emphasized the fact that different ericoid mycorrhizal fungi can be found in close proximity within a small segment of salal root. This finding is quite understandable considering the fact that in forming ericoid mycorrhizae, the fungal hyphae form a loose weft on the root surface and penetrate inside the cortical cells with a single entry point, without entering adjacent cells (Harley and Smith 1983; Xiao and Berch 1996). In ectomycorrhizae, on the other hand, the fungal mantle provides protection from root pathogens and other mycorrhizal fungi by forming a mat around the plant root and so the chances of recognition and penetration by other fungal hyphae or root pathogens become very slim. 75 Observations on ericoid mycorrhizal fungi that suggested high diversity on a small scale (Monreal 1996; Perotto et al. 1996) led to my next experiment, where I mapped colonized roots and identified the fungal isolates with regard to their location on the map. Bonfante-Fasolo (1980) used transmission electron microscopy and observed that fungal hyphae colonizing some of the cortical cells of Calluna vulgaris have dolipore septa and the hyphal wall consists of a single dense electron layer, indicating the presence of a basidiomycete. Other cortical cells in the same root were colonized with an ascomycete. This thesis presents the first fine scale analysis of the distribution and colonization patterns of ericoid mycorrhizal fungi in culture. In general, the results did not show any specific patterns and it was obvious that, i f the fungal hyphae were in close proximity to the uncolonized cortical cells, they were able to recognize, penetrate, and proliferate inside the cells. Almost 81% of the roots collected from under both inocula were mixed cultures, indicating that, given the same opportunity, both fungal endophytes can colonize a small root segment. The competition studies I performed showed deadlock between all three ericoid mycorrhizal fungi, due possibly to competition for nutrients, or release of antagonistic compounds (Cooke and Rayner 1984). However, mixed inoculation culture studies showed that none of the fungi prevent the entrance and colonization of the other fungi in adjacent cortical cells. Almost 65%> of the fungi isolated from roots under Oidiodendron griseum were mixed cultures, confirming the fact that different ericoid mycorrhizal fungi of salal can cohabit a small root segment. In contrast, almost 89% of the isolated endophytes from seedlings inoculated with S9 and S246 appeared to be pure cultures of S9. One possible explanation is that S9 grew faster than S246, which resulted in more cortical cells being colonized by fungal hyphae from S9. The results of competition studies showed that the growth of the entire colony of S9 was favored in 76 the presence of S246. The growth of S9 was significantly increased both towards and away from S246, indicating that S246 might release compounds or secondary metabolites in the media that favors the growth of S9. The increase in fungal growth of S9 could result in more recognition between the fungal hyphae and the cortical roots and hence, more colonization. However, the more probable explanation is that we were not able to recognize all the mixed cultures of sterile fungi. Molecular methods showed that, in 38% of the PCR amplifications, both sterile fungi were detected. The isolation by culturing, on the other hand, suggested that only 12% of the isolated fungi were from mixed cultures of sterile fungi but that 70% were from mixed cultures when a sporulating and a sterile fungus were used as inoculum. Comparing the two very different results from isolation by culturing clearly shows that the number of mixed cultures estimated by molecular methods is the one closer to reality. Again, there was no indication of any particular root colonization pattern in salal seedlings inoculated with S9 and S246. In fact, in a single salal root it was found that the first 5-mm segment was a mixed culture of the two sterile fungal isolates, the second and third 5-mm segments were pure S9, and the fourth 5-mm segment was again a mixed culture. Basically, in a salal seedling inoculated with two different fungi A and B, the roots that were located under inoculum A were not specifically colonized with fungus A . The three possible outcomes were: (1) pure cultures of A ; (2) pure cultures of B; or (3) mixed cultures of A and B. The roots that were located under both inocula with the same opportunity for colonization by both fungi often turned out to be mixed cultures. There is at least one problem associated with isolation by culturing and that is the low recovery rate of ericoid mycorrhizal fungi from highly colonized roots. Observations using light microscopy revealed that, although 91% of salal field roots were colonized (Xiao 1996), there 77 was only a 15% recovery rate of ericoid mycorrhizal fungi in culture. According to this study, isolation by culmring is good for identification of sporulating fungi such as Oidiodendron griseum but it is not ideal for identification of sterile fungi. Evidently, relying only on cultural studies would bias our final results. Molecular studies provide an alternative method for identifying nonsporulating fungal isolates and are also less time-consuming compared to cultural isolation techniques. The advantage of using molecular methods is that mycorrhizal fungi can be amplified directly from plant roots, although molecular approaches do have their own limitations. The results of the present study showed that Oidiodendron griseum D N A could be amplified and detected when present in a D N A mixture or when it was present in isolation in salal roots. 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Department of Botany, University of British Columbia, Vancouver, B.C. pp 137. 84 Xiao, G. and Berch, S. 1995. The ability of known ericoid mycorrhizal fungi to form mycorrhizae with Gaultheria shallon. Mycol. 87: 467-470. Xiao, G. and Berch, S. M . 1996. Diversity and abundance of ericoid mycorrhizal fungi of Gaultheria shallon on forest clearcuts. Can. J. Bot 74: 337-346. Zar, J. H . 1984. Biostatistical analysis, second edition. Prentice-Hall, Inc. Englewood Cliffs, New Jersey. 85 APPENDICES Appendix A. 1 Measurements of mycelial growth of Oidiodendron griseum (O.g) away from interacting colonies. Species MMN MES O.g.-O.g. 11 9 10.5 11 10 9 0.g.S9 9 9 8 11 9 11 Q.g.-S246 9 11 10 9 9 10 Anova: Two-Factor With Replication SUMMARY MMN MES Total O.g-O.g Count 3 3 6 Sum 31.5 29 60.5 Average 10.5 9.666666667 10.08333333 Variance 0.25 1.333333333 0.841666667 0.g-S9 Count 3 3 6 Sum 26 31 57 Average 8.666666667 10.33333333 9.5 Variance 0.333333333 1.333333333 1.5 0.g-S246 Count 3 3 6 Sum 28 30 58 Average 9.333333333 10 9.666666667 Variance 0.333333333 1 0.666666667 Total Count 9 9 Sum 85.5 90 Average 9.5 10 Variance 0.875 1 ANOVA Source of Variation SS df_ MS F P-value F crit Sample(Species) 1.083333333 2 0.541666667 0.709090909 0.511594 3.885290312 Columns(Gel) 1.125 1 1.125 1.472727273 0.24826188 4.747221283 Interaction 4.75 2 2.375 3.109090909 0.081669009 3.885290312 Within 9.166666667 12 0.763888889 Total 16.125 17 86 Appendix A.2 Measurements of mycelial growth of Oidiodendron griseum (O.g) towards interacting colonies. Species MMN MES O.g-O.g 9 8 9.5 9 9.5 6 O.g-SP 10 10 10 9 11 10 0.g.-S246 6 8 8 8.5 6.5 7 Anova: Two-Factor With Replication SUMMARY MMN MES Total O.g-O.g Count 3 3 6 Sum 28 23 51 Average 9.333333333 7.666666667 8.5 Variance 0.083333333 2.333333333 1.8 0.g-S9 Count 3 3 6 Sum 31 29 60 Average 10.33333333 9.666666667 10 Variance 0.333333333 0.333333333 0.4 0.g-S246 Count 3 3 6 Sum 20.5 23.5 44 Average 6.833333333 7.833333333 7.333333333 Variance 1.083333333 0.583333333 0.966666667 Total Count 9 9 Sum 79.5 75.5 Average 8.833333333 8.388888889 Variance 2.8125 1.736111111 ANOVA Source of Variation SS df_ MS F P-value F crit Sample (Species) 21.44444444 2 10.72222222 13.54385965 0.000837231 3.885290312 Columns (Gel) 0.888888889 1 0.888888889 1.122807018 0.310177822 4.747221283 Interaction 5.444444444 2 2.722222222 3.438596491 0.065987699 3.885290312 Within 9.5 12 0.791666667 Total 37.27777778 17 87 Appendix A.3: Tukey test for mycelial growth of Oidiodendron griseum (O.g) towards interacting colonies. Calculations presented in Appendix A. l and A.2 showed that there was no significant difference between the two different gel types therefore the data from each interaction were pooled together. In appendix A.2 there was a significant difference between mycelial growth of Oidiodendron griseum towards different species; in order to pinpoint the source of difference Tukey's test was performed on the pooled data. Numbers 1 to 3 are the means of data summed across or pooled together with respect to their interactions but regardless of the gel type. SE is calculated as the root of the s2 (the error mean square from the analysis of variance) divided by n (the number of data compared). (1)10 (2) 8.5 (3) 7.33 SE= (s 2 /n) 1 / 2 0.g-S9 O.g-O.g 0.g-S246 SE= (0.791/6 ) 1 / 2 = 0.36 Comparison differences SE q q0.05,12,5 1-2 10 - 8.5 = 1.5 0.36 4.16 4.508 (il= u2 1-3 10 -7.33 = 2.67 0.36 7.42 4.508 Reject ul= u3 2-3 8.5 - 7.33 = 1.17 0.36 3.25 4.508 u2= u3 88 Appendix B.l Measurements of mycelial growth of S246 away from interacting colonies. Species MMN MES S246-S246 22 25 25 18 24 17 S246-S9 19 23 22 22 22 23 S246-0.g. 23 22.5 22.5 22 21 23 Anova: Two-Factor With Replication SUMMARY S246-S246 MMN MES Total Count Sum Average Variance 3 71 23.66666667 2.333333333 3 60 20 19 6 131 21.83333333 12.56666667 S246-S9 Count Sum Average Variance 3 63 21 3 3 68 22.66666667 0.333333333 6 131 21.83333333 2.166666667 S246-0.g Count Sum Average Variance 3 66.5 22.16666667 1.083333333 3 67.5 22.5 0.25 6 134 22.33333333 0.566666667 Total Count Sum Average Variance 9 200.5 22.27777778 2.944444444 9 195.5 21.72222222 6.569444444 ANOVA Source of Variation SS df MS F P-value F crit Sample (Species) Columns (Gel) Interaction Within 1 1.388888889 23.11111111 52 2 1 2 12 0.5 I. 388888889 II. 55555556 4.333333333 0.115384615 0.320512821 2.666666667 0.891999979 0.58173267 0.110101933 3.885290312 4.747221283 3.885290312 Total 77.5 17 89 Appendix B.2 Measurements of mycelial growth of S246 towards interacting colonies. Species MMN MES S246-S246 12 11 12 12.5 12.5 13 S246-S9 13 13 13.5 12 12.5 13 S246-0.g. 14 12 16 14 15 13.5 Anova: Two-Factor With Replication SUMMARY MMN MES Total S246-S246 Count 3 3 6 Sum 36.5 36.5 73 Average 12.16666667 12.16666667 12.16666667 Variance 0.083333333 1.083333333 0.466666667 S246-S9 Count 3 3 6 Sum 39 38 77 Average 13 12.66666667 12.83333333 Variance 0.25 0.333333333 0.266666667 S246-0.g Count 3 3 6 Sum 45 39.5 84.5 Average 15 13.16666667 14.08333333 Variance 1 1.083333333 1.841666667 Total Count 9 9 Sum 120.5 114 Average 13.38888889 12.66666667 Variance 1.923611111 0.8125 ANOVA Source of Variation SS df MS F P-value F crit Sample (Species) 11.36111111 2 5.680555556 8.891304348 0.004278692 3.885290312 Columns (Gel) 2.347222222 1 2.347222222 3.673913043 0.079387551 4.747221283 Interaction 2.861111111 2 1.430555556 2.239130435 0.149148618 3.885290312 Within 7.666666667 12 0.638888889 Total 24.23611111 17 90 Appendix B.3: Tukey test for mycelial growth of S246 towards interacting colonies. Calculations presented in Appendix B.l and B.2 showed that there was no significant difference between the two different gel types therefore the data from each interaction were pooled together. In appendix B.2 there was a significant difference between species; in order to pinpoint the source of difference Tukey's test was performed on the pooled data. Numbers 1 to 3 are the means of data summed across or pooled together with respect to their species regardless of the gel type. SE is calculated as the root of the s2 (the error mean square from the analysis of variance) divided by n (the number of data compared). (1) 14.08 (2) 12.86 (3) 12.16 SE= (s2 / n) 1 / 2 S246-S246 S246-S9 S246-0.g SE= (0.638/6)"2 = 0.32 Comparison differences SE q qO.05,12,5 T 2 14.08 - 12.86 = 0.32 3.906 4.508 ul= u2 1.25 1- 3 14.08 - 12.16 = 0.32 6 4.508 Reject ul=u3 1.92 2- 3 12.86 - 12.16 = 0.32 2.093 4.508 u2= u3 0.67 91 Appendix C.l Measurements of mycelial growth of S9 away from interacting colonies. Species MMN MES S9-S9 20 20 21 17 20 18 S9-S246 20 23 22 22 20 23 S9-0.g. 22 18 20 20 19 18 Anova: Two-Factor With Replication SUMMARY MMN MES Total S9-S9 Count 3 3 6 Sum 61 55 116 Average 20.33333333 18.33333333 19.33333333 Variance 0.333333333 2.333333333 2.266666667 S9-S246 Count 3 3 6 Sum 62 68 130 Average 20.66666667 22.66666667 21.66666667 Variance 1.333333333 0.333333333 1.866666667 S9-0.g Count 3 3 6 Sum 61 56 117 Average 20.33333333 18.66666667 19.5 Variance 2.333333333 1.333333333 2.3 Total Count 9 9 Sum 184 179 Average 20.44444444 19.88888889 Variance 1.027777778 5.361111111 ANOVA Source of Variation SS df MS F P-value F crit Sample (Species) 20.33333333 2 10.16666667 7.625 0.007292701 3.885290312 Columns (Gel) 1.388888889 1 1.388888889 1.041666667 0.327570178 4.747221283 Interaction 14.77777778 2 7.388888889 5.541666667 0.019737691 3.885290312 Within 16 12 1.333333333 Total 52.5 17 92 Appendix C.2 Measurements of mycelial growth of S9 towards interacting colonies. Species MMN MES S9-S9 11 11 12 11 12 11 S9-S246 12 13 14 10 12 11 S9-0.g. 14 12 15 12 13 11 Anova: Two-Factor With Replication SUMMARY MMN MES Total S9-S9 Count 3 3 6 Sum 35 33 68 Average 11.66666667 11 11.33333333 Variance 0.333333333 0 0.266666667 S9-S246 Count 3 3 6 Sum 38 34 72 Average 12.66666667 11.33333333 12 Variance 1.333333333 2.333333333 2 S9-0.g Count 3 3 6 Sum 42 35 77 Average 14 11.66666667 12.83333333 Variance 1 0.333333333 2.166666667 Total Count 9 9 Sum 115 102 Average 12.77777778 11.33333333 Variance 1.694444444 0.75 ANOVA Source of Variation SS MS P-value F crit Sample (Species) Columns (Gel) Interaction Within 6.777777778 2 9.388888889 1 2.111111111 2 10.66666667 12 3.388888889 3.8125 9.388888889 10.5625 1.055555556 1.1875 0.888888889 0.05226723 3.885290312 0.00695643 4.747221283 0.338407793 3.885290312 Total 28.94444444 17 93 Appendix C.3: Tukey test for mycelial growth of S9 towards interacting colonies. Calculations presented in Appendix C.2 showed that there was a significant difference between species as well as gel types therefore the data from each interaction were used separately with respect to their interactions as well as different gel types. Tukey's test was performed on the data. Numbers 1 to 5 are the means of data with respect to their different interactions and different gel type. The specific interaction and gel type is mentioned under each data. SE is calculated as the root of the s2 (the error mean square from the analysis of variance) divided by n (the number of data compared). (1)14 S9-Q.g (MMN) (2) 12.66 S9-S246 (MMN) (3) 11.66 S9-S9 (MMN) S9-0.g (MES) (4) 11.33 S9-S246 (MES) (5)11.0 S9-S9 (MES) SE= (s2/ n) 1 / 2 SE= (0.888/3)"2 = 0.54 Comparison differences SE q qO.05,12,5 1-2 14.0 - 12.66 = 1.34 0.54 2.48 3.082 1-3 14.0 - 11.66 = 2.34 0.54 4.33 3.082 Reject ul= u3 1-4 14.0 - 11.33 = 2.67 0.54 4.94 3.082 Reject (xl= u4 1-5 14.0 - 11.0 = 3.0 0.54 5.55 3.082 Reject ul= u5 2-3 12.66 - 11.66 = 1.0 0.54 1.85 3.082 u2= u3 2-4 12.66 - 11.33 = 1.33 0.54 2.46 3.082 ]x2= u4 2-5 12.66 - 11.0 = 1.66 0.54 3.07 3.082 u2= u5 3-4 11.66 - 11.33 = 0.33 0.54 0.61 3.082 u3= u4 3-5 11.66 - 11.0 = 0.66 0.54 1.22 3.082 u3= u5 4-5 11.33 - 11.0 = 0.33 0.54 0.61 3.082 u4= u5 94 Appendix C.4: Tukey test for mycelial growth of S9 away from interacting colonies. Calculations presented in Appendix C.l showed that there was no significant difference between the two different gel types therefore the data from each interaction were pooled together. In appendix C.l there was a significant difference between species. In order to pinpoint the source of difference Tukey's test was performed on the pooled data. Numbers 1 to 3 are the means of data summed across or pooled together with respect to their interactions but regardless of the gel type. SE is calculated as the root of the s2 (the error mean square from the analysis of variance) divided by n (the number of data compared). (1)22.7 (2)20.7 (3)20.3 S9-S246 (MES) S9-S246 (MMN) S9-S9 (MMN) S9-0.g (MMN) (4) 18.7 (5) 18.3 SE= (s2/ n) 1/2 S9- O.g (MES) S9-S9 (MES) SE= (1.333/3)1/2 = 0.67 Comparison differences SE q q0.05,12,5 1-2 22.7 - 20.7 = 2 0.67 2.98 3.082 u.l= u2 1-3 22.7 - 20.3 = 2.4 0.67 3.58 3.082 Reject u.l= u3 1-4 22.7 - 18.7 = 6.4 0.67 9.55 3.082 Reject ul= u4 1-5 22.7 - 18.3 = 4.4 0.67 6.57 3.082 Reject |i.l= u5 2-3 20.7 - 20.3 = 0.4 0.67 0.60 3.082 u2= u3 2-4 20.7 - 18.7 = 2 0.67 2.98 3.082 u2= u4 2-5 20.7 - 18.3 = 2.4 0.67 3.58 3.082 Reject u2= 3-4 20.3- 18.7 = 1.6 0.67 2.38 3.082 u.3= u4 3-5 20.3 - 18.3 = 2 0.67 2.94 3.082 u3= (J.5 4-5 18.7 - 18.3 = 0.4 0.67 0.60 3.082 u4= (i5 95 Appendix D.l Measurements of shoot dry weight of salal seedlings in culture. Salal shoot dry weight Species MMN MMN (PG) S9 - S246 3.7 10.8 5.5 3.8 4.7 11 O.g - S9 3.8 14.7 4.7 11.8 4.3 5.2 O.g - S246 4.9 18.7 4.2 4.7 4.6 7.3 Anova: Two-Factor With Replication SUMMARY S9 - S246 MMN MMN (PG) Total Count Sum Average Variance 3 13.9 4.633333333 0.813333333 3 25.6 8.533333333 16.81333333 6 39.5 6.583333333 11.61366667 0.g-S9 Count Sum Average Variance 3 12.8 4.266666667 0.203333333 3 31.7 10.56666667 23.70333333 6 44.5 7.416666667 21.46966667 O.g - S246 Count Sum Average Variance 3 13.7 4.566666667 0.123333333 3 30.7 10.23333333 55.45333333 6 44.4 7.4 31.864 Total Count Sum Average Variance 9 40.4 4.488888889 0.313611111 9 88 9.777777778 24.88444444 ANOVA Source of Variation SS df MS F P-value F crit Sample Columns Interaction Within 2.723333333 125.8755556 4.641111111 194.22 2 1 2 12 1.361666667 125.8755556 2.320555556 16.185 0.084131397 7.777297223 0.143376927 0.919847841 0.01638154 0.867889689 3.885290312 4.747221283 3.885290312 Total 327.46 17 96 Appendix D.2 Measurements of percent root colonization of salal seedlings in culture. Species MMN MMN (PG) S9 - S246 57 71 85 70 71 82 O.g - S9 71 63 56 62 45 72 O.g - S246 77 78 60 68 58 83 Anova: Two-Factor With Replication SUMMARY MMN MMN (PG) Total S9 - S246 Count 3 3 6 Sum 213 223 436 Average 71 74.33333333 72.66666667 Variance 196 44.33333333 99.46666667 0.g-S9 Count 3 3 6 Sum 172 197 369 Average 57.33333333 65.66666667 61.5 Variance 170.3333333 30.33333333 101.1 O.g - S246 Count 3 3 6 Sum 195 229 424 Average 65 76.33333333 70.66666667 Variance 109 58.33333333 105.4666667 Total Count 9 9 Sum 580 649 Average 64.44444444 72.11111111 Variance 154.0277778 57.36111111 ANOVA Source of Variation SS d[_ MS F P-value F crit Sample 425.4444444 2 212.7222222 2.098082192 0.165430057 3.885290312 Columns 264.5 1 264.5 2.608767123 0.13224433 4.747221283 Interaction 49 2 24.5 0.241643836 0.789066497 3.885290312 Within 1216.666667 12 101.3888889 Total 1955.611111 17 97 

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