ROOT-ASSOCIATEDORGANISMS OF THE CYPRIPEDIODEAE
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
In partial hilfilment of requirements
for the degree of
Doctor of Philosophy
December, 200 1
O Caria Zelmer. 200 1 National Librery Bibliothèque nationale 1*1 atcando du Canada Acquisions and Acquisitions et BiMiogrsphic Sewkes services biûliographiqubs
The author has granted a non- L'auteur a accordé une licence non exc1usive licence aiiowing the exchsive permettant à la National L1hry of Canada to Bibliothèque Pationale du Canada de reproduce, loan, distriibute or seil reproduire, prêter, distribuer ou copies of this thesis in rnicroform, vendre des copies de ceüe thèse sous paper or electronic formats. la forme de microfiche/fdm., de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or othenÿise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.
Canada ABSTRACT
ROOT-ASSOCIATED ORGANISMS OF THE SLIPPER ORCHIDS (CYPRIPEDIODEAE, ORCHIDACEAE)
Carla Dale Zelmer Advisor: University of Guelph, 200 1 Dr. R. L Peterson
AI1 orchid species are associated with fungi and other organisms during their life cycles. A review of the literature on genera in the Cypripedioideae (Slipper orchids) indicated that few species of Cypriipdium have been studied in terms of root-associated organisms and features of root anatomy that may be related to colonization of roots by these organisms. Likewise, there are few data on the microorganisrns associated with the roots of Pqhiopedilum and Phrugmipedium, two subtropicaVtropical orchid genera in
Cypripedioideae. Field samples of roots of five Cyprirpdim species fiom Manitoba and
Ontario showed high levels of colonization by mycorrhual fùngi. Microorganisms isolated into stenle culture included several fungal species known to be mycorrhizal with orc hids, demat iaceous fùngi, including PhiaiocephaIaforri>rii, cyanobactetia. aigae and bacteria. Roots of al1 Cypripedium species had an exodennis of suberized ce11 walls t hat may limit the ingress of most microorganisrns into the cortex. Isolates of Epforhia sp., a known mycorrhizal fùngai genus, affected colonization of protocoms of Cypripdi~ircm reghue Merently depending on cuitun substrate. A large expenment to test the effects of various root-associatecl organisms on the growth of nerile-raid C. regime seedlings showed that Epulorhi:a sp. increased shoot phosphorous and the number of roots. One bacterial isolate showed positive growth effects similar to those caused by plant growth promoting rhizobacteria (PGPRs). Many organisms, including mycorrhual fungi, other hngi, cyanobactena, algae and ciliated protozoans were isolated fiom within the multi- layerad epidermis (velamen) of roots collected fiom greenhouse-grown species of
Pa~~hioptidi/~~mand I ~hragntijw(Ii11m. M yconhizal tùngi were also localized wit hin the cortex. The diversity of root-associated organisms show in this study should be taken into consideration dunng the commercial propagation of these genera and the re- introduction of seedlings into field sites during conservation programs. When 1 began my Ph. D.. 1 was often askd why 1 had undertaken this task. 1 had the answer then (there were several). but looking back now at the completion of this research, one answer eclipses the others. The best answer was 'for the joumey.' Along this path 1 have had the honour of leaming fiom a multitude ofpeople. 1 hope that those that t have not named here will forgive me for the ornmission. 1 am gratehl for dl of your contributions.
Thank you to my Advisor Lany Peterson, who helped me map out the temtory and taught me to find my own way. 1 have leamed fiom you more than you will know.
Thank you, Randy Currah. Joe Gerrath and Melissa Farquhar. the members of my advisory cornmittee. You were willing to guide me through some unfamiliar terrain.
Thank you to those that panicipated in my research as surnmer students and Research Assistants, including Lara Armstrong, Jen Pnce. Orie Berlasso. Tony Smrnigalski, Chessie Pottier, Karrie Thomas, Kevin Burgess, Greg Vamier, Fengshan Ma and Chris Peterson. You made my load lighter, and your interest in my research quickened the pace.
There were others on the trail. I thank the members of the Root Biology and Mycorrhizal Associations lab (Shawna Carneron, Ron Deckert, Ryan Geil, Kevin Stevens, and Trevor Yu) for their companionship, their curiosity and for sharing parts of their joumeys with me. A special thank you to Lewis Melville, a world-class traveller.
Thank you to George Barron, Bud Ewacha. Dons Ames. Laune Winn, Shemy Haii. Franliea Ailen, lain Johnston, Allan Anderson, Peter Crozen, Carole Ann Lacroix, Demis Lynn, Al Bickell, Austin Hill, Grace Hill-Rackett, Michael Yee, NSERC, OGS for helping me obtain the resources 1 needed to complete this work: schoiarships, lab space, equipment, books, plant specimens and knowiedge. 1 couldn't have accomplished this without you.
Thank you to my family. You are always there for me, and 1 know 1 will always have a place to corne home to.
Finaily, thank you to those who never needed to ask me why, and wdked with me much of the way: Kirsten Müller, Dimaris Acosta, Alex Morisson, Yukari Uetake and Tony Smmigalski. 1 continue to be inspired by your joumeys and your discoveries. TABLE OF CONTENTS
Acknow ldgcmcnts ...... i
List of Tables ...... vi .. List of Figum. Illustrations and Plit~memmem.mmee.mee~~eee~mem~m~eemmmmmeetemeee~me~eeeee~e~~emem~em~~~~~~~o~~~
List OC Appendicts ~~~~~~~~o~~~~~~~~~~~~~~~~~~mmmmmmmee~emmeeeemmmemmemeeemeeeemex
Introduction...... 1
Chapter 1. CypripeJim mots and mycotrhizai associations .a review ...... 3
1ntroduction and Clarification of Terminology ...... ,...... -...... 3
Root Anatomy and Morpho10gy ..,e.....e..mm..e.e..e...... t.me.e...... m...... ~...... 5 Gene ral ...... 5
Epidcrmis ...... -...... 6 Hvpodermis ...... 7 Corticai parenchym ...... 8 Endodermis...... 9 Periqcle ...... 10 Xylem ...... 11 Phlocm ...... 11 Pith ...... 11 Roots and Cypri'dium Tuonomy ...... ,...... 12 CypRpcdium Mycarrhizas and Symbiotic Fungi ...... ,...... 12
Roots and Cj'pripedium Conservation ...... 17 Concluding Rcmarks ...... ,...... t.m...... t...... t...... 18
Chqpter 2 :Cypripediuni mycorihi~lrr. unotomy and extent of coloni&on ...... 20
Introduction ...... 20
Met ho& ...... ~...~~~~~~~~~~~~~~~H~~~.~~t..~W~~"~~~~~..~.~~L~~~~~~.~~~~~..ee~~~~~.e~~~~~~~~~.~.~~~~~I~~~~~~~~~~~~~t~~~~~~~~.~t.~~.*O 21 Tissue preparation ...... --7? Clearing and staining for e.dent~fcolonizationassessmenis ...... 2.1 Scoring of coloniation ...... 75 ...... 28 Concs...... 28 Endodermis...... 31 Stele ...... 32 Emnt of colonialion ...... 32 Discussion...... 34 Conclusion ...... 39
Chapter 3. Cypripedinm mt-msaciated orgunism. inchdïng mycowkiznlJiungL.... 63
Methoâs...... 65 Fungi ...... 65 Bacteria ...... 69 Algc ...... 72 Ciliates ...... 73 Rtsults ...... 74 Fungi ...... 74 Bacteria ...... 76 Algae ...... , ...... 77 Ciliates ...... 78
Conclusions ...... *...... 85
Chapter 4: Symùiosîs between Cypr@edluni reginae prutucorms and fungi: subshrrte and isolate dependence ...... loi
Discussion: Ehpcrimcnt 1 ...... 106
Methods: Expriment 2 ...... 111 Assessments...... 112 Rcsults: Erpcriment 2...... ,....o....-....m...... Il2 11 days ...... 112 80 Days ...... *...... *...... 113
Diïcussioa: Erpcriment 2 ...... l.ll...... 115
Ciliaics ...... 166 Distusion...... 167 Corclusions ...... 173
Chaptet Z Generul Conclusiutt~...... 202
introduction ...... *...... 1...... 1...... t...... 202 Root anatomy relative to colonization by mycorrhizal and otber root-associated orginismr ...... 202 Root-associated organisms of wild Cypriprdium plants ...... 203 Root-usocirteâ orgrnisms of greenhouse specimcns ...... 204 Root-associated organisms cminterad with sterileraised seedlings ...... 205 Implications of this work ...... 206
Litemîure Cited ...... 213 List of Tables Cpripedium specimens examined. Summary of percent of mot length colonized for four C-dium spp. Fungi isolated from the rwts of Cypripedium species. Bacterial isolates tested for ability to grow on nitrogen-free media. AIgae isolated from the mots of C'ripedium species. Ciliates isolated from the roots of C'ripedium species. Treatments used in inoculation experiment. Kruskal Wallis test results for the cornparison of non- and inoculated plants with the fun@ isolate (Epulorhicr sp.). Harvest 3 (12 months). Shoot phosphorous concentration at Harvest 3 (12 months). Mean rank by treatment. Kruskal Wallis test results for the comparison of non- and inoailated plants with the bacterial isolate 3 16b-1. Harvest 3 (1 2 months). ffiskd Wallis test results for the comparison of non- and inoculated plants with the bacterial isolate 3 18b-6. Harvest 3 (1 2 months). KNskal Wallis test results for the comparison of non- and inoculated plants 4ththe cyanobacterium Nostoc hum~figa. Harvest 3 (12 months) Paphiopedilum and Phragnipedium collections. Fungd isolates from the rwts of Paphiopedlum and Phragmipedium species. inoculation of Puphiopdiinni seedlings with Rhizuctonia s.L isolates Algae and cyanobaaeria isolated fiom the mots of Puphiopedifum and Phragmipedium spacies. CTated protozoans isolated corn the roots of Pqhiop4dium and Phragmipedium species. Figures, Illustrations and Plita
New peloton. Intact peloton. Degraded peloton. Digested peloton with "suspensor hyphae" radiating from hyphal mass. Extemal hyphae on root epidemiis. Recolo~zationof root cortical ceii. Epidermd cell outer tangential wall. Epidermal cells showing thickened outer tangential wall. Living and collapsed epidemial celis of C. urietimrn. Monilioid cells within an epidermal ceIl of a C. reginw root. Hyphal cells in epidennis. Hyphae in passage cells of exodemis. Dark septate hyphae. possibly of Phiulocepiwia fortinii in sulcus between epidermal cells and passing thfough to exodermai cells. Rhizuctonia-like hyphae and monilioid cells in root hain. Colonkation of root cona by myconhiliil hgusvia penetration of epidemal cell. Hyphae exiting root epidemiis. Root-associated organisms of C. acut.de. Root exodermis of C. acatrle. Exodennal cells wail lignification. C. arietimm root cross section. Structureci proniberances in the intercellular spaces of a C. regrule root. intercellular space. TEM view. Starch deposits in the root cortex of C. amie. Non-synchronous devdopment of pelotons in the root cortex of C. regime. Formation of separate digested pelotons (hyphal masses) representing tluee colonization events. Dolipore septum in a peloton hypha. Broad hyphae fonning a peloton in a root cortical celi. Narrow hyphae (possibly of Qmlorhizo sp.) in a rwt cortical ceil. Hyphae with clamp connections forming pelotons Ïn a C. regime root. Detail of Fig. 28. TEM view of a minute digested peloton surrounded by starch grains. üetd of Fig. 2.3 1. Hypertrophied and lobed mclei in colonized ceUs of C. acaule. Root cortex of C. acaule viewed wiih fluorescence microscopy. Endodermis and stele of CI candidrcm root. Endodemis and portion of stele of C.acarrle. Graph: Colonization of individual Cypripediurn roots - new pelotons Graph: Colonization of individual C'n'pdÏum mots - intact pelotons Graph: Colonization of mdividual C'ripedi~m rom - degraded pelotons. Graph: Colonkation of individual Cypripudium mots - collapsed pelotons. Graph: Peloton stages present per species. Graph: Colonization (al1 stages) along a root of C. repurr specimen 390. Graph: Colonization (dl stages) along a root of C. regiwe specimen 408. Isolate 227.1 Epulorhiza sp. colonial morphology . lsolate 227.1 l"pdorhiza sp. hyphae and monilioid cells. Isolate 209.2 Epulorhira sp. colony morphology . Isolate 209.2 Epidorhi=c~sp. hyphae and monilioid cells. Isolate 220- 1 .1 Epiclorh sp. wlony morphology. Isolate 220- 1 .1 Epiriorhiza sp. hyphae and monilioid cells. Isolate 220-1.1 Epdorhiza sp. hyphae and monilioid cells. lsolate 3 12 Epdorhiiu repens monilioid cells. Graph: Status of lettuce seedlings inoculated with bacterial isolates. Graph: Size categories of seedlings inoculated with bacterial isolates. Chloridellu ~ieglectuisolated tiom a root of C'r~pdium regilme. Eliipsoidion stichococcoides isolated corn C. ucaufe. Stichmtxcus bacillaris isolated fkom C. acmde. Stichococcus bacilluris isolated from C. reginae. Xauithonenio monfamm isolated fiom C. reginae. Nostuc sp. isolated from C. ucde. Oscilla!oria sp. isolated fiorn C. acuule. Lyngbyu sp. isolated from C. regim. Synechococctls Synechocystis sp. isolated from C. regme. Dilab rfilm arrhropyr~niaeisolated fiom C. regnue. Fottea stichococcoides isolated from C. regme. Rotifer isolated from a root of C. regme. Coipoda (culcullus-like)isolated fiom a mot of Cypfipediurnacmle. Coiporki steinii isolated from Cpripediun acuule. Coipodri (maupsi-like)isolated from Cypriipediurn acrnrle . CoIpado infata isolated from Cypnpdium acaule. Coi'sfeinii isolated from C@ripdium acuule. Col@ steinii isolated f?om Cypnpedium reginw. Fungal isolate 2709.2 (Qmlorhizu sp. ). 3 mo., Potato Dextrose Agar. C. regmue protocorm at approximately 4 mo. post seeding. Fungal isolate 2209.2 fo-8 pdotons in the cells of a C. reginoe protocorm on starch medium ( 1Oa). Fungal isolate 2227.1 in C. reginue protocorm cells on Sphagrtum medium. 1 1 days after contact. Early peloton formation in C. regznae protocorm cells with isolate 2220-1.1 on Sphrrpm CASYE medium. Monilioid tels withïn the pdotons of a C. regime protoconn colonized by 2209.2 on Rye CASYE medium. Graph: Experiment 1. Protocorms forming pelotons with isolate 2209.2 on 7 media types. 121 Graph: Experiment 2,80 days post inoculation. Compacison of numbers of protocorrns fonning pelotons with &ngal isolates 2209.2.2227.1 and 2220- 1.1 on each of 1O media types. 122 Graph: Experiment 2-80 days post inoculation. Comparison of numben of protocorms with digesteci fungal pelotons after inoculation with isolates 2209.2.2227.1 and 2220-1.1 on each of 10 media types. 123 Graph: Experiment 2.80 days post inoculation. Comparison of nurnbers of protocorms with monilioid cells within fûngal pelotons afler inoculation with isolates 2209.2,2227.1 and 2220- 1.1 on each of 1O media types. 124 Graph: Experiment 2,80 days poa inoculation. Comparison of numbers of live protoconns following inoculation with fungal isolates 2209.2.2227.1 and 2220- 1.1 on each of 10 media types. 125 Graph: Experiment 2,80 days post inoculation. Comparison of numben of parasitized protocorms following inoculation with isolates 2209.2. 2227.1 and 2220- 1.1 on each of 10 media types. 126 Epuforhiza repens isolated from Pqhiopedilunr hirmi~isimurn.On potato dextrose agar (PDA),3 months post inoculation. M, monilioid ceU; H. vegetative hyp hae. E. albertiensis isolat4 Phragnipdium equadoreme. P DA medium. 3 months. E cf: inquilim isolat ed From PoprhiopediIum charIesworfhti. PDA medium, 3 months. E. cf: iqtîilir>o monilioid cells isolated fiom Pujvhioprdhm chfesworthii.PDA medium, 3 months. E. cj: inquilino monilioid ceUs isdated fiom Puphiopedilum calIosum. PDA medium, 1 month after isolation. E inquifinumonilioid celis isolated Pciphiopdifum cafIosum. PDA medium &er 4 years of serial transfers. Colonization of cortical cells of Pphiqupedilum sanderimrn by an Epîlorhîta-like fiingus. Resin embedded; laser swuiing confocal microscopy; stained with Sulf'arhodamine. Monifiopsis mimi isolated from PqhiopediIum esqrcirofeii.PDA medium, 5 months. Stained with acid fùchsin in lactoglycerol.
T. penrtums anamorph fiom Ptphiopedilm cbfesworfhiii. PDA medium, 6 months. Stained with acid fûchsin in lactoglycerol. Moniliopsis-like fungus (arrows)coloniring mot cortical cells of Pqhiopdilwn henvamrm. D, dolipore septa. Resin embedded; stained with toluidine blue O. Apparent mycorrhizal colonization of a T. pt~riattts-likefungus. Phragrnipedium boi&unum. Resin embedded; stained with toluidine blue O. Parasitism of Phrugmtpedtutn boisemtamrm by a T. penna~rir-like tùngus. Resin embedded; stained with toluidme blue O Connection between velamen and cortical hyphae Ma passage cells. Paphiopediium lmrencianum. Coiled cells of fungus in velamen of Pqhiopedilum primulinum.. LSCM image, reconstnicted Z senes resin embedded material stained with sulforhodarnine G. Velamen ce11 of Puphiopedilum primulinum containhg coiled fungal cells. TEM microscopy. Detail of laminated wall in coiled hyphae.TEM microscopy. Larninate walled hyphae in peloton of Paphiopedi/irrn primulinum. Imperforate parenthome (arrow) in dolipore septum of coled hyphal ce1 ls fiom velarnen. TEM microscop y. Paphiopedlum primulinum. Daail of imperforate pwnthosome (Fig. 6.18). TEM microscopy. Paphiopedilwn primirliiium. Hypha with laminated wall passing into an adjacent velarnen cell. TEM microscopy. Paphiopedium primulim~m. Hyphal cell wall of similar hyphae in the peloton of P. primuiinum. TEM microxopy. Graph: Isolation of major fungal groups from velarnen, whole rwt, and cortex samples fiom the roots of Paphiopediilum and Phragmipeditrrn species (Johnston ûrchid plants). Pestalo~iopsissp. conidia. lsolated fiom Pqhiopedilum victoria-regrnw . S tained wit h trypan blue; light microscopy . Dendkosporium lobatum conidia (mws) and conidiophores. lsolated from Paphiopedii~mwilheimrniue. Stained with acid fuchsin in lactoglycerol; light microscopy. Bacterial cells in velarnen in close contact with exodermal and passage cell t ilosomes. Phragmipdium boiswïmm. Resin embedded, stained with toluidine blue O; light microxopy. Nosroc sp. fiom Paphiopedilum pcvishii . Live; unstained; iight microscopy . AmbaeM sp. from Puphiopdilum insigne. Live; unstained; light microscopy . Scy~onemusp. hmP The subfmily Cypnpedioideae (Orchidaceae) contains four genera: Cypriped~~tm Limaeus 40 spp.. Pqphiopedilum Pfitzer 60 spp., Yhrapijwdium Rolfe 16 spp. and Srferi~pednmRolfe 6 spp. (Dressler 1993). The subfamily is widespread, with members in Nonh America, Asia and Europe (Cypripeditïm), Southeast Asia (PaphioprliIt~m). Central Amenca (Selenipedium) and Central and South Amenca (Phragmipdium) (McCook 1 989). In Canada, the Cypnpedioideae is represented by 1 1 species of Cypripedium. The Cypripedioideae are oAen showy plants. consequently. they are wlnerable to collection as well as the habitat destruction that threatens many orchid populations. One species (Cypripedi~mcandidtm) is listed as endangered in Canada (DePauw and Remphrey t 993); status reports on other species are being undertaken. Habitat loss and collection pressures have sewreiy afTîxted PuphiopdiIum and Phragmipedium species, some are now extinct or very rare in their countnes of ongin (Cash 199 1 ). Consequently, al1 Pop>hiopedilum and Phrogniipdium species are iisted under the Convention on International Trade in Endangered Species (CITES) Appendix 1, which remicts the impon and expon of these plants. Conservation arategies for many species in the Cypnpedioideae wiU involve a combination of ex situ cultivation, the relief of collection pressures for showy species by providing a source of cultivated plants (Steele 1996) and perhaps the restoration or supplementation of populations in appropriate habitats through in vin0 plant production (DePauw et al. 1995). This will require more idormation than is currentiy available on the desand identities of mycorrhizai and other root-associated organims. There is evidence that rwt-associated organisms are important to the health and su~valof orchid seedlings. MycorrhiPl orchid seedlings appear to have higher rates of su~valthan axenically-grown xedlings when planted into non-sterile environments (Anderson 1992. Clements et al. 1985). The cornmon recommendation to orchid growers to incorporate media fiom a mature plant into the poning medium used to pot sterile- grown seedlings may also have arisen from a need to inoculate the plants with other beneficial rnicroorganisms. Unfortunately. effective symbiotic germination and culture methods for the Cypripedioideae have not ôeen developed. in part due to a lack of knowiedge about the fingal participants in the syrnbiosis. In order to understand, and perhaps utilize, root-associated organisms in the culture and conservation of orchids in the Cypripodioideae, an examination of the organisms associated with these plants is critical. The overall thesis of this audy is that root-associatecf organisms are present and perhaps important throughout the He histories of plants in the Cypripedioideae. To assess thisTiiis thesis represents an exploration of the roots and root-associated organisms of wild Cypripeditm species, and, due to export restrictions, cultivated specimens of Paphiopedilum and Phrugmipedium. The chapters that follow document the locaiization, culture, and identification of root-associateci organisrns, and expenments to characterize their interactions with orchids in the subfamiiy Cypripedioideae. Chapter 1. Cypripedium roots and mycorrhizal associations - a review Introduction and Clarification of Terminology The genus Cypripedirrm is composed of approximately 47 species of perdal herbaceous orchids native to the Amencas, Europe and Asia (Pridgeon et al. 1999). Cyprijxd~min its cunent usage refers to orchids with mealy pollen produced by two fertile anthers, the third one modified to fonn a shield-like starninode. Their pliutte leaves have sheathing bases and newly emerging leaves are rolled (convolute) rather than folded (Pridgeon et al. 1999). The flowen typically remain attached to the hit. However, it was the prominent pouch or slipper-like lip fiom which the genus narne was derived: 'Kjpris' refemng to the goddess Aphrodite and 'pedih' meaning slipper (Pndgeon et al. 1999). They are commonly known as the lady slipper orchids. This genus has a broad appeai and a large commercial potential. Considering the size of the genus root anatomy of relatively few C'dhm species has been examined in detail. Also, the extent of mycorrhizal coionization in mature autotrophic plants is controversial and requires clarification. Syrnbiotic culture methods have aided the sunival of orchid seedlings of other species (e-g. Jorgensen 1994), but Iittle is known about the mycorrhizal fun@ associated with any deveiopmental nage of C'ripedium plants. idormation on the development, morphology and anatomy of C'npedium mots and their association with mycorrhizal h@has potential to aid the conservation of plants in this genus. This review highlights major published research on Cjpri'dium root anatomy and associations with various organisms. Where the origin of the plants used in the research could be determinecl, the current species narnes have been indicated. Species concepts within Cyprïpudircm have evolved over time. For example. Cjpriiprdium caiceoius until recently inciuded both the European and Nonh American yellow ladyslippers. However, this species is now resemed for the European yellow ladyslippers, and C. paw~,jor-umhas been proposed to contain the Nonh American yeilow ladyslippers and their varieties (Cribb 1997). Orchid mycorrhizal associations are common to many of the Orchidaceae. These associations are here defined as symbioses between an orchid and root-inhabiting hngi that result in the formation of characteristic orchid myconhital structures, such as pelotons. Pelotons are coiled and branched masses of fungal hyphae that. while inside the root cortical cell wall. are excludeci frorn the cell's cytoplasrn by a perifungal membrane (Peterson et al. 19%). Mer their formatio~pelotons are lysed and their contents released to the intedacial matnx, where they are absorbed by the root cell. The term symbiosis as used here does not imply mutual benefit to the interacting organisms. Mthough there are demonstrated benefits of mycorrhizal syrnbiosis for the orchids. at least at a juvenile stage (see Rasmussen 1995). there are as yet no confirmeci advamages of the association for the t'wigus. This review covers mycorrhizal associations of the roots only. Fungal interactions with seeds of C'dïumspecies are beyond the scope of this paper and information on these interactions is summarized in Peterson et al. ( 1998) and Rasmussen ( 1 995). Root Anatomy and Morphology General The roots of Cypripedizim species, like those of other orchids, are produced adventitiously fiom stem tissue (Dressler 1993). Curtis ( 1943) noted tbat new roots of C. acaule formed just below the sale leaves of the underground stem (rhizome) at the rate of 2 per year. According to Stoutamire (1991), C. cdidum roots are initiated from the base of the sympodial bud near the end of May and reach 5-1 O cm in length by October. A pair of roots forms at the base of the reserve and replacement buds, sometimes followed by an additional 1-3 roots opposite these buds later in the season (Stoutamire 199 1). C. regirae roots in active development in early July reach their usual length of greater than 30 cm by the end of August (Harvais 1974). In Iowa, C'diumplants initiate root growth near the rhizome apex between late lune and August depending on species (Whitlow 1983). The older portion of the rhizome does not initiate new roots. but the roots attached to it remah alive for several years (Whitlow 1983). Old C'dimroots remain functional for many yean, perhaps as long as 19 for C. pantflomm var. pubescens (Curtis 1943) and 1O years for C. cmdihm (Moller 1968. MoUer 1977, Stoutamire 1991). Roots of C. ucauie plants are probably also long-lived. In a 20 year study of two populations of C. ucde (Gd 1996). sunival rates of mature plants were 98% per year, and most of the plants marked at the beginning of the study were ail1 alive 20 years later. C. acauIe rwts are persistent on the rhizome and continue to grow annually until reaching approltimetely 20 cm in length (Curtis 1943). Constrictions are produced annuaily on the non-contractile roots (Hoh 1904) due to the narrowhg of the rwt dimeter during wimer growth cessation. These constrictions are not reliable for aging individual mots beyond 3-4 years of age, however, as they become obscure &er this time (Stoutamire 199 1). Root systems of Cypripdi~mare ofien extensive. Four to five meters of root may support each flowering stem of C ~'(~did~rm,and individuai roots may increase by 10 cm per year to a maximum length of 40 cm (Stoutamire 1991). Roots of C. crnddum are shallow (in the top 5 cm of soil), sinuously curved and extend radially from the rhizome (Stoutamire 199 1). This horizontal radiation of roots and shallow rooting depth appears to be cornrnon to C. acmde. C. arietinum and C. panflomrn as weli (persona1 observations). C. repue may have geater root penetration, estimated by Harvais ( 1974) to be 30-50 cm in depth. The lengh of mots appears io be restricted by their inability to branch or to replace damaged apical meristems. Few roots of C. cmdidum had active apical meristems beyond 2-3 years due to darnage or senescence of the meristems in the population studied by Stoutamire (1991). Roots of C. regme, C. cuiceoIus, C. candirh~mand C. accrule dl produce an unusual and characteristic odor. which is present even in the mots of stenle-grown seedlings (Hdman 1983). Holman found that the scent was strongest in C. ccmdâùm and C. regime, Iesser in C. acmle. The aromatic compound was related chernically to methyl anisole isomers and most closely matches pmethylanisole (1 -methoxy-4-methyl benzene) (Hoiman 1983). The role of this substance in the roots is unknown. Epidcrmis According to Holm ( 1904). al1 species observed possessed a singie, living epidemal layer, wbich was thin-waüed in C. acaule. C. montmm, C. ficic11Iaturn, C. calijomict~mand C. urietinun, but had a thickened outer wall in the roots of C. gutîafurm. Root hairs were described as abundant on these species. In contrast to Holm (lm), Cunis (1943) stated that the first roots of these species produced sparse root hairs, and later roots were ofien free of root hairs. However. Rosso (1966) dso observed persistent unicellular root hairs in Cypripdium species. On C. caiceolus roots, these reached a maximum length of 2300 pm. C. irupemn~mis unusual in the genus in possessing a single layered. dead velamen with persistent root hain (Rosso 1966). These lose their protoplasts as the rwt ages (Rosso 1966). The tiinction of the velarnen is unknown for this temestrial species. Dressler ( 1 98 1 ) suggested that in the Orchidaceae, the "ongin of the tissue [velarnen] may be in some way related to the mycorrhizal relationship." Hypodetmrs Hypodennis and exodemiis are the two rnost cornmon tems used to describe one or more layers of modified outer cortical cells in roots. According to Esau ( 1977). a hypodennis consists of ceUs that differ 6om underlying ground tissue celis. Peterson and Pemmalla ( 1990) agreed with this, but suggested that if hypodennal cells develop a Casparian band then the tenn exodermis should be used. The literahire on orchid root hypodermis/exodermis was published before this distinction was made. It is therefore unclear whether Casparian bands have been detdin the hypodermises of Cypripdh~ species. The presence of Casparian bands can have a regdatory effect on both water uptakdoss and mycorrhizal colonization by lirmting the passage of water and organisms to and fiom the cortex to specific, less suberized cells. Holm (1904) noted the presence of a hypodermis in C+'ripedium roots, which was thickened oniy in C. ptatfum. Rosso (1966) examinecl rwt specimens of C. acaule. C. arieti~n~rn.C. caIceolus*C. cdceolus var pubuscens (now C. purv~jlorumvar. pubescem), C. caI$tonticum. C. candidum, C.f~sciculatum, CC. guttahm, C. irqpemrn, C. rnacranihum. C. ntoIt!amrn, and C. regmae for the presence of a hypodermis. Like Holm, Rosso alw observed in some species of Cypripdiurn an underlying laye? of smaller cells that had similar staining properties and textures as the epidennis (Rosso 1966). Although Holm (1904) considered this subepided layer distinct enough to be called a 'hypodenn', Rosso considered only C. irupeanim to possess a true exodennis consisting of long (dead) and short (live) cells altemating in longitudinal rows. The outer tangentid wdls of the short cells bore obvious pits, so were termed passage cells. The long cells of the C. irapanlm exodemiis were prorninently thickened dong the radiai and imer tangentid wall (U-type exodemiis) (Rosso 1966). From Rosso's description, it is probable that the subepidennal layer of C. irupemm roots conforms to the present usage of the term exodemis. However, it is unknown if the 'hypodermal' layers of the other species contain Casparian bands. C'm*cd porcnchym In al1 species, the cortex is composed of approximately 6-8 layers of large parenchymatous cells that store starch (Hoim 1909, Rosso 1966). The parenchyma ceils were descnbed by Holm (1904) as compact in moa species, but "'thickened and porose" in C. pubescens (now C. parv~flommvar. pukscem). The cortical parenchyma ceUs of old and young rwts of C. cdidum were uniforrn in the amount of starch they contained, but a yearly decline in aarch content of both ocnvred during Apnl and May (Stoutsmire 199 1). Starch levels were Iowest in newly-formed mycorrhizas, and 1-3 cm behind root apical meristems (Stoutamire 199 1). Starch was present in dl areas of the roots with the exception of those that had active or degraded hngal pelotons (Stoutamire 1991). Alexandrow (1 925) examined a young root of a C. cakeolus seedling. Noting that the cortex was relatively wide. Alexandrow hypothesized that it fbnctioned in water storage. The cells of the root cortex fiom this plant were filled with fùngal hyphae. Alexandrow (1 925) believed that the "saprophytic" life of the plant was dependent on these fun@. The rhizome. though continuous with the roots, was not colonized by mycorrhizal fùngi. EnclOdrrnùi The endodemis. the imermost layer(s) of cells in the conex (Esau 1977). can pass through a series of wall modifications, depending upon the plant species and the distance fiom the apical meristem. Peterson and Enstone (1996) surnmarke these as follows: state I - Casparian bands present; state II - Casparian bands plus suberh lamellae present; aate III - Casparian bands, suberin lamellae and a secondary ce11 wd present. Endodermal cells in orchid roots nodly pass through these three stages during ontogeny. The Uuiermost layer of the Cjpripediuum root conex is an endodermis cornposed, at mawity, of state III cells adjacent to the phioem and thin-walled cells next to the xylem (Holrn 1904). The endodermis is a single layer in aii species examineci except for C. irvantlm. which has a mltiiayered endodermis (Rosso 1966). The roots of C. acaule, C. gututtum, C. arietinum each have 3-4 state iII endodermal cells per xylem arc. The endodemal ceus of these specîesare U-type,meaning that they are thickened on the inner tanpntial and radial wails (Holm 1904). C. caIifomiçimr also has U-type endodemal cells but an average of 6 cells per xylem arc is typical for this species. U- type endodenniil cells are present also in C. reginae and C. irapemm (Rosso 1 966). Two to 4 evenly-thickened (O-type) endodemal cells were found adjacent to the xy lem in C. ptd be.sccm (now C parwj7ondm var. p~be.vcens)and C. montamm. State 11 I cells were not seen in the endodemis of C. fascia~ia~umwhen exarnined by Holm ( 1904), but O-type cells were observed in this species by Rosso ( 1966). O-type endodermal cells are also present in C. C'LI~CYO~IIS,C. cacdich~m and C. macranth~m. Those observed by Holm probably were immature, because state 1 endodemal cells in young roots usudly develop to state III as the roots mature (Rosso 1966). The endodemal cells are living at maturity ( Alexandrow 1925). Pdqcle The pericycle of mon Cyprpditim species is single layered (Holm 1904. Rosso 1966). According to Rosso, thick-walled cells form next to the phloem, while thin-wailed cells are found adjacent to the xylem. However. C. irupunzim has consiaently thickened ce11 walls in its pencycle (Rosso 1966). Holm ( 1904) also noted a thick-walled pericycle in C. arierimrm. As quoted by Rosso. Meinecke (1894) found thick-walled pericycle cells only in C. regrme and C. cdceoius. Alexandrow (1 925) also observed thick-walled pericycle cells adjacent to the phloem in the third and subsequent roots produced by C. cuic~oltrs seedlings. Rosso (1966)saw both thick and thin-walled pericycle ceiis in alrnoa al1 the species examined, and thickened cells in ail specimens to differîng degees. The thickness of pericycle ceiI walls may be relateâ to the maturity of the roots in C'ripdî~~rn (Rosso 1966). Xy lem In the developing roots of C. culceoIus seedlings. xylem tissue was poorly developed and parenchymatous phloem tissue was present (Alexandrow 1925). Development of the xylem was concurrent wiih ihai of the endodennis (Alexandrow 1925). A polyarch stele with 5- 10 archs is usual for Cypripediiun, species. but the stele characteristicaily has 8- 10 archs in C. montamm (Holm 1 904). Metaxylem usually extends to the center of the root. but pit h is sometimes found in the roots of C. irqpeanum and C. caiifrnicum (Rosso 1966). Vessels are present in Cypripedium but not in the closely related Paphiopediium (Rosso 1966). Cypripedium vessels have simple pertoration plates with tapered ends and scalarifom pits, while tracheids have bordered scalarifonn pits (Rosso 1966). Phlocm The observation of phloem tissue is rarely reported. Phloem is well developed in C'npedium species and alternates with the xylem archs. Protophloem is fomed as part of, or adjacent to the pericycle. Rosso (1966) reports that the sieve elements are elongate with somewhat oblique transverse walls and simple sieve plates. They are associated with cornpanion celis and phloem parenchyma (Rosso 1966). A'th In most species of C'rip4dium. rnetaxylem extends to the center of the root, but pith is sometimes found in the roots of C. irapeumm and C. cuiiforni~tlm(Rosso 1966). C. caifornicum possesses a thick-walled pith. Roots and Cypripedium Taxonomy Cypripeditim roots have received Iittle attention fiom taxonomists. Although Atwood ( 1984) used morphological characters in his phylogenetic analysis of the Cypripedioideae. extemal root features were not considered idormative. He stated: "Aside fiom differences in size. the roots of most slipper orchids appear externally sirnilar." Differences in the intemal structures of the roots were not exarnined, which is surpnsing given the information about taxonomically significant root characters provided by Rosso ( 1966) and Holm (1 904). Albert's ( 1994) cladistic -sis of Cypripedioideae used Rosso's ( 1966) root characten - absence of vessels in Paphiopedilr~m.simple perforation plates in Cypriipdum. Sufenipudiuium and Phrugmipdiz~rn.ût her characters used in this analysis included the fom of the pencycle, velamen, and the abundance of root hairs. The most ment monographs of the genus Cpripdium (Cribb 1997, Pridgeon et al. 1999) do not indiate the anatomid differences in the root systems of the species. Cypripedium Mycorrhizas and Symbiotic Fungi Holm ( 1904) wrote "the roots of our tenestriai Orchidaceae very often represent mycorrhizae, but not al1 the roots of the same species, nor of the same specimen." This aatement may clarify the apparent controversy over the mycorrhizal status of mature Cypriipedum plants. Holm recorded the presence of Nngal hyphae in the cortex of both C. pubescens and C. faciiculuhm, and in the endodermis and pencycle of C. guttatum. This latter observation probably represents parasitism by a fungus, since fungi are nodyexcluded fiom the endodermis and stele in typical orchid mycorrhiral associations (Rasmussen 1995). Bernard (1904) also noted mycorrhual fùngi in the cortex of Qpriipdium insigne, however. this record actually refers to the species now knom as Paphiopudiium insigne. The root of a young Cypripedium calceolus seedling illustrated by Alexandrow ( 1925) displayed a cortex heavily colonized by mycorrhUal fiingi. Fungi were also present in the root hairs of this plant. Stages of peloton formation and breakdown were also seen in the third root of C. calceoius seedlings, production of which also coincided with the production of the tim green le& This finding indicates that mycorrhizal hngi are therefore not elirninated as soon as the plants become autotrophic. According to Alexandrow ( 1925). older roots possessed larger vascular cylinders, which he ükened to the myco~hizomes(mycorrhiza-like rhizome tissue) of the wholly mycotrophic subterranean orchids Cwufforhizaand Neottia. These achlorophyllous orchids depend upon mycorrtuzal fungi for their carbon sources, and so are usually heavily colonized (Rasmussen 1995). This com parison suggests that Alexandrow observed extensive coloniration of older roots, however, he later stated that the mature root was fungus - fke. Fuchs and Ziegenspeck (1926) also illustrated a heavily colonized seedling of C. caiceoltist but noted that new roots possesseâ no fungal pelotons. Curtis (1 939) succeeded in isolating severai potentiaily mycorrhiral fungi from the roots of Cjpripedium species. Rhizuctoni~subrilis cultures were O btained from the roots of C. caiceoIus, CC.cdhm and C. pw~jlorurn.Rhizoctonia sclerotica was obtained from C. regime roots. Although Rosso ( 1966) midiecl the root anatomy of twelve species of C~ripedim. observations of fiingal hyphae in the cortex of the roots is not mentioned. The lack of information on mycorrhizai colonkation could indicate that mycorrhizal hngi were not seen, or that Rosso excluded colonized areas to avoid descnbing any effects of the colonization upon the anatomy of the root. Harvais ( 1974) suggested that mature plants of C. repue rnight be completely autotrophic, resistant to c~lonization~and mostly free of mycorrhizal fun@ except where the defenses of old, weak roots had been breached. He observed that old roots had "occasional restricted paths of infection dong their lengths where pelotons were small, amorphous and in an advanced stage of digestion." The pelotons of these roots occurred in paiches, interpreted by Hamais to mean that colonizing hngal hyphae were quickly controlled and prevented fiom laterai expansion. Young roots of mature C. regrnw plants were uninfectecl. This implies that colonization of the roots was therefore from the surrounding mil. a process Harvais believed was dependent on many factors, including rate and degree of digestion, the pattern of colonization and the degree of heterotrophy exhibited by the plant. In an expriment to determine if mycorrhizal fùngi were present in the rhizosphere of mature plants. Harvais baited soi1 samples with commeai. Rhizoctonia- like hyphae were recovered fiom the rhirosphere samples. There have been strong opinions voiced in the literature regarding the mycorrhizal status of Ctpripeditm plants at maturity. For example, Whitlow (1983) wrote, " Thou$ the beneficial fun@ are critical to seed germination and deveiopment. once the plant becomes photosynthetic, their deliterally ceases." A similar statement appears in Durkee (2000). However, both of these papers focus on the growth of asymbioticaiiy- germinated Cjpripedium plants in vitro, and neither present evidence in support of this position. Symbiotic seedlings have a survival edge over asymbiotic seedlings in other species (Jorgensen 1994). ln an effort to find suitable symbiotic fiingi for CJpipedium seed germination, attempts have been made to isolate mycorrhizal fungi fiom the roots of mature plants. Clements et al. ( 1985) reported the isolation of two symbiotic fungi from Cypripedium but did not record any success at symbiotic germination using these isolates. An "intensive study of the species in Britain and Europe" also ended without a symbiotic partner for C calceoius ((Mitchell 1990). However this statement referred to seed germination attempts, and does not imply that isolates were not obtained from mature plants. A low level of mycorrhizal colonization in a C. cd&m population was indicated by research by Stoutarnire ( 199 1 ). Mycorrhizal coionizations in the roots of ihese plants were patchy in distribution, with individual patches extending axially 1 - 10 mm. Patches were usuaily dixrete, but overlapped in older, highly colonized roots. Colonization levels were low at both the proximal and diaal root ends, reaching a maximum at 1 5-22 cm away from the rhizome. Stoutamire (1 99 1 ) estirnateci that less than 1% of each root was colonized per year, and believed that active colonizations of the root were short-lived. Usually more than 50./0 of each root was fungus fiee. The method of determining percent colonization is not elaborated on in this paper, and so it is unclear if the assessrnent of mycorrhizas includes all stages of colonization or just new or active colonizations. The fe~turesof mycorrhizal colonization recorded for C. candidum (Stoutamire 199 1) were similar to that for many terrestrial orchids. There were no extemal indicators of mycorrhizal colonization. Fungal hyphae were absent from the innemon cortical cells. Ceiis with pelotons generally lacked starch accumulations. Individual root cortical cells could be colonued by hingal hyphae two or more times and digested pelotons were tan to dark brown and irregular in shape. bornes were not colonized despite colonization of the attached roots. Stoutarnire obtained five fun@ isolates fiom the roots. unfortunateiy. t hese fungi are not characterized or identified. Zelmer (1994) and Zelmer et ai. (1 996) attempted the isolation of fungi associatecf with the roots of mature specimens of 6 species of Cypripdium. This work resulted in the isolation of Epulorhiza, Ceratorhi= and Moniliops~sspecies (dl Rhimcto~~iaS. I.) from C. cakeolus (now C. purv~jhmrn).Epulorhiza sp. from C. candihm and Cerutorhiza sp and Moniliopsis sp. frorn C. pmeri~ttlm.Isolates were not obtained from the roots of C. acai~le.C. montïzm~mand C. reginw. Recently Vujanovic et al. (2000b) described Phtalucephalo victorinii sp. nov. as an endophyte of C. purwifromm. The role of this tiingus in the roots is unknown. Phialocephaa/orrinii, w hic h has also been isolated €rom the roots of C'ripedium spp. (Currah et ai. 1997a). is a common root endophyte of a broad range of plants. P. jortinii has been suggested to occupy many positions on the parasite-mutualia continuum in its association with host plants (Jumponen and Trappe 1998). Writing about a 20 year study of marked C. acaule plants. Gill(1996) noted that marked plants occasionally would rernain underground and then reemerge, usualiy the next growing season. He believed that a srnaIl portion of the population could remain underground for up to 2 decades. A recently published 5-year study (Shefferson et al. 200 1) recorded the typical subterranean donnancy of C'ripedium calceohs ssp pun@nïrn to last up to 2 y- but dormancy as long as 4 years was observed. While not direct evidence of myconhual dependency. such information suggeas that a completely heterotrophic existence is a possibility for mature C'ripedium plants. C~vpripedizimRhizosphere Harvais ( 1974) estimated that the continuous rhizosphere of a C. reghae plant at his study site was ai least 50 m2.It is at the level of the rhizosphere that substrate qualities become cntical. Wherry ( 1 9 1 8) characterized the soils supporting Cypripdium species. C parvrjorum. C. regtiiae. C. pubcscem nonnally occur in soil of aikaline pK whereas C. acaule inhabits substrates with a pH in the acidic range, such as roned pine needles and Sphugtnm hummocks. Apparently contradictory soi1 types sometimes support plants of these species. but the root zones of the plants are ofien in microsites with a substrate of a different composition than the surrounding area. Orchids rooted in Sphognm hummocks may be in highly acidic substrate, even though the water in the holiows may be al kaline (Hamais 1974). Roots and Cypripediurn Conservation C~pripeditmspecies are becoming increasingly rare over the last several decades due to a combination of collection pressures and habitat destmction (Chu and Mudge 1994). Do root characteristics have any importance to conservation? Little is known of the importance of root features to in si& conservation but several studies have pointed out the importance of root characteristics in transplanting mature plants to safe sites. C'rijxdï~irn, roots often radiate horizontally in the upper layer of soil. Given the dimensions of the root systems mentioned in the previous section of this review. moving a single plant would require the removal of an unrnanageably large area of soil. Root damage therefore often accompanies transplanting. Since lost root apical meristems are not repaireâ or replaced, and the roots can not branch, transplants need to fom new roots to replace the old, darnaged root system (Stoutamire 199 1 ). Only a few new roots are produced each year, so this can be a slow process. Cjprijxdium plants lack storage organs, such as tuberoids or psuedobulbs, that are fomed by many other orchid species (Dressler 1993). Therefore, the bulk of starch storage probably resides in the roots. Loss of a major portion of the root syaem due to poor transplantation techniques obviously deprives the plants of energy reserves. Root- damaged plants may still flower in the spring due to the initiation of a flower bud the previous year (Stoutamire 199 1). resulting in hrther resource depletion. The role of mycorrhizal fùngi in the roots of mature Cypnpedircm plants is unknown. but may be important to their sumval. Root removal also may separate these plants fiom the resources of myconhital fungi, w hich are not present as inoculum in the rhizome. Concluding Remarks Considering the size of the genus, the root anatomy of relatively few Cjprrjwditrm species has been exarnined in detail. The lack of data concerning some of the Asian Central and South Amencan species is panicularly noteworthy. There appears to be species or &on differences in root anatomy that may be of interest to taxonomists. however, matornical re-emnhations are necessary for the clarification of some characters. Some features of the root. such as the presence of an exodermis, may aect mycorrhital coionization and protect the root during periods of soi1 drying. The extent of mycorrhizal colonization in mature autotrophic plants requires clarification. Additionally, isolation and identification studies should be undertaken to determine the identities of the symbiotic fungi. As well, examination of cleared whole roots of many species of Cypripditm may give a better indication of the extent of myconhizal colonization and the hngi involved than isolation work. Success in the isolation of mycorrhizal hngi from Cyprïpedtum appears to be relatively low. It will be important to clarify the roles of symbiotic hngi (and perhaps other organisms) in Cyptipedium. If mycorrhital symbiosis proves to be common among Cypripedium species, the requirements of the syrnbiotic organisrns will need to be taken into account by those wishing to conserve or restore species, or to propagate them for commercial purposes. Chapter 2 : Cypripedium mycorrhizas - aoatomy and extent of colonization Introduction The role of mycorrhizas in the biology of Cypripedium species is poorly understood. While there is general agreement that the seedlings in their achlorophyllous early growth stages are supponed by myconhllal hngi (Rasmussen 1995). the persistence of this association afier matunty is in doubt. Many authors (e.g. Alexandrow (1925). Durkee (2000). Hamais ( 1974). Whitlow ( 1983)) have stated that the extent of the mycorrhizal association declines in the leaf-bearing stages of the plants, and that at maturity mycorrhizal fùngi are rareiy present. Although this is a common view. 1 have frequently observed mycomhiral colonization in species of Cypripedium. Further. there is evidence that mature Cypripdium plants can remain underground for 1 to severai growing seasons (Gill 1996, Sheviak 1974). during which tirne they may be dependent upon nutnents provided by rnycorrhizai fungi. Rasmussen (1995) pointed out that new roots are produced by C'ripedium species in the late summer and auturnn, at a time when water requirements are low and leaves are dying. She suggested that this timing could imply an "initially mycorrhual" role for these roots. Clarifjmg the role of mycorrhizal fungi in the biology and ecology of the Ladyslipper orchids is of criticai importance to restoration, protection and propagation efforts. As C'ripediwn species become easier to propagate on a large scale, nich basic biological information on the species may help horticultunsts undemand the requirements of the Ladysli pper orchids. There are few published studies of the mycorrhuas of Cypripdium species. Many of the early observations were made during comparative anatomical studia (Holm 1904. Alexandrow 1925, Fuchs and Ziegenspeck 1926). These studies used live, sectioned root material. primarily of C)pri/wJit~mca/ceoI~~.o, C. regiitae and C. acm~fle.The use of modem preparative (eg. resin embedding) and rnicroscopical techniques (eg. laser scanning confocal and transmission electron microscopy) may offer bhinsights into the anatomy of CypriiPrJwn mycomh. Stoutamire ( 199 1) and Zelmer et ai. ( 1996) evaluated the mycorrhizal colonization of C'ipdiumspecies in roots and seedlings made transparent by clearing. The application of this technique to a variety of species. sites and collection dates would be valuable in detennining a pattern of colonization, if one exists and to evaluate the overail fiequency of mycorrhiza formation in the species of the genus. The objectives of this research were to examine anatomical feanires relevant to mycorrhi2al symbiosis of several Nonh Amencan C'ip~diumspecies (C. acuuk. C. mietimm, C. cmdiiium, C. pvijlorum, ami C. regme),and to determine the extent of mycorrhizal colonization in the roots of 4 of these species. Methods To avoid damage to the sampled plants, a single root was removed fiom each plant listed in Table 2.1 . Plants collected represent populations in the provinces of Ontario and Manitoba, Canada and were coiiected over a 4 year period at varying times of year to account for variations due to season, habitat and climate. Indicated by an asterisk are those collections that were cleared to assess extent of colonization. AU other collections were prepared by resin ernbedding for microscopy (see betow), and for isolation of associated orga~sms.The isolation/identification of mycorrhital fùngi from these roots is covered in Chapter 3. Provenance of the Cjpripudircm specimens are found in Table 2.1. Plants were sampled non-destmctively using the following method. On each plant, the position of the latest shoot or winter bud was detennined. The rhizome was located by gently peeling back the substrate behind the current shoot. The extent of the rhizome was noteâ, and then a single root was severed from the middle of the rhizome and followed through the substrate to the tip. For C. arietimm and some C. pmv~flomrnplants, the shon vertical rhizome made the determination of root placement impossible without uprooting the plants. For these plants, the roots were chosen on a random basis. The substrate covering the rhizome was restored. Each root was packeû in local soi1 in a small piastic bag labeled with a unique plant number and nord in an insulateci box at ambient temperature for transport. On retum to the lab. the roots were processed immediately or held at 4°C for up to 48 lus until processed. Tissue prepamtion Segments of root tissue ( 1- 10 mm) to be thin-sectioned and observed with light rnicroscopy were fixed in 2.5% glutaraldehyde in O. 1M HEPES buffer at room temperature for at least 8 hrs. Roots collected under very dry conditions were allowed to rehydrate for 1 tu in deionized water before fixation. Following glutaraldehyde treatment, the root segments were ~seû&ce in O. 1M HEPES buffer, and dehydrated in a graded ethanol series. Tissue was thm gradually infiltrated wîth LR White resin (London Resin Co. Basingstoke, Ln<) and embedded in pure resin in stacked flat aluminum weighing trays. or in gelatin capsules. Samples were polyrnerked at 60°C for 24hrs. For samples observed using transmission electron microscopy (TEM), specimens were post-fixed with 2% osmium tetroxide following initial fixation as above, prepared for embedding in Spurr's resin (Spurr 1969) using the transition solvent propylene oxide, gradually infiltrated with resin, and heat polyrnerized in flat aluminum trays at 60°C for 4 hrs. Sections of the samples for light and TEM microscopy were cut with glas knives using a Reichen OMU-3ultramicrotome. For general Mewing. sections were stained with toluidine blue O (0.5% in 1% sodium borate solution). For use with the laser scanning confocal microscope. resin embedded tissue was mounted with epoxy ont0 microscope slides, the surface of the tissue exposed with a razor blade and stained with suiforhodamine G (Melville et al. 1998). The stained blocks were then viewed under oil without a coversiip, using a BioRad MRC-60 LSCM, equipped with a krypton-argon Mxed gas laser, utilizing a 568 nm excitation wavelength . the KIMfiher block, and photomultiplier (PMT)1 detector (for emission > 560nm). For TEM gold sections were mounted upon fonnvar-coated grids and stained with lead ciirateluranyl acetate. They were observed with a JEOL100 CX- TEM. Stains employed to highlight structural or histochemical feanires of fiesh or embedded, sectioned materials included toluidine blue O for polychromatic general naining (O'Brien and McCully 198 1), phloroglucinol for lignin, I2KI for starch (Jensen 1962). berbe~efanilineblue for aiberinfcallose (Bnuidrett et al. 1988), fluor01 yeUow (Brundrett et al. 1988) and Sudan III-IV for lipids (Jensen 1962), trypan blue for ceU walls and general staining and calcofluor for cellulose (O'Brien and McCully 198 1). Images of the sections were captured digitdly using a light microscope mounted video camera driven by Northem EclipseO software. Photographie plates were prepared using Corel ~raw~version 7 & 8 or Adobe c hot os ho^@ version 5. Clcarhg ord stainingfor -nt-of-~oIoni~on assessincn h Each whole root (rhizome end to apex) was immersed in a 10°h w/v potassium hydroxide solution in a glass jar labeled with the root's source number. The source number linked the root to information about the plant from which it was collected. including species. habitat, collection date, reproductive nate, etc. The roots were heated at 60°C for 24-48 hrs. until the stele was clearly visible through the translucent tissue. Mer two rinses in deionked water, a final acidified water soak (pH approx. 4-5) removed remaining traces of the potassium hydroxide, and prepared the roots for naining. Trypan biue (0.05% in I : 1 : I giycerin: lactic acid: water) was used to stain fungal ceIl wall material in the roots. A minimum 30 min soak in the stain solution at 60°C produced hi&-contrast naininp. On occasion, dark pigmentation of the epidennis obscureci interna1 detail. These roots were bleached (following clearing and rinsing) in a 1% household bleach solution untii the surface pigment was removed. The roots were thm rinxd and stained as above. Stained roots were aored in a mixture of equal paris water:lactic acid:glycerine until processed for observation. For mounting roots were laid out on a plexiglass sheet previously marked ho1 cm lengths. The whole root was then cut with a razor blade into 1 cm segments. Root pieces were numbered relative to distance from the rhizome (Le. the proximal end of the root was designated segment 1). The segments were mounted two to a glass slide, rnaintaining their orientation and position relative to the original rwt. Polyvinyl alcohol (PVA ) (Salmon 1954) was useâ to mount the segments permanently under glas covenlips. Gentle pressure on the coverslips was used to squash the root segments, aliowing observation of al1 the cells of each segment. Pnor to or following assessment, the slides were placed in an oven for a week or more at 60°C to harden the PVA For storage. Scoring of colonizution For each 1 cm segment of each cleared root, continuous fields at lOOx were observeci by light htcroscopy. The presencelabsence of five features were scored for each 1 cm segment: new peloton formation (Fig. 2. l), intact pelotons prior to obvious breakdown (Fig. 2.2), degraded pelotons in which individual hyphae could nill be discemed (Fig. 2.3). collapsed pelotons (Fig. 2.4) (usually aggregated hyphal remains) and surface hyphae (Fig. 2.5). Features such as recolonization events (Fig. 2.6) and moniiioid cells in the epidermis were also noted. To provide a fine scale percent-colonization assessment for each segment the nurnber of contiguous fields parallel to the stele of the root displaying fungal pelotons in any state relative to the total number of fields per segment was also scored. Diversity of endophytes was exarnined in the cleared sarnples at higher magnifications (250- 1000~).Data were graphed using SPSS" version 10. Results Myconhizai rooi anatomy offiw Cypripcdium species Epidermai, cortical and stelar features of roots of relevance to mycorrhizal colonization were noted Ui specimens of C. acmde. C. metinum, C. cddtrm, CC.pmvijotwm and C. regime. W~ththe exception of C. candidurn, which is now iisted by the Convention on International Trade of Endangered Species (CITES)as endangered, specimens of each species represent more than one collection site. Mycomtnzal fiingi (live or digested peloton remains) were present in almost al1 roots sarnpled. but were of patchy distribution in some cases. Roots collected from mid-rhizome on ail species were pale arnber to brown-black in colour, due to the deposition of pigrnented material on the surface of epidermal cells (Fig. 2.7). Roots of C. arietinum were generaily of thi~erdiarneter than those of the other 4 species. EpidnnUs A single epidermai layer of usually living, or in older pans of the root, dead and collapsed cdls was common to al1 species. The outer tangential wall of epidemal cells was often thickened relative to the i~ertangential and radiai walls. In C. candidum. laminations of wail material were present in the outer wall (Fig. 2.8). The epidermises of C. candid~imand C. parv~flonmwere similar, formed of celis that were rounded in cross- section, producing a deep sulcus between the cells. Despite this. the sufiace of the epidennis remained nearly flat due to a thick deposit of material, possibly phenoiics, on the epidermal surface. In addition, there was some evidence for lignification of the epidermis of C. acaule and C. pwtflomm. C. acuule and C. regme also possessed thickened outer tangential ceil waiis and dark deposits on the epidennal surface. However, in cross section the epidennal cells of these latter huo species were 4- 6 sided and angular, with a flat outer tangentid surfiace and an inner tangential prouusion between the underlying exodennai cells. A neariy 1 :1 ratio of epidermal to exodermai cells was seen in al1 four of these species. In con- the epidemiis of C. urietimim was quite different. C. urietinum was distinctive for its large epidermal ceUs that were convex to concave on the outer wall and convex on the inner wall in cross section (Fig. 2.9). Each epidennal celî spanned the width of 3 to 4 exodemal cells. In older roots, the epidermal cells were oflen collapsed. With the exception of C. arietimm, epidermal cells in tangentid view were rectangular in shape, oriented parallel to the long êrs of the root, and of similar size as the underlying cells of the exodermis. Each epidennal ce11 was in contact with several exodermal cells. In C. arietimm, the epidemal cells were considerably larger than the underlying cells, and tended towards being square in shape. Again, several exodermal cells were in contact with each epidennal cell. Live epidemal cells were often plasmolyseci, ah hou@ underlying cells remained intact. There was evidence of fungal and bacterial colonization of epidermal celis. Although some of the colonized cells contained intact nuclei, it was difficult to ascertain in al1 cases if cells remained dive after such colonization. Monilioid cells of Rhizoctot~-likehyphae (monilioid ceUs and hyphae; Figs. 2.10-2-12). hyphae bearing clamp connections and hyphae resembling those of Phiahcephalafortinii (Fig. 2.13) were sometimes seen in epidennal cells. Long, sparse, unbranched root hairs were present on many roots. These were mostly free fiom intemal colonization but occasionally fungal hyphae were present (Fig. 2.14). Mycorrhizal fiingus colonization in many roots seemed to be achieved by direct penetration of the epidermal cell wall (Figs. 2.15.2.16). Appressona were not formed by these fùngi. The epidermises of dl species aamined had patches of debris. bacterial colonies and possibly other organisms attached to the surface (Fig. 2.17). Algal cells were present on the surface of root hairs in some Iiving matenal. Erodermis The exodemis of al1 species consisted of a nearly regular pattern (rnost reguhr in C. arietimm) of altemating long and shon cells. In cross section, the size and shapes of both long and shon cells were similar. In cleared preparations stained with trypan blue, the more darkly stained walls of short cells suggested direrences in the composition of the cell walls. The presence of Caspanan bands (Fig. 2.18) was continned in ail five species, and therefore the term exodermis is appropriate. Lignification of the radial and outer tangentid wdls of the exodemd cells was also demonstrated using phloroglucinol HCL staining (Fig. 2.19). However. lignification was reduced or absent in the tangentid walls of some cells. suggesting their roles as passage cells for substances and organisms moving between the epidermis and the cortex. Both long and shon cells remained dive at matunty. but did not store aarch. No teniary cell wdl development was noted in the exodermal cells of these species. Co~et: The cortex of al1 species was composed of thin-walled, parenchymatous cells, often with large intercellular spaces. In al1 species, ceIl wails stained pink in TBO,indicating a richness of pains in the walls and rniddle larnellae. Cross sectional ce11 diarneter was greatest in the central area of the cortex, decreasing towards both the exodermis and endodemis (Fig. 2.20). These narrower outer and imer cortical «Us aiso tended to be much longer than wide. while cells in the central cortex tended towards a lower lengtldwidth ratio. In C. arietimm, centrai conical ceus were dmoa cuboidal in shape. In al1 species, abundant, large prhary pidields were apparent in the cell walls. Mvcorrhital fungal hyphae were observai on several occasions in primary pitfields. Large intercellular spaces were present between cortical cells. In many roots, structured (almost bacteria-like) intercellular protuberances (Butterfield et al. 198 1) projected into these spaces Qig. 2.21 ). The staining reaction of these structures was similar to those of the pectin-rich cell walls to which they were attached. TEM observations showed the protuberances to be composed of cell wall components (Fig. 2.22). Myconhizal fiingal hyphae were rarely found in the intercellular spaces, despite the apparent usefulness of such a space to colonizing organims. AU cortical cells (exclusive of idioblasts and exo- and endodermal cells) were capable of noring large arnounts of starch (Fig. 2.23). In many cases, aarch in fresh material obmred the view of hyphae within the cells. Starch grains were reduced or absent in cells contai~ngintact or degraded fiingal pelotons. but were ofien present during early colonkation of a cell, and afier the collapse of pelotons. The single layers of cortical cells adjacent to both the exoderrnis and the endodermis often contained large amounts of starch, even in highly colonized roots (Fig. 2.24). Extensive mycorrhizal colonkation (e.g. Fig. 2.20) was evident in some specimens of al1 species examineci. Frequently, several stages of formation and breakdown were seen in a single resin section of a root. indication that these processes are not synchronous within patches of colonization (Fig. 2.24). There was also little evidence for an outer cortical layer of live hyphae with more central 'digestion' cells. However, digested hyphae in the outer cortical cells often coliapsed and were bro ken down in sinr. rather than behg aggregated into large peloton masses like those of the central conex. Recolonizations of cells afler digestion of earlier pelotons were fiequent. Two patterns of recolonization were noted (Fig. 2.25). In the recolonizing hyphae mp around the old peloton. As breakdown and digestion occur, these hyphae form a shell around earlier pelotons. OBen three or more layen of pelotons could be discemed in a single cell. Some of these large. multilayered pelotons appeared to be separated fiom the cytoplasm by wall material. In the second pattern. recolorüzing hyphae did rot interact with older pelotons. and fonned discrete clurnps at breakdown. Five or 6 pelotons were seen in the cells of some Cjpr~pdiiumroots. This pattern appeared to be more common in C. urietimm, but ofien both patterns could be seen in the same 1 cm segment of a root in al1 specia. In ail cases, during later aages of digestion, the peloton appeared suspended by hyphae connecteci to the ceil periphery (Figs. 2.4, 2.25). These 'hspension hyphae" often linked across cell walls. and perhaps across pnmw pit fields. to those of adjacent cells. Since these hyphae were not noted in such abundance in early aages of colonization, it appears that they were rapidly produceci, perhaps at the onset of digestion. Suspension hyphae may become incorporated in the hypM rnass of very old pelotons, because they were not present at this late stage. Several differmt hyphd types forrd pelotons in the mycorrhizas of Cypripditm species. These dEered in diarneter and in septation. Dolipore septa (Fig. 2.26) were seen in some hyphae of al1 &es. Broad (7-1 1 pm) (Fig. 2.27) and finer diarneter hyphae (2.5- 4.Spm) (Fig. 2.28) with dolipore septa resembling those of the orchid-associated Basidiomycetes, Moniliopss and Epdorfika respectively. were moa commonly recorded. Also frequently seen were several types of hyphae bearing clamp connections (Figs. 2.29,2.30). There appeared to be a difference in the extent to which these various hyphae could be degradeci. The foms of large hyphae were still recognizable in old pelotons. Fine, unclarnped hyphae often becarne morphous even before they were aggregated into a hyphal mass. TEM obsavations of an apparently uncolonized area of a C. regrnue mycorrhiza revealed diminutive hyphai wall masses that were not seen in the light microscope (Figs. 2.3 1.2.32). These tiny peloton masses were generally smaller than the starch grains of the same or neighboring cells, and may represent cross sections of the "suspensor" hyphae of old, well-degraded pelotons. Nuclear hypertrophy of colonized and adjacent cells was a common feature of dl Cjpripedium species observeci. Several prominent nucleoli per edarged nucleus oflen were apparent (Fig. 2.33 ). Nuclei becarne lobed, so t hat a thin section of a ceIl appeared multinucleate. Colonizing hyphae were often associated with the nucleus. perhaps even passing through it . Endodnmis State III endodemal cells only were formed adjacent to the phloern in dl species. Those abutting the xylem remaineci in state Il. with suberized (Fig. 2.34) and lignified (Fig. 2.36) radial walls. With the exception of a single specimen, they possessed no secondary wall thickenings. C. candiidun, and C. pamioonun endodemal cells were thickened on both radial and tangentid walls (the O-type endoddcelis: Fig. 2.35). The secondary waüs did not nain for lignin, despite strong staining of the pnmary wail with phlorogiucinol HCI. These two species appeared to mer in the number of endodemal celis abuthg each xylem arc. In C. parvrfrlwm, there was typicdy 5-7 state ceus per arc. C. dichrm produced only 2-4 cells per xylem arc. However, only a single specimen of C. cdidum was obtained for observation, so this will require funher snidy. The endodermal walls of C. ocmie. C. rcgime and C. arietinuni had U-type endodernid cells meaning that the secondary wall thickenings were laid dom on the radial walls, and the inner tangentid wall (Fig. 2.36). The secondary wails stained for pectin, or lignin, or both. Where both histochemical reactions were seen, the newest (i~ermost)wall layen stained with toluidine blue O for pectin, while oldet. outer layers stained for lignin. This suggests a secondary wall that is ongindly nch in pectic substances. into which lignin is gradually incorporated. Despite the presence of the thin-walled passage cells in the endodermis. the hyphae of mycorrhizal îùngi were never observed in the stele. Stele Although a pericycle of thin to thick-walled cells was discernable in al1 species. no laterai roots were initiated ffom it, nor was branching seen on the roots of any of the Cjpripeditrm specimens. Phloem was abundant in the roots of dl species, but lignified cells of the metaxylem dominateci the aele. Scalariformly pitted vesse1 elements with simple perforation plates were observed in C. acaule and C. regmue. Tracheids with bordered pits were cornrnon. The aele of these species contained no pith nor were starch-containing ceUs observed in the aele. Extent of cdoni&un .4 surnrnary of the extent of colonization for the cleared specimens is provided in Table 2.2. Four species, 63 specimens and more than 8 meters of mots were examined. Of the specimens assesseci, only one lacked evidence of past or current association with mycorrhizal hngi. In each case, the percemage of the total rwt segments occupied increased fiom the 'new' to the 'collapsed' peloton stages. With the exception of the C. parv~florwrncollection, dl collections had some roots with new mycorrhizal colonization stages. Percentage of segments with new pelotons was highest in the mid-summer collections of C. reginae. The percentages are very similar for the two populations of C. acatde, despite differences in collecting time and location. While individual roots fiom a population varied in their colonizatioa none of the populations had an average less than 75% of segments showing evidence of previous colonization (al1 stages). If [ive colonization is defined as the 'new' and 'intact' categories, none of the populations had a lower colonization rating than 4.5% (C. pm~jiorum)),and some are at least as high as 75.7?!(C. regnae, Purdon, ON). C. acmde had the highest overall level of coioniAon. These data were not analyzed statistically due to the diffecing collection times and the small number of collection sites. which couid have afkted trends and colonization levels. However, as mentioned previously, the colonization levels for aü stages were similar for the two populations of C. acaule representing different provinces and seasons of collection. Colonization levels of each sage for al1 roots examinecl are shown in Figs. 2.37-2.40. It is evident fiom these figures that the colonization stages overlap in many roots, and that there is a steady increase in the fiequency of the stages moving from new to collapsed pelotons. The shortest roots, while free of early colonization stages, generally already had been extensively colonized, as evidenced by the presence of collapsed pelotons in the roots. The presence of the four colonization stages was recorded in each of the four species (Appendix 2.1-2.4). Although C. regime and C. acaule had a greater percentage of segments with new, intact and degraded peiotons than do C. mierimm and C. pan,~f!omm,the percentage of segments with collapsed pelotons is similar for the four species. The degree of peloton stage overlap for the C'ripdium species is illuarated in Fig.3.4 1.. At the time at which the plants were collecteci. the C. arierimrni and C. paniflonim roots were dominated by single stages. However the profiles for C. regrnoe and C. acm11e are dominated by three stages of colonkation per segment. C. ocoule also has the lowest percent of segments without evidence of currmt or previous fungal colonization. C. mietinuni and C. parwifloruun, were similar in the amount of uncoionized segments: just under 25% of the segments had no history of myconhual colonitation. Colonization along two individual C. regimp roots fiom the Purdon Natural Area population can be seen Ui Figs. 2.42 and 2.43. Note that 'new' colonization stages are rarely unassociated with 'intact' and 'degraded' ones. New pelotons are found in patches, and do not appear to be correlated with position dong the root axis. Discussion C'ripdiuin roots have several features of relevance to mycorrhizal symbiosis and its formation. The epidermd cells would appear to present a banier to colonizers of the root. Epidermal cells may form thickened and perhaps ligninecl outer tangentid ce11 wds. As well, deposits of pigmented materials (possibly phenolics) accumulate on the outer surface of the epidermd cells. C'diurnroots are in contact with a large number of soi1 organisms, yet few are able to colonize the epidennis, and fewer stiU pas into the cortex. The exodemis, reported hem as possessing a Casparian band and 'long' and 'short' cell differentiation, appears to control the ingress of organisms into the cortex. Cdonizing hyphae in arbuscular rnycorrhizal systems are known to utilize the more easiiy traversed short cells of the exodemiis to gain entry to the cortex (Matsubarn et al. 1999). This has also been observed for the orchid mycorrhizal fun@ that inhabited greenhouse plants of three orchid species with velamenous roots (Esnault et al. 1994) and for the wild orchids of this study. Since the outer wall of the shon cell is the last barrier to access the cortex. these cells could be the sites of symbiont seleetion by the orchids (Esnault et al. 1994). In C. acairk, C. cadidum. C. purvîflomm and C. re-e. each epidermal ce11 interfaced with a few exoderrnal cells, at least one of which was a shon cell. In C. arietirium, the lower number of short ceils in the nmow diarneter roots of this species may be cornpensated by increased access to shon cells through the larse epidermai cells. Many organisms, including bacteria, fùngi, algae and protozoa exined on the surface or within the epidermis of Cypripediium roots. Ahhough death and/or phenolktion of epidermal cek were occasiondy observed. there was no evidence of plant defense responses to mycorrhiral fungal colonization as the hyphae passed through the passage ceils of the exodermis. This may be similar to the colonization processes of arbuscular mycorrhizai fûngi, where plant defense responses are triggered but then down-regulated during fiingal penetration (Dumas-Gaudot et al. 1996, Mohr et ai. 1998). Once inside the exodemiis, fiingai hyphae move intracdularly from cell to cell. This is pecutiar considering that mature regions of the root cortex have large interceiiular spaces through wfüch fûngal hyphae easily could penetrate. It is possible that this inability of the hyphae to travel intercellularly is under the control of the host plant. Coraihrhizz mfiah,a mostly achlorophyilous orchid is mycorrhizal with a fùngus that cm also forrn ectomycorrhizal associations with lodgepole pine (Zelmer and Currah 1995). In it association with the orchid, the mycorrhizal fungus is restricted to the root cortical cells, while in the ectomycorrhizal association, it penetrates the pine root only intercellulary. Root cortical cells in al1 species contained large amounts of starch. When live Cwipediun, roots were hand-sectioned, the abundance of aarch often obscured the presence of pelotons, especially in newly colonized ceils. Although starch typically disappears in colonized orchid cells (Rasmussen 1995), this process may be quite gradua1 in Cyprpdiium species. Under LSCM colonited cells that still possessed starch grains were commonly observed. The results of this audy cleady indicate that myconM hngi are frequent endophytes in the roots of mature plants of several Cjpripediium species However. the literature contains many negative statements regarding the mycorrhizal status of mature C'ripedium plants. Alexandrow (1 925) iliustrated the roots of a young C. cuiceolus plant heavily colonized with mycorrhizal hingi but aated that the roots of mature plants were fungus-6ee. This assessrnent was liiely based upon the observation of hand- sections of live material. Harvais (1974). noted that young roots of mature C. regïme plants were always uncolonireci. Old roots of these plants occasionally possesseâ patchy and "restricted paths of infection dong their lengths where pelotons were srnail, arnorphous and h an advanced stage of digestion." He concluded that mature C. regme plants actively rsisted colonization, but the defenses of old, weak roots were sometimes overcome. Whitlow (1983) betieved that the usefiil role of mycorrhizal hngi in Cypnpedium ceadat the onset of photosymhesis. In an intensive midy of C. candidum. Stout amire ( 1 99 1 ) found only 1 û??colonization in white, healthy mots of C. candidm Recent orchid propagation literature also contains statements on the relative unimportance of mycorrhkal fun@ to mature plants of C. acmle (Durkee 2000). These views are in opposition to the findings of this study. The individual roots sampled corn mature CI.prpr!Jium plants varied in extent of colonizatioa but overall, mycorrhizai colonization was both cornmon and as extensive as reponed for many other species of orchids (Zeimer et ai. 19%). The morphological and anatornical investigations may provide ches to the discrepancies between the results of this investigation and the literature. The roots of C'ypripedium species are perennial (Whitlow 1983). Individual roots can persist in a functional and healthy state for up to 19 yean in C. pubescens (Curtis 1943). and 10 for C. cmtdidtrm (Stoutamire 199 1 ). Those of C. candiduun, increase in length by 5- 10 cm each year for as long as the root apical meristem remains undamaged (Stoutamire 199 1 ). By selecting roots fiom the middle of the rhizome, roots several yean old were collected for observation in this study. Newly-fomed roots are light in colour, aarchy and seneraliy free of fungus (Harvais 1974). Ln contrast, the older roots selected for study here were pale amber to dark brown-black due to pigrnented deposits on the wface of the epidermis and lignification of the epidermis and exodennis. These darkened roots may have been unattractive to those collecting roots for study because they have the appearance of king dead or senescent. Funher, the relationship between darkening and morbidity of roots is generaliy tme for other terrestrial orchid species. The lack of colonkat ion re pond by other researchers may, therefore. be due to sarnpling error. Colonization in many orchid tissues is prevented by the production of phytoalexins, inducible fiingicides produced by the plant (Rasmussen 1995). This may be true for the new, lightly coloured roots as well. The protection against invading organisms afforded by the materials deposited on the epidermis may ailow for a reduction of phytoalexin production and subsequent colonization of the roots. The lower colonization rates in this study are ofien associated with shoner roots, suggesting that these roots may be relatively younç. Not al1 roots on a given yearly rhizome increment are forrned simultaneously. One to 3 roots may fom later on the opposite side of the rhizome to the pair normally initiateci at the base of the sympodial bud (Rosso 1966). The rate of tumover of pelotons rnay also affect the impression of mycorrhizal importance to ~vpripediumspecies. In this nudy. new pelotons were rarely unaccompanied by intact and degraded sages, suggesting that there may be rapid formation and degradation of the pelotons. Stoutamire ( 199 1) also beiieved this to be true for C. cmuiidrm. If this rapid turnover of new hyphal coionkations occurs throughout an extended part of the growing season., the imponance of new peloton formation will be greatly underestimated. Old peletons are observed more cornmody because of the persistence of hyphal wall remains. Several hyphal types were observed in pelotons of the roots. The fonn of the hyphae and monilioid cells (ofien in the epiderrnis) moa frequently resembled those of the orchid mycorrM fun@ Epulorhizu and les ofien Moniliopsis. Both of these fun@ are members of Rhtzucto~~iaS. I. Of interest is the fiequent observation of hyphae bearing clamp connections. Since Rhizuctotiia S. I., the most comrnon group of 'orchid mycorrhiral fungi' does not by definition bear clamp connections on their hyphae (Currah and Zelmer 1992). these f'ûngi are noteworthy. Clarnped hyphae are known hom the myconhizas of several orchids (Currah et al. 1997a). including the holomycotrophic orchid Cdlorhiza tr@& (Zelmer and Currah 1995). Those associated with C. 1rtj7cda are also ectomycorrhizal with several tree species. fonning a conduit for tree-fixed carbon to the orchid. As many ectomyconhizal fungi are dependent upon their plant host. they are difficult to isolate and axenically culture. The low number of isolates reporteci fiom Cypripedium species rnay be related to this. Clamped hyphae also have been found in the donnant and germinating seeds of 0p@edi11m species placed in natural habitats in mesh packets (Zelmer er al., 1996). Speculatively. the low su~valrate of relocated Cvpripediurn plants and difficulty in symbiotic gemination in culture may also be explained by a requirement for a third syrnbiotic panner. Colonization of individual roots is patchy and seemingly without pattern fkom the base of the root to the tip. Since the fùngi colonize directly through the epidennal cells. their appearance in a particular segment relates more to the resource base for the extraradical hyphae outside the root than to the construction of the root. Conclusion The roots of Qpriipediurn acmle. C. repue. C. candichm. C. pant~jlon~rnand C. urietimm are similar in respect to several anatomical features related to mycorrhizal symbiosis. AU possessed darkiy pigmenteci deposits on the outer tangentid walls, and an exodermis (complete with Casparianband) composed of 'long' and 'short' (or passage) cells. In some species, a thickened and lignified outer tangentid epidemal ceIl wall was also seen. Epidemal cell shape and sire relative to the underlying exodennal celi layers was similar in four of the species but difkent in C. arieii~tt~m.Although mycorrhizal fungi passed through the epidermis without the formation of appressuria, colonization of the cortex generally occurred only via the short cells of the exodermis. Cortical cells in al1 species accumulated large amounts of starch, which tended to decrease after colonization. However, colonized cells, pmicularly in early peloton formation, ofien possessed starch grains. The Cjpripeditm species examined in t his study were associated wit h mycorrhizal fun@ at maturity. Although colonitation of individual roots varieci, overall colonization levels for many collections were quite hi&. Roots with no evidence of fungal colonkation were rare. Former reports of low colonjzation levels may reflect a sarnpling aversion to the darkly pigmented older roots and the extensive starch reserves, which can obscure observation of the fungi in live roots. There is a diversity of fungi fonning pelotons in the roots of mature C'npeditm specimens. Most peloton-fonning hyphae resemble those of Eworhiza and Moniliopsis, both Rhi:uctonia S. 1. members. However, hyp hae bearing clamp connections kequently were seen in the pelotons of Cyp~pediumroots. Tabk 2.1. Cypripedium specimens examiad Site Collection Date Number C. acarrie St. Williams, ON June 23, 1997 Ci uca~de Brereton L., MB July 23, 1997 c'. trca~dtr St. Williams, ON Oct. 3, 1997 C. awle Waterloo Reg., ON Aug. 23, 1998 C. acat11~ Brereton L., MB Sept. 12 1998 C. acaule Near Clifford, ON June 24, 1999 C. acaule Belair Forest, ME Oct 16, 1999 C. acaule Belair Forest, MB Nov. 5, 1999 C. acuule * St . Williams, ON May 17,2000 C. acade* Belair, ME3 July 18, 2000 C. ariefirt~m Near Hamilton, ON June 1 1, 1997 C. arietinirm * Woodridge Rd., MB Sept. 2,2000 C cardid~m Cuhivated, MB luly 30, 2000 C. parvrjlwum Crief, ON June 1 1, 1997 c. pantlfl~ruum Near Hamilton, ON June 1 1, 1997 C. parvrfort~m Bruce Peninsula, ON June 18, 1997 C. parvrjunm Anola, MB July 23, 1997 C. parvflonm Crief, ON luly 29, 1998 C. prvtflorum Hwy 66, MB Aug. 20,2000 C. regiwe Crief, ON lune 1 1, 1997 C. regrurp Anola, MI3 My27, 1997 C. repe 21 k wofElma, MB July 23, 1997 C. regime Crief, ON Sept 14, 1997 C. reginae Crief,ON July 28, 1998 C. reginae Purdon N. A., ON July 25, 1999 C. reginae* Crief, ON May 16,2000 Samples used for extent of colonkation assessrnent Table 2.2. Summay of percent of roo4 length coloniacd for four Cypnpedkm spics Smcies Site Plant Date New Intact Dard Clpsd Seg. Ave.1. C. acaule St. Williams, ON C. acaule St. Williams, ON C. acaule St. Williams, ON C. acaule St. Williams, ON C. acaule St. Williams, ON C. acaule St. Williams, ON C. acauie St. WiHiams, ON C. acaule St. Williams, ON Total % C. acaule Belair, MB C- acaule Belair, MB C. acaule Belair, MB C. acaule Belair, MB C. acaule Belair, MB C. acaule Belair, MB C. acauîe Belair, MB C. acaule Belair, MB Total % Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON Purdon, ON C. regiiiae Purdon, ON 415 253ul-99 1 10 14 28 29 C. regnnae Purdon, ON 416 2S-Jul-99 1 3 7 6 15 Totrl % 3&1% 58.8% 75.7% 80.1% 342 22.8 C. regrnae Crief, ON 432 1CMay-00 10 19 22 21 34 C. n?@nae Crief, ON 433 16-May-00 O 1 3 3 20 C.#ginae Crief,ON 434 16-May-00 1 6 t6 27 32 C. regrregrnae Crief, ON 435 16-May-O0 O 8 21 23 23 C. regiiiae Cnef, ON 436 16-May-00 O 4 6 9 17 C.mghae Crief,ON 437 16-May-00 1 24 28 31 31 C. risgriiae Crief. ON 438 16-May-O0 O 19 27 27 27 Total % 6.5% 44.0% 66.8% 76.6% 184 26.3 C. ahbtinurn Woodridge Rd, MB C. atbtinum Woodridge Rd, MB C. anétinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. arietinum Woodfidge Rd, MB C. arEetinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. &tinutn Woodridge Rd, MB C. mtinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. an'etinum Woodridge Rd, MB C. arietinum Woodridge Rd, MB C. anètinum Woodridge Rd, MB 486 2-Sep00 O O 1 7 8 Total % 1.3% 2.6% 30.3% tt.636 f 52 1 1 -7 Columns Nm.Intact, Dgrd @egnded), and Clpsd (Collapxd) indicate the number of I cm segments of each root containhg thai stage of peloton development . Seg. is the total number of segments per root. Totals are given for each stage as a percentage of the total number of root segments assessed from that population. Figures 2.1 to 2.6. Peloton stages and extemal hyphre: Cypnpedum ~inaemots cleared in KOH and stiintd with typm blue. Fiy. 2.1. New peloton. Hyphae have just entered cell wall and are coiling. 200x. Fig. 2.2. Intact peloton. Maximum extent of colonization appears to be reached but hyphae are ail1 intact and usually aain darkly with trypan blue. 200x. Fig. 2.3. Collapsing peloton. Hyphae within peloton begin to lose their contents and cell wall integrity. Note collapsed hypha (arrows). 2ûûx. Fig. 2.4. Digested peloton with "suspensor hyphae" radiating fkom hyphal mass. Hyphae become indistict, and are gathered into a mass of cell wall material. 100~. Fig. 2.5. Extemal hyphae on root epidennis. 100~. Fig. 2.6. Recolonization of root cortical cell. Note intact hyphae surrounding digested hyphal mas. 200x. Figures 2.7 ta 2.12. Featum of' Cyprfpcdium root epidermis and esodermis. Fig. 2.7. Epidermal cell outer tangential wall. TEM microscopy. C. acaule. Arrows indicate deposits of matenal (probably phenolics) responsible for the dark appearance of mature roots. 45,000x. Fig. 2.8. Epidermal cells showing thickened outer tangential walls. Light microscopy, hand section stained with Sudan IVN. C. candidum. .rlrrows indicate laminations. Ep, epidennal cell; Ex, exoderrnal cell. 500x. Fig. 2.9. Living and collapsed epidennal cells of C. arirt;mm. Ep, epidermal cell; Ex, exodemd cell; C. cortical ceII. Resin embedded, thin sectioned and stained with toluidine blue O. 200x. Fig. 2.10. Monilioid cells (arrow) within an epidemal ceIl of a C. rtpioe root. Tissue cleared in KOH and stained with trypan blue. 200x. Fig. 2.1 1. Hyphae in epidermis. C. reginae. Tissue cleared in KOH and aained with trypan blue. Ex, position of exodermai passage cells seen through epidemal ce11 layer. 50'r. Fig. 2.12. Hyphae in passage cells (Ex) of exodemiis. Lower plane of focus than Fig. 1 1 . Tissue cleared in KOH and stained with trypan blue. 50x. Figures 2.13 to 2.18. Featum OC Cypnpe&um raot cpidermis and exodemis. Fig. 2.13. Dark septate hyphae, possibly of Phialocephalafurti~tii. in sulcus between epidermal cells and passing through to exodemal cells (arrows). C. acarrk Cleared in KOH and stained with trypan blue. 45Ox. Fig. 2.14. Rhizocto~~ia-likehyphae and monilioid cells in root hairs of C: ocaide H, hypha, RH, root hair; M. monilioid ceil. Cleared and aained matenai.20ûx. Fig. 2.1 5. Colonization of root cortex by myconhUai fungus via penetration of epidermal ceIl (arrow). C. cortex; Ep. epidermal cell; Ex, exodemal cell; Fp, fungal penetration point. C. regnue. Resin embedded, thin sectioned. aained with TB0 200x. Fig. 2.16. Hyphae exiting root epidemis (arrows). C. regimre. Cleared and stained material. 200x. Fig. 2.17. Root-associateci organisms of C. acuule. H, Hypha; B. bacteria; M mycorrM colonkation; C, cortex; Ep, epidermis; Ex, exodertnis. Resin embedded. thin sectioned, stained with TBO. 200x. Fig. 2.18. Root exodennis (Ex) of C. acurule. Fluorescence microscopy, berberinelaniline blue staining. Casparian bands (arrows) fluoresce brightly; arrow heads indicate positions of passage cells. 100x. Figs. 2.19 to 2-24. Fcrtures of Cypnpedium root cortices. Fig. 2.19. Exodennal cell wall lignifications (arrows). C. Cortex; Ex, exodemis; Ep. epidennis. Hand section; phloroglucinol staining. C. purv&mm. 250x. Fig. 2.20. C. arietimr~root cross section. Intensive colonkation of cortex showing many stages of peloton formation and breakdown. Resin embedded. stained with TB0 50x. Fig. 2.2 1. Stnictured protuberances in the intercellular spaces of a C. regime mot. C, cortical cd; IS. intercellular space. Resin embedded. stained wit h TBO.250x. Fig. 2.22. Intercellular space. TEM view. Protuberances are continuous with the middle lamella of adjoining cells. C, cortical cell; IS. intercellular space. Resin embedded, viewed with TEM. 27,000~. Fig. 2.23. Starch deposits in the root cortex of C. acaule. Starch grains (arrow) are stained darkly by I:Ki in a hand section. 100~. Fig. 2.24. Non-synchronous development of pelotons in the root. conex of C. regpitue. DP, digested peloton (hyphal mas); N, hypertrophied nucleus; H. vacuolate hyphae; R, recolonizing hyphae. Resin embedded. stained with TBO. 100x. Figum 2.252.32. Mycorrbiul colonization of Cypnpedium reginue roots. Fig. 2.25. Formation of separate digested pelotons (hyphal masses) representing three colonization events (1. 2. 3). Cleared in KOH and stained with trypan blue. 200x. Fig. 2.26. Dolipore septum in a peloton hypha (H). Resin embedded, stained with TBO. 25h. Fig. 2.27. Broad hyphae fonning a peloton in a root cortical cell. Cleared and stained material. 200x. Fig. 2.28. Narrow hyphae (possibly of Epuforhiza sp. ) in root cortical cell. Cleared and stained material. 200x. Fis. 2.29. Hyphae with clamp connections (arrows) foming pelotons in a C. regnue root. 2m. Fig. 2.30. Detail of Fig. 28; arrow. clamp connection on hypha. Cleared and aained material. 500x. Fiym2-31-2-36. Featum of Cypripldium rmt cortex and stck. Fig. 2.3 1. TEM view of a minute digested peloton (P) surrounded by starch grains (SG). C. reginae. 3 500~. Fig. 3.32. Detail of Fis. 3 1. CH. collapsed and aggregated hyphal walls. C regi~lae. 1500ûx. Fig. 2.33. Hypertrophied and lobed nuclei (N) in colonited cells of C. clcoule. P, digested peloton; NO, nucleolus. Resin embedded. thin sectioned, TB0 staining. 200x. Fig. 2.34. Root cortex of C. acmde viewed with fluorescence microscopy. Berberinehiline blue aain under blue light. DP, digested peloton; arrows, Casparian bands of endodermis. Live tissue, hand section. 250x. Fig. 2.35. Endodennis and stele of C. candidum root. Endodermal cells adjacent to the phloem have eveniy deposited secondary wall thickenings (long arrow). Only the pnmqwaii shows Trypan blue aaining C, cortical cell; P. pericycle cell; En, endodermis; shon mow. primary cell waii. Live tissue. hand section. 500x. Fig. 2.36. Endodennis and stele of C. acaule. Ody the primary wall stains for lignin using the stain phloro&xinol (arrows). Unusually, this endodds has cells with well developed secondary walls adjacent to both the phloem and the xylem and may represent a vety old mot. Ph phloem; X, xylem; En, endodermis; Casparian band; arrow to lefi: secondary wall thicke~ngs.arrow to right. Live tissue, hand section. 400x. Fig. 2.37. Colonization of individual Cypripndium mots by mycorrbizal huigi: new pelotons. Each spechen number represents a mot of a single plant of C. acczuie. C. pmiflorum, C. urietinum or C. parvijlorum. Bars represent the number of 1 cm root segments present in the root. Darkened bars indicate the number of segments possessing new pelotons. Fig. 2.38. Cobnization of individuil Cypripedium mots by mycorrhuil fin@: intact pelotons. Each specimen number represents a mot ofa single piant of C. acaule, C. purwziflorum, C. arietinum or C. parv~jlorurn.Bars represent the number of 1 cm root segments present in the root. Darker bars indicate the number of segments possessing intact pelotons. Fig. 2.39. Cobnization of individual Cypripdium mots by my corrhhl hingi: degraded pelotons. Each specimen nwnber represents a root of a single plant of C. acaule. C. parvflomm, C. arietinum or C. pmviflorum. Bars represent the nurnber of 1 cm root segments present in the root. Darker ban indicate the number of segments po ssessing degraded pelotons. Fig. 2.40. Colonizstioo of individual Cypripdium mots by mycorrhiul tùngi: cohpsed pelotons. Each specimen number represents a root of a single plant of C. acaule. C. parvzflorurn, C. arietinum or C. pamiflorum. Bars represent the number of 1 cm root segments present in the root. Darker bars indicate the number of segments possessing coilapsed pelotons. Fig. 2.41. Ovedap of peloton stages in the mots of four Cypipedium spd~.Each pie chari represents aii the 1 cm root segments sampled for that species. Slices indicate the percentages of those segments coloaized by I,2,3 or 4 peloton developmenial stages (new, intact, degraded or coiiapsed). The percentages of segments lacking colonkation are labelled O. Numbers of stages indicate the presence of any combination of peloton stages (eg., new and coliapsed or intact and degraded = 2 stages; new or degmied = 1 stage). cm Colonurtion stages !y!?' 31 12 .a --B4 Fig. 2.42. Peloton frequency along a sine root of C. reginae specimen 390. Each section represents a 1 cm section of root. l= 1 cm segment of mot adjacent to the rhizome; 25 = mot tip. Peloton frequency is expressed as the ratio of the number of microscope fields at lOOx in which pelotons at any developmentd stage were present to the total number of fields per 1 cm segment of root. Fig. 2.43. Peloton brquency aloy a single mot of C. nginae specimea 408. Each section represents a 1 cm section of root 1 = 1 cm segment of mot adjacent to the rhizome; 25 = mot tip. Peloton fiequency is expressed as the ratio of the number of microscope fields at lOOx in which pelotons at any developmental stage were present to the total number of fields per 1 cm segment of root. Chapter 3. Cypripedium root-associated orgaaisms, including mycorrhizal fungi Introduction Several species of Cy-ditm can now dependably be genninated and raised in asyrnbiotic culture (Steele 1996). However, seedlings grown in a sterile environment eventually must be introduced into the diverse biotic environment of potting medium. The huent high losses of dlings at this time, combined with the documenteci hiçher survivai rates of dlings (e.g. (Anderson 1992)) raid in symbiosis with mycorrhUal fungi, has sparked interest in finding symbiotic partners for Cjpripediium species. A search for Cypriipedm symbioms mua reasonably begin with the collection of wild plants and an exploration of their associateci organisms. Although mycorrhizal fùngi are well known symbionts of orchids, there is little hown about the identity and ecology of fun@ symbiotic with Cypripeditm species. In addition, tripartite myconhizai symbioses exist between some orchids (e.g. species of Corolwhiza (Taylor and Bruns 1997, Zelmer and Currah 1995). other plant species and shared mycorrhizai fiingal symbionts. C'ripedittrn species. with t heir subtenanean juvenile Wods and occasional retum to a subterranean habit at maturity rnay dso have the ability to tap imo existing nutritional rnutuaiisms to meet their nutnent requirements. If the fungi that fonn myconhkas with Cjpripudium species are determineci to belong to known ecto- or encoid mycorrhiral taxa, Mertesting for uipartite symbioses would be appropriate. Based on studies with nonsrchid plants, there may dm be reasons to isolate and identifi other types of organisms associateci with the roots of C'jpnpudiiun,species. Plant growth promoting rhizobacteria (PGPR) are well documented, and aid the growth of plants by providing fixed Ntrogen. chelating iron in a forrn available to plant roots, producing plant growth hormones and decreasing levels of growth-reducing plant hormones in the root environment (Glick 1995). Although they can be free living, they are oflen closely associated with the root surface, embedded in a mucigel that is produced jointly by the plant and bacteria. in Tillandsia remmatu, a bromeliad, nitrogen-fixing bacteria can be found within the tissues of the plant (Puente and Bashan, 1994). Nitrogen- fixing bacteria have ken found aiso in the intercellular spaces of stems (Dong et al. 1994) and in roots (Bellone et al. 1997) of sugarcane. Cyanobacteria may also participate in the promotion of plant growth. While cyanobacteria are moaly fiee-living Gunneru, cycads and Azollo (Bergman et al. 1992) provide structures to house these important symbiotic organisms. The Ritrogen content of mangrove seedlings (Toledo et ai. 1995b) and wheat (Obreht et al. 1993) irnproves when roots are colonized by filamentous cyanobacteria. Plant growth may also be affecteci by soi1 protozoa, which tend to be abundant in the rhizosphere of plants. Protozoa play important roles in mineralization, and increase the nitrogen content and biomass of piants with which they are co-culhired (Ekelund and Ronn 1994). There have been few reports of isolations of fungi or bacteria fiom the roots of C'rip4dium species (see Chapter 1). and nothing is hown about the algae, cyanobacteria and protozoans in the rhuosphere of C'r@edium,species. The objective of this research was to localize, isolate and, where possible, to identify fun@, bacteria, algae, cyanobactena and ciiiated protozoans king on or withui the roots of wild C)pripedÏum species. Methods Roots of wild plants of cvpripediitrn reginoc, C. acmle. C. panvjlon~rmand C.arietirnrm were collected (see Chapter ? for methods and collection sites) in Ontario and Manitoba, Canada. Fungi, bactena algae and ciliates were isolated fiom the roots using the following methods: Fungi Fungi were isolated by transversely sectionhg I cm surface aerilized root pieces into approximately 1 mm discs and placing the discs. 4 to a Petri plate. on each of 3 agar based media (Corn Meal Agar (CMA). Potato Dextrose Agar (PDA) and Tap Water Agar (TWA). Hyphal tips emerging from the roots were removed to fkesh media. The isolates were subcultured untii pure cultures of each fungus were obtained. Fungal isolates were aored at 4°C on PDA slants. Colonial morphologies of many isolates were recorded by cornputer scanning Petri plates of 3 month old PDA cultures of the fun@. Samples of each fungai isoiate were stained with 1% acid fuchsin in equal parts lactic acid, glycenn and water and observed by Iight microscopy. Isolates were identified to genus where possible using light microscope-visible morphological feanires of the isolates on corn meal agar (CMA) and potato dextrose agar (PDA).Identifications of the aerile dark septate and Rhizoctonia-like isolates were confimecl by Dr. R. S. Currah. University of Alberta. Localization of fungi was accomplished ushg LR White resin (London Resin Co.. Basingstoke, WC) -embedded root material sectioned for light microscopy or viewed as a block using laser scanning confocal microscopy (LSCM). Root sarnples were fixeci, dehydrateci and embedded following the protocol outlined in (Melville et al. 1998). Thin sections were cut with glas knives on a Reichert ultramicrotome, stained with 0.5% toiuidine blue O in O. 1% sodium borate solution, and viewed with a Leitz Orthopian compound microscope. For LSCM. approxirnately 1-mm root mples embedded in resin were attached to glass microscope slides with epoxy. A two-sided razor blade was empioyed to remove excess resin from the upper surface of the resin blocks to expose the root tissues. After staining with a 0.5% aqueous solution of suiforhodamine G (Melville et al. 1998) for 3 min on a warm heating plate. the blocks were thoroughly rinsed with deionized water and allowed to dry. Root pieces in the blocks were examinecl under oil without a coverslip using a Bio Rad MRC-600 laser scanning confocal microscope equipped with a krypton- argon mixed gas laser. utilizing a 568 nm excitation wave length the KI/'filter block and photomultiplier (PMT) 1 detector (for emission > 560 MI). I. Tests Tor ericoid mycorrbizal ability Several isolates of a aerile fungus resembiing "Variable White Taxon" (Hambleton and Currah 1997). a yet un-narned ericoid mycorrM fingus, were isolated from the roots of Cypr~ipdïumacm~le. Seeds of blueberry ( Faccinium sp. ) were used to test the ability of wo of these "Variable White TaxonVike ftngal isolates to form ericoid mycorrhizas. Seeûs were surface aenlized by 2 bleach rinses (lm with a drop of dish soap for I hr, then 20 mins). nnsed in aerile distilleci water and placed on the sufiace of sterilized vermkulite in Phytatrays (Sigma). The venniculite was moistened with K strength Long- Ashton's micronutrient solution (Hewitt 1966). They were maintained in an illuminated incubator at 26°C. 16 hr light per day for 7 weeks. At this time the seedlings were removed to a sterilked agar medium in Petri plates. The plates were placed vertically, and the upper half of the medium removed to provide a space for the three to six leaves on the plants. The roots (4 or 5 'hair roots' per plant) were positioned in contact with the cereal agar medium (distilled water lL, HeinzTMPablurn Cereal 3% gellan Qum 2.2%Murashige & Skoog basal salts with minimal organics; pH 5.5). Petri plates containing 3 seedlings were each inoculated with one of two "Variable White TaxonV*-like(Hambleton and Currah 1997) isolates. Two triangles (2 mm per side) of agar cut from actively growing colonies of the fin@ were placed alongside the roots of each plant. Control plants were mock-inoculated with stenle agar medium. The plants were incubated in a lighted growth charnber under the sarne conditions used for germination. Three weeks der initial contact. the roots of the seedlings were harvested. cleared, stained with trypan blue and ob~rvedunder the Iight microscope to detennine if hyphal coils characteristic of encoid mycorrhual associations had formed in the epidermal cells of the biueberry roots. 2. Test for ability to fonn ectomyco~izasin viho Several fùngi isolated from the roots of C'ripedium species were tested for the ability to form ectomycomhizas. These hngi were selected for testing a) because of their similiarity to "Variable White Taxon", at the suggestion that fun@ siniilar to these may aiso fonn ectomycorrhizas (Vralstad et al. 2000). or b) the isolate possessed clamp connections. Clamp connections are reiativeiy uncornmon among orchid mycorrhizal symbionts, and could indicate a hingus capable of ectomycorrhizal symbiosis, as for the "Yellow Clamped Isolate" symbiotic with Cordorhizu 1r1pci4(Zelmer and Currah 1995). The sarne two "Variable White TaxonW-likeisolates used to test for ericoid mycorrhizal ability were also tested for their ability to fom ectomycorrhizas. The clamped füngus was isolated fkorn a rwt of Cypripdium reghue in Ontario. Testing was accomplished by inoculation of the fungal isolates ont0 the roots of pouch-grown Piinm srrobr~~L. seedlings. Five month old seedlings (one seedling in each of 5 pouches per isolate) were inoculated with 1 cm squares of agar cut from actively growing PDA cultures of the fiingi. Additionally. 2 pouches were inoculated with same-sized pieces of stenle PDA as controls. mer 4 weeks, dichotomuing short roots (ofien an indication of ectomycorrhual syrnbiosis in pine) were excised, fixed, dehydrateci and embedded in LR White resin. Thin se*ions stained with 0.5% toluidine blue O in 0.1% sodium borate were exarnined under light rnicroscopy for the presence of a hyphai mantle or Hartig net. structures comrnonly fomed by ectomycorrhiral root tips (Smith and Read 1997). 3. Tests for ability to lyse bacterial and a1ga.l cds A single fungai isolate was tested for the ability to utilize algai and bacteriai cells as nutnent sources. The carbon sources for orchid mycorrhiral fungi are not well known. Some basidiomycetes have the ability to utilize algai and fimgal colonies as nutrient sources (Barron 1988, Hutchison and Barron 1997). A single fungal isolate was tested for the ability to utilue bacterial and cyanobacteaiai colonies (later used to inoculate seedlings in the Chapter 5 expenment). These same kgai, bacteria and cyanobacterial isolates were later used in the Chapter 5 inoculation experiment. Fungai isolate 23 12 (E. rupm) was combined on ASM-3D (a mineral medium - ASM-3 - modified by the addition of 5gR dextrose) medium with each of 2 bacteriai isolates (3 16b- 1 and 3 18b-6). and the cyanobacteriurn NOSIOChrrmrfgu. Three Petri plates (1 00mm size) of each fùngus/oqanisrn combination were prepared. Controls were inoculated with the Fungal isolate and the respective carrier media. Each plate was inoculated centrally with a 5 mm cube of PDA agar cut fiom the edge of a 2 month old culture of fungal isolate 23 12. Four drops of the inoculum and camer medium (tryptic soy broth for the bacteria and liquid ASM-3 medium for the dgae) were placed around the tùngal inoculum equidistant between the cube and the plate edge. Plates were examhed after 3 weeks in weak daylight at room temperature. The plates were inverted and placed on the stage of a compound microscope at low power. They were examined for directional growth of the fungus, ramification of hyphae within the colonies. and breakdown of algal and bacterial cells by the fùngi. These features have been noted in saprophytic hngi that are capable of degrading and utilizlng algal (Hutchison and Barron 1997) and bacterial cells (Barron 1988). Bacterio Bacteria were iwiated concumently with the fbngal isolations. particularly on PDA media plates. Bacterial colonies formed on the cut airfaces of the orchid roots were removed using a flamed wire hop and were areaked onto PDA plates. Mer 3- 10 days incubation in the dark at 22°C. colonies were sdected for restreaking on PDA based on colony texture and color. This nep was repeated until pure cultures representing the diversity of colonial types found idon each plant root were obtained. Gram aain reaction and shape of bacterid cens were observed by Iight microscopy using a 1OOX oil immersion lens. LIVE/DEAD@ BacLightm Bacterial Viability Kit (Moleailar Probes L-7007) was used to assess the viability of cultures afler dorage using LSCM. Thin sections (for light microscopy) and gold sections (for transmission electron microscopy) of LR White resin-embedded material were examined for the presence of bacterial cells on the surface and within the cells of the Cypripednrni roots. 1. Test for the ability to Tir atmosphcric nitmgen Bacterial cultures isolated from the roots of Cypripdium spp. were screened for N2 fixation by inoculation to a nitrogen-free liquid medium. Prior to screening. bactenal cultures to be tested were subcultured to fiesh tryptic soy media to determine the purity of the culture. Due to the thick mucilage produceci by the cultures, it was not always possible to obtain single-species colonies, but there was usually a very arong dominance of one type of bacterium. Nitrogen-free liquid medium (10 rnL per tube) was inoculated with a stenlked loop fkom colonies on tryptic soy plates, taking care not to introduce agar into the tubes. Tubes were incubated for 26 hrs and assessed for growth (evidenced by a turûidity of the medium). Where turbidity of the medium was detmed, 500 pL of media fiom the fim tubes was used to inoculate a second set of tubes containing the same nitrogen-free medium. These tubes were incubated for 48 hrs and reassessed for turbidity. 2. Scmning for Plant Growth Pmmoting Rhïzobacterù (PGPR) Bacterial isolates from the mots of C'r@edilcm spp. were also bioassayeà for plant growth promoting abilities (PGPR) using aerilegrown seedlings of lettuce (Lacttcca sotBa var. longifoIia). For indation of plants, 50 rnL Basks of tryptic soy broth were inoculated with each of 16 bacterial cultures and incubated at room temperature on a rotational shaker. Arnong the 16 isolates were 15 isolates (al1 Gram negative bacilli) fiom the roots of Cypripedium species, and 1 known PGPR reference bacterium (isolate P W2R a rihycin resistant arain of Bacilhs polymyxu) provided by Dr. C. Chanway, University of British Columbia. Forty-eight hrs &er the inoculation of the liquid media, the bacteria were applied to the roots of aseptically geminated 3-daysld lettuce seeûlings. Lettuce was chosen for its rapid growth, small space requirements and ease of culture in the growth pouches. lnoculation was accomplished by soaking the roots in drops of bacterial suspension (1 mL per 25 seecilings) in sterile Petri dishes for six houn. Control seedlings were soaked in aerile tryptic soy broth. To observe the effects of the bacterial arains on seedling growth and survivaf, ten sterile growth pouches. each containing two randomly chosen seedlings soaked in the liquid inoculum were prepared for each bacterial arain tested. Pouches were maintained under continuous fluorescent light in a laminar flow hood at approximately 20 OC. Seedlings were piaced with the cotyledons near the top of the pouch. The pouches were initially watered with Vi strength Long Ashton's micronutrient solution (Hewitt 1966). Thereafler. sterile, deionized water was added as required to maintain the hydration of the wick et the mouth of the pouch. AU seedlings were harvested at 2 1 days der inoculation. Each seedling in each pouch was categorized into 1 of 3 'aatus' and 1 of 3 'ske' categories. Status categories were live (healthy seeûlings), moribund (seedlings dominated by decaying tissue although some üve tissue remained). and dead (those entirely brown with watery tissues). Sue categories for live seedlings reflected the development of the seedlings. 'Small' seedlings possessed only cotyledons or cotyledons and an emerging first le& 'Medium' plants were approximately I Sx the length of small plants and had two well-developed tme leaves. 'Large' plants were approximately 2x the length of the small plants. and bore at least three leaves. The size and status treatment was compared to the control and to the known PGPR isolate using SPSS version 10. Kniskal Wallis and Chi-square tests. AIgae Algae were isolated fiom the Cypripedizïm roots using both liquid and agar solidified ASM-3 media (Gerrath et d. 1995, Gerrath et al. 2000). For isolation on solid medium, roots were removed from wild plants, sealed individually in small plastic bags and retumed to the lab. Segments of approximately 2 cm in length were removed aseptically. shaken vigorously in stenle distilled water and drained in a stede Petri plate. Each cleaned root piece was then rolled on the surface of a ASM-3 agar plate. The plates were kept in a 26OC. illuminated incubator that provided 16 hr days. Mer 3 weeks. individuai colonies were removed to fiesh media. For isolation of algae on liquid medium, stenle. capped 50mL centrifuge tubes filled with 20 rnL of ASM-3 liquid medium were taken to the root collecting sites. Immediately upon rernoval from the substrate, the srnall amount of adherent material was removed fiom the roots by forceps, then 2 cm pieces were added individually to the media in the centrifuge tubes. Upon retum to the lab, the tubes were placed in the lighted incubator under the sarne conditions as the isolation plates. The tubes were subcultured 3 weeks afler collection by agitating the tubes and then removing approximately 5 mL by aerile pipette to a new tube of medium. For identification, algal ceils 6om the plates and the liquid medium were examined using light rnicroscopy. Individual colonies from the solid medium were mounted on a glas siide in water for Mewing. Liquid cultures, which contained a mixture of algae, were obsewed by gently agitatins the tubes. and then pipetting a small amount of media onto a glass slide. Images of each al@ species were captured using a microscope- mounted digital camera and Northen Eclipse imaging sofiware. Aigal species were identified by Dr. J. Gerrath (University of Guelph). Algae cell localization on the roots was attempted using LR White resin-embedded root samples. Root pieces in the blocks were examined for algal cells under oil without a coverslip using laser xanning confocal rnicroscopy (as for localization of fungi). Ciliates Ciliates were cultureci from the liquid medium algal isolation samples. Approxirnately 1 mL of the ASM-3 media into which the orchid root segments (3 weeks previously) had been placed was dispensed into 60 mm Petri plates or plastic storage containers. Three boiieâ barley caryopses and 10 mL of EviarP or Aberfbyle SpringsN minera1 water were addeû to each dish. After 3-5 days, this was repeated with the first culture acting as the inoculum. 3-5 days later. 1 rnL of the second generation cultures in which ciliates were seen with a stereoscope was added to 5 mL Bouins Fiive for aorage. Ciliates were not seen in the uninoculated control cultures. For identification, specimens were prepared using quantitative protargol staining (QPS) following the methods of Montagnes and L~M(1987). Images of ciliates prepared in this manner were captured using a Li@- microscope mounted video carnera ushg Nonhem Eclipse imaging software. Ciliates were identified fiom these images by Dr. D. Lynn and D. Acoaa. Roots embedded in resin blocks were acamined ushg LSCM for the presence of ciliates (see algae. above). Results Isolates of al1 five groups of organisms (fungi. bactena. algae. cyanobacteria and ciliates) were obtained fiom the mots of the wild Cypripedium plants. The isolates for each group are discussed in tum below. Fungi 1 06 isolates were obtained from Cypripedium roots. These are sumrnarlled in Table 3.1. Of these isolates 7 belonged to the form-genus Rhîocronia, a taxon containing several orchid mycomhizal fungi (Cunah et al. 1997a) (Figs. 3.1-3.8). An 8" potentiallv mycorrhual isolate, a hyaline to yellowish isolate of unknown identity bearing clamp connections, was svnilar in appearance to those seen in the pelotons of Cjywipdium mycorrhitas (see Chapter 2). Despite the heavy mycorrhizal colonization seen in the roots of C. regme collected at Purdon Natural Ar- ON, and C. acmle at Brereton Lake, MB (see Chapter 2). hngi were not isolated from these root samples. Al1 other collections yielded hngal isolates. Sterile, dematiaceous, septate fun& many of which were identifiecl as Phialocephaia fortinii were the moa cornmon isolates. At least one isoiate of P. foriinii was cultureci fiom each of the four C'ripedium species, and at five sites in two provinces. However, P. fortÎnii was isolated moa fiequently fiom the roots of C. acaule. The C. acaule roots dso yielded many isolates of a steriie ascomycete ref'ierred to here as "Variable White Taxon," &ter their resemblance to the descriptions of a yet unnarned group of fùngi derived from ericoid mycorrhizas (Hambleton and Currah 1997). The cornmon identity of these ericoid mycorrhizai fungi and one of the Cjpr&wiurn isolates has been confinned Pr. S. Hambleton, personal communication). A single isolate of this fungus was also obtained from C. parv~~oluumin Manitoba. Two isolates of "Variable White Taxon" were tested for ability to fonn ericoid mycorrhizas. Ahhough the fun@ grew directionally toward the roots. and heavily colonized the surface of the i accin~~cmroots. the formation of mils in the epidermai hairs could not be contïrmed. Due to the similarity of these isolates to those described and provisionally narneâ (Vralstad et al. 2000) as ectomycorrhizal fun@ (and a possible ectomycorrhizal rnernber of the Hymemxypphtrs ericae assemblage), these metwo "Variable White Taxon" isolates were also tested for the ability to form ectomycorrhizas in vitro. Dichotomous lateral roots were not formed by the Pinus strobus seedlings, and the isolat es did not appear to form a mantle or Hartig net on the root tips closest to the points of inoculation. The clamped fungal isolate encouraged the growth of dicotymous root tips on the Pinus strobus seedlings but despite hyphae seen on and between some root epidermal ceus. an organited Hartig net andior mantle was not fonned. It is unknown whether this indicates an incompatability between the pine and fungus, a lack of momycorrhizal symbiotic ability in this fùngi. or culture conditions that did not support the development of an ectomycorrhizal symbiosis. 1. Fungd interactions with a cyanobacterium and 2 bacterial isolatts The Epulorhizo isolate tested (Z3 12) showed no evidence of directional growth toward or utilization of the bacterial and aga1 cultutes. However, the bacterial isolate 3 16b-1 affécted the branching pattern and therefore the radial expansion of the fungus on the agar plates. On the control plates, and on those inoailated with isolate 3 18b-6, the fùngal isolate was highiy branched and formed monilioid cells close to the point of inoculation. At the end of the trial, fungal colonies on control plates had not extended more than half the distance between the point of inoculation and the edge of the plate. In contrast. hngi co-cultured with isolate 3 16b-I were much less branched, and so extended across the suface of the medium at a much faster rate. These fungal colonies had aiready reacheâ the edge of a 100 mm Petri plate by the end of the experiment. Bacterial colonies on plates containing fun@ tended to be less homogeneous in structure than those on axenic plates. A watery, clear liquid. not present on the axenic plates, surrounded the more opaque centen on the fungus-containing plates. Bacterial colonies appeared to expand at similar rates with and without fun@. &ctenà Seventy-six bacterial cultures were obtained from Cypripedium roots. moaly fiom root segments plated on PDA medium. The cultures were cream, paie yellow or pale ochre on PD& with the exception of one isolate that produced dark brown pigment. The cultures were sirnilar in their production of a thick viscous mucilage which did not disperse readily when added to liquid culture media. Almost all isolates containeci Gram-negative rods or short rods, usually with a few coccoid cells intersperd. The thick, cohesive mucilage of the colonies prevented the elirnination of these other bactena when streaked ont0 agar. When inoculated into Ntrogen-fiee liquid medium, severai isolates Uiitiaiiy grew well (Table 3.2). However, there was no growth of any of the isolates after repeated subculturing. Subsequent subculture of the original isolates also failed to grow on PD& tryptic soy agar, and nutrient agm, even though the isdates were live when tmed with BacLight LIVEIDEAD aain. It is, therefore, unclear whether the faiiure of the bacteria to proliferate in the second transfer on nitrogen-free medium was due to an unsatisfied requirement for nitrogen or for sume other compound or condition. Substrate utilization tests were not conducted for the identification of the bacteria because sufficient ceii concentrations could not be achieved using standard culture media. 1. Effkcts on lettuce secdlings There were significant differences in the growth and survival of the lemice seedlings among seeâlings treated with the bacterial isolates (Kmskal Wallis test, p <0.0 1 ). Some isolates appeared to affisurvivaî, while othen seemed to encourage growth of the seedlings (Figs 3.9-1 0). These tests would need to be repeated to CO& the trends seen in this study. The mean ranks (survival) for the seedlings inoculated with isolates 3 16b-1, 3 lob-2 3 18b-2, 320b-6 and 3 18b-6 were above that of the PGPR test isolate, which was similar in rank to the controls. Survival of Kedlings with isolate 324b-6 was ranked lowest. There was iittle difference between the PGPR, control 1,3I6b-l and 3 18b-6 mean ranks for seedling size. However, these 4 treatments produced the largest seedlings. The lowest mean rank (seedling sue) was for sdlings inoculated with isolate 324b-6. Algue Twenty-one genera of algae and cyanobacteria were identifieci hmthe rwts of Cjpripudium species (Table 3 -3). An additional 8 isolates, including diatoms, green algae and colonial and unicellular cyanobacteria were recorded but not identifieci due to the lack of important taxonomie fegtures in the cultures. The divenity of dgae and cyanobacteria isolateû from the rwts of C. regme was greater than that of C.acaule roots. While many different algae and cyanobacteria were isolated. most taxa were isolated frorn a low nurnber of plants. The mou comrnonly isolated algae were Chiorldeiia spp. (Fig. 3.1 1). Stichucoccus spp. (Figs. 3.12-3.14) and Xmthonema spp. (Fig. 3.15). Nostoc spp. (Fig. 3.16), Oscillatoria spp. (Fig. 3-17), Lynglyu spp. (Fig. 3.18) and S~~~~~~OC~ÇCIIS~S~IWC~OC~OC~S~~~spp. (Fig. 3.19) were the moa commonly isolated cyanobacteria. The latter two genera could not be differentiated due to a lack of some taxonomically important features in the cultures. Although rnos of the algae and cyanobacteria isolated were common soi1 or fresh water taxa, others, algae such as Diiabflium dopyreniae (Fig. 3.20) and Fottea stichacoccoides (Fig. 3 -21 ) are not commonly isulated (J. G. Gerrath, personal communication). Diiabifhm ~171hopyreniue is usually found as the algai component of a lichen association. Rotifers (Fig. 3-22), flagelates, ciliates and other small organims were ofken seen in the algae cultures when fim isolated fiom the roots. Of these, only the cilates were cultured and identifieci. Ciliates Species in the genus Colpoda were the only ciiiates isolatd fiom the algal samples (Table 3.4). While 6 types of Coipoda were obtaind from 8 plants of C. acuuie, Co@& spp. were isolated only once from the roots of 7 specimens of C. regme. Although Protargol stain is the common method for staining ciliates Colpodo species ofien sain pooriy with this and other methods @. Acosta personal communication). Consequently, many of the prepared samples contained celis ihat were difficult to identify. Six groups of Colpairr were recognized (Figs. 3.23 to 3.28): C.iflutu, and C.steinii, ciliates resembling C. ~~arflusand Cv. muupasi but requiring Lrther characters for positive identification, ciliates resembling C.stei~~ii. but slightfy outside the size range for this species, and an unidentified Colpub species. Discussion The methods used in this research resulted in the isolation and culture of a diversity of organisms fiom the roots of Cypripdim species. If a broader range of isolation media and methods had been used. an even greater diversity undoubtedly would have been obtained since al1 culture media and methods are selective to some degree. This investigation must therefore be considerd exploratory rather than definitive. None-the- less. it is apparent that the organisms identified in this study represent the many trophic levels present in the rhizosp here of C'ripedium species. Algae. cyanobacteria fun@. bacteha, ciliated protozoans and other organisms were isolateci from the surfaces and interiors of C'ripdium roots. Most of these organisms (at least at the genenc level) are comrnon to soi1 or fieshwater environments, but it is unknown if they are comrnon in the somewhat challenging environments in which the orchid roots were collected. For example, C. repmroots at the Cnef site were collected fkom the boundary between aikaline groundwater-saturateci humus and living, acidic Sphrgnîm moss. C. acmrle plants at the St. Williams site were rooted in a very dry duff cornposeci almoa entirely of pine needles ïhe recovery of 'ordiiary' rhizosphere organisms fkom such extreme habitats suggeas that the orchid root and rtiizosphere offer a moderated environment for root-associated organisrns. More research wili be necessary to detemine if the sarne species are found in sidar substrates that are not iduenced by the orchid roots. Many genera of tùngi were isolated fiom C'ripdium roots, however, moa, including the potentially mycorrhizai hngi, were isolated from a low number of plants. In contrast, Phiaimephalaforfi~~ii and other dematiaceous aenle fungi were isolated from many plants. as was the unnamed fiingus resembling the "Variable White Taxon" from ericaceous roots (Hambleton and Currah 1997). While Phialmephlafortinii and the sterile dematiaceous fun@ were isolated from 4 species of Cypripedium, "Variable White Taxon"-like hngi were isolated only fiom C. acaule (16 plants) and C parviflonm ( 1 plant). Phialocephla fortinii, dong wit h several ot her sterile dematiaceous fun@, is known to occur on the roots of many plant species (Jumponen and Trappe 1998). hcluding temperate orchids (Currah et al. 1988, Currah et al. 1997a Zelmer et al. 1996). Their high rate of isolation may be due to their ability to colonize the epided and possibly more interior cells of Cypripdium roots (see Chapter 2). thereby avoiding the surface nerilants used during the isolation process. The isolation of "Variable White Tauon*'-like organisms (VWT-like) is interesting in light of their original description as endophytes of ericaceous plants (Hambleton and Currah 1997). There is research linking similar cuhum to the provisionally-named "Piceirhizu bicoloratu" ectomycorrhizas formed on the roots of coniferous trees (Vralstad et al. 2000). Two isolates of W-like fùngi were tested for ericoid and ectomycorrhîzai abiiities. The isolates were strongly attracted to roots of the ericaceous plant, forming wefts upon the sudace and trachg the growth of the root. Hyphae were not seen within the haïr roots when cleared and stained. However, roots of ericaceous plants can be resistant to stainhg (R. L. Peterson, personal communication), and so the penetration of the stain into the root epided cells is in question. Ectomyconhuas were not fonned by the two VWT-like isolates. The frequency of isolation of this hngus fkom the roots of C. accnrlr warrants further investigation. Basidiomycetes bearing clamp connections at their septa are seen frequently in the cleareà and stained roots of Cypripedium species (see Chapter 2) and they have also been reported from the geminating seeds of Cypripedium species incubated in the natural habitats of the plants (Zelmer et al. 1996). A clamped isolate was obtained fiorn C. reghue roots, the fira from Cypripedium to date, but it is unknown if this isolate is the sarne as those fohng pelotons in the roots. Some clarnped isolates fiom the mycorrhizas~mycorrhizomesof non-photosynthetic orchids are ectomycorrhizal associates of trees (Taylor and Bnins 1997. Zelrner and Currab 1995). thus conducting the flow of photosynthate fiom the trees to the orchids. The isolate tested for ectornycorrhizal symbiosis with Phsstrobus did not fonn classic ectomycorrhizas in growth pouches, but did encourage dicotominng of the short roots. It was also able to colonize both the sufice and the intercellular spaces of the epidermis and cortex to a lirnited degree. It is possible that the conditions of this experiment were not appropnate for the development of an ectomyconhizal symbiosis. Ectomycorrhital symbiosis of clamped orchid mycorrhizal fûngi is therefore still in need of exploration. Although Cpripedlum roots are often colonized by mycorrhizal fùngi (see Chapter 2), the number of isolates obtaineâ from the many isolation attempts was very low. ûthers have also noted a paucity of isolates hm the roots of Cypripdhiunr species. Although this could be due to non-viability of the fiingi in the rwts, or the patchiness of the fiingai colonïzation, it probabiy is also due to the nutritional needs of the fungi. Isolates of these tiingi initially grew weli, but dersubculturing they declined in vigor and eventually died out even on Potato Dextrose Agar. Orchid mycorrhiral fiingi, particularly Epdorhiza spp.. can be heterotrophic for vitamins and other substances (Hadley and Ong 1978. Hijner and Arditti 1973). Investigations into the nutritional requirements and the usual carbon sources of Cjptipedium-asmciated myconhùal tùngi could lead to more successfùl isolation media and in vitro symbioses. Root-associated bacteria, while more easily isolated than mycorrhizal fungi. followed the wne pattern of declining vigor when subcultured. The overall similarity of the isolates suggests that either the rhizosphere of Cypripedium is e~chedwith a panicular type of bacterium, or that strong selection was imposed on the bacteriai commwiities of the roots by the medium used. Several of the bacterial isolates influenced the growth of lettuce seedlings. Isolates 3 1 6b- I and 3 18b-6, when inoculated ont0 the seedling roots, promoted both greater su~valand increaseû size of the seediings. Inoculation with isolate 324b-6 resulted in low suMval of the seedlings. However, this experiment requires repetition to clariQ the effects of these isolates. Representatives of at least three divisions of aigae were isolated fiom the roots of both C. regme and C. acmle. However, more isolates were obtained from the roots of C. reginw. This is a reasonable result when the habitats of the plants are considered. C. acaule roots were collected fiom deep, dry pine needle iitter ovedying a sandy mil. In contrast, the roots of C. regmue plants were collected fiom a continuously mois fen area. The constant moisture may permit a greater richness and abundance of algal types than that of the periodically moi* pine needle litter. Cyanobacteria were represented in the C>pripdium isolations by species of NOSIOC, Osciliatoriu, Ly~gbyaand several other genera. Heterocyst-producing cyanobacteria such as NOSIOCspp., are capable of fixing atmosphenc nitrogen in the presence of oxyçen and may contribute a fixed nitrogen source to plant roots. ûther cyanobacteria may fix nitrogen under reduced p02. Puphiopedihm and Phragmipedium. genera closely related to Cypripedit~m,also have cyanobacteria associated with their roots (See Chapter 3). However. these lithophytic, epiphytic or terrestrial orchid roots possess an open, sponge- like multiple epidermis (velamen) in which the algae live. A velamen surrounding the root may moderate rapid drying of the algae on the root surface of plants in well drained areas. and encourage root colonization by nitrogen-fixing cyanobacteria. The contribution of cyanobacteria to the nitrogen budgets of Cypripedium. Pqhiopediitm and Phragm>pediumspecies is not known. Ciliated protozoans. or ciliates, are mostly camivorous, feeding on bacteria and other microorganisms (Anderson 1988, Nisbet 1984). They are ohmore abundant in the rhizosphere than in the bulk soi1 due to the e~chmentof bacteria in the rhizosphere. The only ciliate genus isolated fiom the roots of C'ripdizm species was Colporki, a comrnon genus from soil. CO/'s@es were more frequently isolated from the roots of C. acaule than fiom those of C. regitiw. Col* have a resistant resting aate that may be desiccated and rehydrated without hm.In vitro, they often rapidly emerge £tom cyas (Nisbet 1984) in the presence of moimire and bacterial prey, feed and multiply for several days and then re-encyst. This cycle of activity is well suited to the sporadic moisture of the piFe litter duE In addition, since the litter was dry when the C. acuzde roots were coiieaed, Colpu& species were probably encysted on and around the roots. aiding their isolation. In the moist root environment expenenced by C. reginae plants, the ciliates were less likely to be encyaed on the roots. This may account for the differences in frequency of Coljmd~species isolations between the two orchid species. Root -associatecl CoiPUJa species may also be influenced by m yco rrhizal format ion. There is evidence that Coiph isolations from amund ectomyconhizas fonned in vino with a nurnber of coniferous species was more iduenced by the species of ectomycorrhizai fungus colonizing the mots than by the host plant species (lngham and Massicotte 1994). The isolation of only Colpoda species probably does not reflect the richness of ciliate genera on Cypripediurn roots. A geater diversity of ciliates was recovered from ectomycorrhizal conifen grown in pots in a greenhouse study (Ingham and Massicotte 1994) than were isolated from the wild collecteci roots in this experiment. Since the sources of inoculurn were quite restricted in the greenhouse study compared to the environment of the C'pedlumroots it seems probable that the culture and assessrnent met hods used here mua have selected against many species of ciliated protozoans. Many ciliates are delicate and short-lived in vitro. and often appear in a succession. As well. soap residues even on well-rinsed glassware can inhibit the growth of ciliate species (D. Acoaa, personal communication). In order to obtain the maximum richness of ciliates, daily sampling for 1-4 weeks after the roots were coilected would have been necessary (D. Acoaa, personal communication). Since the root cultures were sampled and assesseci only once, and the organisms cultured using wetl-rinsed but ongïnally soap-washed vessels, it is probable that moa species of ciliates were lost before the cultures were sampled. Of the organisms associated with Cypripedimr rwts. most are in contact with the root surface. Fungi and perhaps some bactena were isolated fiom within the roots, but aside fiom the mycorrhizai fbngi, they may only be present in the epidermis (see Chapter 2). This means that the root surface is an important interface between the orchid and the root-associated organisms. and increases the probability of interactions behveen the organisms. For example, in vitro, bacterial isolate 3 16b-I influenced the branching pattem and rate of radial expansion of an endophytic fbngus. If this occurs in vivo as well. this bacterium could alter the foraging pattern of the fungus, and therefore also the orchid with which it is mycorrhizal. Attempts to fonn growth-enhancing mycorrM symbioses with Cypripdium seedlings in vitro have been unsuccesshil. It is possible that interactions between soi1 organisms are necessary to provide the conditions for symbiosis in C'pedium.A suite of interacting organisms, rather than a single fingal isolate with a lirnited range of carbon sources. may be required for succesfil colonization and gro wt h of Cypripedium species. Conclusions The rwts of Cypripedium species are associated with a diversity of organisrns representing several trophic levels and ecological fùnctions (e-g. nitrogen fixation). Fungal isolates included an abundance of darkly pigrnemecl sterile isolates (Phidocephalafortinii and others), "Variable White Taxonn isolates (Hambleton and Cunah 1997) and potentiaily mycorrhizal species, including Eplorhizu spp. Bacteria, almost exclusively Gram-negative rod-shaped ceus forming rnucoid colonies were also isolated. It is uncenain whether any of these isolates were diazotrophs. Bacterial isolates fiom C'n'pediun, roots af'fécted the growth and survival of lettuce seedlings in vitro. Survival of the lenuce seedlings was higher when inoculated with each of five of the C'pripdiutn root-associated bacteria than for the known PGPR test isolate. Lettuce seedlings inoculated with one of two C'priprdium root isolates (3 16b-1, 3 18b-6), or the PGPR isolate had the highest mean ranks for seedling site, along with one of the uninoculated control treatments. URiceIlular and filamentous algae and cyanobacteria were among the many organisms isolated from Cypriipdim roots. The most cornmon genera were Chloridella, Stichococct~s.Xa~ithocwma, Noszuc. Osctllatoria, Lyngbyu and Sjwechococeus/ Synechocystis. Isolated in liquid culture along with the algae and cyanobacteria, ciliated protozoans in the genus Colpuch were identified. Most organisms isolated hmthe roots were cornmon soi1 or water organisms - there was little evidence of a unique community of organisms on the roots. Many of the Fungi and bacteria isolateci from the roots were short-lived in culture, suggesting un-met requirements for nutnents or conditions provided by the orchid rhizosphere. Table 3.1 Fungi isolatcd from the roab of Cypnpsdi~rinspics. No, of No. of Fungal taxon plants isolates Rost orchid spccics Location Acremonium spp. 3 Crief, ON Aspurgillrrs spp. c. acarrfe St. Williams, ON A weobasid~rmsp. C. prv~jrorum Crief, ON CIQdosporium s p. C. parvrflortlm Bnice Peninsula, ON Cladosporit~msp. C. regrrcw Cnef, ON Corticioid (c.f. Sistotrema?) C. acmle St. Williams, ON Hirmicola sp. C. regrime Cnef, ON I.eptOdorttidit~morchi Jico fa C. parv~jlorurn Near Crief, ON Leptdontidhtm orchidicoia C. regnae Crief, ON Mo~~ocilliumsp. C. acmiu St. Williams, ON Oididerrdrot~(maius and other) c. UCQ~I~~? St. Williams, ON Phia/ocepMaforti~tii C. acmle St. Williams, ON Phiaiocephoa /orii c. arivtimrm Near Crief, ON Phialocephala fortinii C. pan,r/iorum Anola, MB Phialmephla fortinii C. parvtrfomm Bruce Peninsula, ON Phialocephara forti~tii C. pant~flonm Cnef, ON PhiaIocephaIa fortinii C. pan,1frowirm Near Crief, ON Phialocepholrcr fortir~ii C. regirme Anola, MB PhialocepMa fortinii C. regiMe Crief, ON Phiafmephalafortittii? C. acaule St. Williams, ON PMixepha/afortinii? C arietimm Near Cnef, ON Phialocephaia fortinii? C. pmvjlorum Near Crief, ON Sterile dematiaceous isolates C. arietimm Near Crief, ON Sterile dematiaceous isolates C. paw~floturn Bruce Peninsula, ON Sterile dematiaceous isolates C. parv~flotum Near Crief, ON Sterile dematiaceous isolates C. regiiiae Crief, ON Sterile hyaline isolates C. acatde St . Williams, ON Sterile hyaline isdates C. parv~jrorurn Bruce Peninsula, ON Tisiasp..fMollsia sp. C. regitwe Anola MB Trickptirm abiehrnurn C. prv~jlornm Anola, MB Trichoderma sp CIacaule St. Williams, ON Trichoderma sp. C. regimr 20k W. of Elma, MB Uhown hyphomycete I C. regnue CRef, ON Unknown hyphomycete 2 CI acm1I4 St . Williams, ON "Variable white taxon" C. acauie St. Williams, ON "Variable white taxon" C. parv~frorurn Anola, MB No. of No. of Fungus plants isola tes Host orchid specics Location Ceratorhim 9. 1 1 C. reginae Crief: ON cf. Ceratorhizu sp. 1 1 C. acatrle St. Williams Eplorhiza cf repens 1 1 C. reghue Crief, ON Qmlorhiza sp. 1 2 C. pavij]rum Bruce Peninsula, ON Epulorhi~uq. 1 1 C. reme Crief: ON Ejaiorhka sp. I 1 C. regiiiae Crief, ON Sterile clamped basidiornycete 1 I C. regiiiae Crief, ON Table 3.2 Bacterial isolata hmCypripdum spp. tatdfor the rbility to grow on nitmgen-free media. (Al1 isoiates Gnm negative bacüli). lsolate Host orchid speciar Subcuîtum 1 Subculhim 2 C. porvpon~m neg CO pawijlomm CT. pan~i/ontrn c: pa~lrf0~4rn C. pUn'l/I0rl4rn C: punptponm C panvflomm C. ponvjontm C. rcgime C. regi~iae C. regi~~ae C. repiae C. reghim C. reprrae C. tegniae C. regime C. rrg»uw C. regime C. acmle C. acuule C. acade C. acaule C. acuule C. acmie C. acmle C. acaule C. acmle CI acaule Ccacaule CI accnrle Ccacde C. acal~lt! C. acuule CI acaule C. acaule C. acaule C. acaule C. acaule C. acaule C acuule C. acaule C. acaule C. acatrle CI acude CI acatde CIacarrle C. acaule C. acatdu C. acmii! C. acaule C. acmde C. acaule C. acuiiie C. acade C. acaule C acaule Ccacairie C. acatde C. acaule C. acatîle C. acaule C. acaule C. acaule C. acauk C. acaule C. acaule C. acmfe C. acaule CI acmle C. acaule C. acaule CI acaule C. acaule C. acauie C. acaule C. acaule C. acaule ** nrongly positive (pos) positive after 3 weeks of incubation Table 3=3Alye isolatcd fmm the mots of Cyp~pdumspecia- From orcbid: C r. Ca Alga Division CaIowis sp. Hetero kontop hyta C'hlorella-like Chlorophyta Chloridella spp. Chlorophyta C'hloridelfa neglecta Chlorophyta Chiortxfoster sp. Heterokontophyta Choricystis mimr Corcomyxa sp. C hlorophyta Dilabtjilum wthopyreniae El~notiasp. Heterokontophyta Eîliproidion sp. C hlorophyta Ellipsoidiott stich0~occoides Chlorophyta Fottea stich0~occoides Kle bsonnidium pseudostichxocc11s Chlorophyta LY@?YQSPP- Cyanophyta Lyngbya perelegm~ Cyanophyta Monoraphidium terrestre Heterokontophyta Nmimla spp. Heterokontophyta Ni~tschiaspp. Heterokontophyta Nostoc hurn~figa Cyanophyta Nosroc @aericum Cyanophyta Nostoc spp. Cyanophyta Oscillaoriu spp. Cyanophyta Pserrhhena catenata Cyanophyta Stichacoccus baci IIms Chlorophyta Stic~occuschlorelloides Chlorophyta S~ichtmcctîssp p. Chlorophyta Spechococcus Spechocystis Cyano phyt a Unknown cyanobacteria - colonial Cyanophyta Unknown cyanobactena - unicellular C~ano~h~ta Unknown diatoms Heterokontophyta Unknown green algae Chlorophyta Xmthonema montmnrnr Heterokontophyta Xat~lhonemuspp. Hetero kontophyta C. r. = Cjp-ipdium regime; C. a. = C. acaule (? Indicates taxonomie uncenainty - not enough chamers to ensure positive ID) Table 3.4 Ciliates isolated Crom the mots of Cypnpedium specia. Ciliate specias C. regin& C. mule Calpoda cucullus- l ike 1 Colpods inflata Colpoda maupasklike Colpoda spp. Colpoda Stein1 Colpoda steinklike 'Nurnber of plants from wtiicti the indicated citiate was isolated (of 7 plants of C. reprnae and 8 plants of C. acaule) Figura 3.1 to 3.8. Fuagd isolates from the mots of Cyprïpedium spics. Fig. 3.1. Mate 227.1 Eprrlurhka sp. colonial morphology on potato dextrose agar at 3 months. Isolated from C'ypripedium parv~florum.0.5~. Fig. 3.2. Isolate 227.1 Epdorhka sp. hyphae and monilioid cells, corn med agar, 4 months. 280x. Fig. 3.3. Isolate 209.2 &dorhi=ci sp. colo~almorphology on potato dextrose agar at 3 months. Isolated from Cjpiipdium reggrme.0.5~. Fig. 3.4. Mate 209.2 Epzdorhiza sp. hyphae and monilioid cells, corn meal agar, 4 months. 200x. Fig. 3 S. Isolate 220- 1 .1 Eplorhza sp. colonial morphology on potato dextrose agar at 3 months. lsolated from C~ripdiliunreginue. 0.5~. Fig. 3.6. Isolate 220- 1.1 Eplorhirrr sp. hyphae and monilioid cells, corn meal agar. 4 months. 3ûûx. Fig. 3.7. Isolate 220- 1.1 Epdorhiza sp. hyphae and monilioid cells, potato dextrose agar. 6 months. Septa (one indicated by arrow) are oflen formed across monilioid cells in otder cultures. 300x. Fig. 3.8. Isolate 3 12 Epulorhi=a repens monilioid cells. corn meal agar. 2 months. Isolated from C'dimregime. 290x. Fig. 3.9. Status of kttuce seedlings inoculited with bacterial isoiates. - T 1-3 Jlbl llbl IIOI DrDi 1-9 ItW PD> P(b? -1 i2AW -1 1 Fig. 3.10. Size categories of reàüngs inocuhted with bacterial isolates. Figures 3.1 1 to 3.22. Algie, ginobactcria and a rotifer Wtedfrom the rootr of Cyp?ip&um spp. Fig. 3.1 1. Chlordefia negiecta isolated fiom a root of Cypripedium regme. 230x. Fig . 3.1 2. Ellipsokihtt stichucoccoides isolated from C. acade 23 0x. Fig . 3.1 3. Stichoc0c:cus bacillaris iisoated fiom C. acaule. 23 0x. Fig. 3.14. Stichococcus baciliaris isolated from C. regme. 200x. Fiç. 3.15. ~at~lhonemanro?ItcImrn isolated from C. reginae. 200~. Fig. 3.16. Nostoc sp. (arrow) isolated fiom C. acaule. 200x. Fig. 3.17. Oscillatoria sp. isolated from C. acoule. 200x. Fig. 3.18. Lyt~gbyasp. isolated from C. regitme. 200x. Fig . 3.19. Sylechucoccus Spechocystis sp. isolated 6om C. reginue . t Oh. Fig . 3.20. Dilabrfllum mthropyreniae isolated from C. regmue. 200x. Fig . 3.21 . Fotfeasticha.occoi&s iisoated from C. regim. 200x. Fig. 3.22. Rotifer isolated from a root of C. regmue. 40x. Figures 3.23 io 3.28. CoIpodp spp. isolited fma the mots of Cypnptdi~mspp. Fig. 3 -23.Colpuda (ct~tcrdl~~s-like)isolated from a root of Cypripedium acade. 86~. Fig. 3 .Dl.Colpodri steinii isolated from Cypripdium acurde. 74x. Fig. 3 -25. Cole(mcnrpmsi-like) isdated from Cpripedium acaule. 74x. Fig. 3.26. Co@ada irflata isolated fiom Cyprirpdi~mamde. 7Sx. Fig . 3 -27. CoIpodo steinii isolated nom Cypripdium acauie.7Ox. Fig. 3.28. Colporki sieinii isolated fiom Cjpripediium regtme. 75x. Cbapter 4: Symbiosis between CyprMedhm reginae pmtocorms and fungi: substrate and isolate dependence Introduction There is much interest in the propagation of Ladyslipper orchids (Cypipediirm spp.) for commercial, conservation and restoration purposes (Steele 1996). Two major approaches have been taken to the in vitro growth of orchids frorn seed. The first. asymbiotic germinatio~involves the sowing of immature or mature, surface-stenlized seeds on a stenlized agar or liquid medium containing mineral salts, sugars and usually. undefined substances such as coconut liquid endosperm, or banana pure. The seedlings are raised in a stenle environment, and then must be introduced to non-sterile conditions as they mature. This is a difficult step in the culture of orchid seedlings (Riley 1983), and ofien results in high monality. The second approach, symbiotic germination, involves the inoculation of seeds with particular fungal isolates on a variety of complex carbohydrate-enriched media (Peterson ri al. 1998). Under favorable conditions, colonization of the seediprotocorms by the fungus is achieved, and the hngus suppons the growth of the orchids thrwgh a mycorrhiza-like symbiosis. Although asymbiotic germination techniques have becn developed for some species of C'ripedium, many do not germinate or grow in sufficient numbers under these conditions to nippon large-scale production of plants. For this reason, there has been interest in the more naturd process of fiingai syrnbiosis to support the germination and growth of C'dtumseediings. Furthemore, there is evidence from studies of other tenestriai orchids that symbiotic seedlings can exhibit rnuch higher growth rates in flasks than asymbiotically grown seedlings (Jorgensen 1994). More imponantly, symbioticaily- grown seedlings appear to have a greater chance of aiMval when planted into favorable habitats in the wild (Markovina and McGee 2000). The hngi used for symbiotic germination trials are oAen isolated fiom the mycorrhizai roots of mature orchids. Few fungi have been isolated fiom Cjpripediurn spp. to date (but see (Clements et al. 1985, Zelmer et al. 1996). Although there have been reports of fungal isolates stimulating the germination of Cypripdium seeds (Light 1993, Vujanovic et al. 2000a). these isolates did not colonize or form the characteristic orchid mycorrhizal structures (pelotons) within the geminating seeds. As yet, peloton-forming, fungal symbioses that stimulate growth of the seedlings have not been achieved in vitro for C'rpedium species (Clements et al. 1985, Jorgensen 1994. Muir 1989. Srnreciu and Currah 1989). In a previous expenment (see Chapter 3). several potentially myconhizal fungal isolates were obtained corn the roots of C'ripedium species native to Ontario and Manitoba, Canada. This presented an opponunity to examine the interactions between the fungi and C. reHtmr protocorms in vitro as intluenced by changes in media, and to compare the responses of the protocorrns to three fungal isolates. To aid the separation of germination factors fiom colonization factors, asymbiotically genninated, growing protoconns were used for al1 experiments. This is important because the seeds of rnany C'ripedium species require treatments such as lenghy bleaching to break the dormancy of mature seeds prior to germination (Steele, 1996). There were three objectives of this research: 1) to detemine the abiiity of aerile- raiseci Cypriipediur re-e protocoms to form pelotons in vitro when inoculatad with a single fungus isolated from a mature Cypripedium plant, 2) to determine the eKeet of undefined additives and levels of supplementation on the formation of pelotons with this sarne ttngus, and 3) to determine if the response of C'riipedum protocom to inoculation and substrate composition is sirnilar for each of three 3 potentially mycorrhizal isolates from the roots of mature C'ripedium plants. The first two objectives helpeâ to assess the ability ofa fungai isolate to colonize protocom afier asymbiotic germination, and the effect of subarate additives on the symbiosis if forrned. The final objective explores the isolate-specificity of the symbiosis in response to substrate composition. To achieve these objectives, two expenments were conducted. The fira examines peloton formation by C. regme protocoms with a single arain of fungus on media supplemented with complex additives ai two different levels. The second experirnent explores the relationship betwem C. regrme protocorms and several fun@ (dl isolated From the roots of mature Cypripediun, plants) as affected by medium composition. The experîments are descnied and discussed sequentialiy below. Methods: Expriment 1 C. regiMe protocorms (4 months from sowing) were combied with fungal isolate 2209.2, an Epdorhiza sp. strain originaüy isolated from the root ofa mature C. regime piant growing in a fen site in Wehgton County, Ontario, Canada. The plant fiom which the seeds were harvested was also part of this population. The protocorms were combined with this hingal isolate on a base agu medium that was supplemented with five different carbon sources at two levels. The base medium consisted of Harvais medium (Harvais 1982) without growth regulaton and dextrose. To this base medium. one of five carbon sources at one of two levels was added, resulting in 10 treatments. The additives to the base medium were dextrose (1 0 gL;20 g/L), cellulose ( 1OgL; 20 gL).starch (1 O&; 20 gk),inositol(10 a;20 g/L)and oat powder (3@; 10 gL).The dextrose at 20gR. corresponds to the level recommemied in the original Hamis medium for the axenic growth of C. regme. and 3gR. oat powder is the level used in several ultrastructural studies using symbiotic Spirunthes sinemis protocorms (Uetake et al. 1997, Uetake and Ishizaku 1995, Uetake and Peterson 1997). For each substrate and concentration, 5 Petri plates (60mm) were prepared, each containing 10 mL of medium. One day der autoclavùig each plate was inoculateci with a small plug of agar taken fiom the colony edge of a 2 month old 0.3% oat powder agar plate of isolate 2209.2 (Fig. 4. 1). These were incubated for 1 week in the dark at room temperature, at which tirne al1 plates showed fùngal growth. Five protocorms of C'pdiumrepe that had ben raised for 4 months on Harvais medium were added to each plate. Most protocorms had developed root primordia Qig. 4. 2). The dual cultures were incubated at room temperature in the dark. Mer 27 days. two protocorms were selected at random from the plates for the assessrnent of hgal colonization. They were hedin 7.5% glutaraldhyde in O. 1M Sorensen's bu* for 24 lus, then nnsed in three changes of deionized water and irnmersed in 5% KOH in shell vials. The KOH was slowiy heated to boiling in a waterbath. Once the protocorms changed fiom opaque to ttanslucent the KOH was removed by several inses in deionized water, with a finai rime of acidified water (pH 3-0)to ensure high contrast staining. Protocorms were stained by placing each in a drop of 0.5% trypan blue on a glass slide for approxirnately 5 min then perrnanently mounted on a giass slide in a drop of polyvinyl alcohol (Salmon, 1954). A coverslip was added and pressure applied to squash the protocorm so that the interna1 cells could be evaluated. Colonization was considered to have occurred if hyphae had penevated into the protowrm and fomed the characteristic pelotons of orchid myconhizas in at least one cell. Peloton formation, presence of monilioid cells, possible parasitism of protocorms by the fungus (assessed by disorganized proliferation of the hyphae throughout the protocom) and frequency of colonization of the protocorms on the various substrates were noted. Results: Experiment 1 The numôer of protocorms fonning pelotons with isolate 2209.2 on each of the media is presented graphically in Fig. 4.7. Supplememation of the medium with dextrose at both levels resulted in little to no colonîzation of the protocom by the [email protected] on base medium with inositol also did not fom pelotons. However, both the hgiand the seedlings grew on the dextrose and inositol-supplemented media. Oat powder in the medium resuited in variable colonization at both the 3 gLand 10 g/L levels. Monilioid cells were present in the pelotons of some protoconns, but only thin, weak-appauing hyphae were present in others. When auch was added to the medium at either level, peloton fonnation in the protocorms was common (Fig. 4.3). There was a restriction of pelotons fiom the shoot and root apical meristems and also from the vasculature. Pelotons consisted of both narrow cyiindncal hyphae and swolien, monilioid cells. Cellulose-arnended media at both levels produced abundant peloton formation of al1 protocoms. Meristematic and vascuiar areas were free of hyphae in most, but not dl, protocorms. Monilioid celis were cornmon in the pelotons of protocorms grown on cellulose media. Diffennces in peloton formation between additives appeared to be greater than between different levels of the same carbon source. There were no obvious growth or developrnental differences in protocorms among the treatments. Discussion: Expennent 1 Many of the protocoms in this experiment became colonized by isolate 2209.2 and formed the characteristic structures (pelotons) associated with orchid mycorrhizal fungi. This experiment establishes that previously-genninated, asymbiotically grown Cypripedium reghue protocorms can enter into symbiosis with a hngus isolated from a mature Cjpripedium plant. This method may be usehl for separating 'germination' fiom 'establishment of symbiosis' requirements for the symbiotic culture of C'ipediccm seedlings. Colonization and peloton fonnation was not achieved on al1 media, however. uncolonized protoconns remained dive. This could be interpreted as the exclusion of the ftngï by the orchid protocomis when exogenous sources of nutrients are adequate. but other interpretatiom are possible. It could be that the duration of the expenment was not long enough to exhaust the remsof the seedlings. Additionaily, chemical signalling between symbionts is known to be important in other symbiotic systerns, such as arbusailar mycorrhizal fun@ and their hoas, and legumes and Rhirohia (Peterson and Guinel2000). If such signalling also occun between orchids and their mycorrhUal fun@, a disruption of the signals by medium components also rnight rewlt in a lack of colonization. Al1 seedlings remained dive for the duration of the experiment, and ttngal colonies grew on al1 media, therefore the lack of interaction in this case is probably not related to the viability of the organisms. There is evidence that addition of sugars to media used in symbiotic trials can result in parasitism of orchid protocorms by the fungal partner (Beardmore and Pegg 198 1 ). In this study, parasitism did not occur at either of the two levels of dextrose added. Parasitism also did not occur when the carbon source was inositol. Although both symbionts remained healthy and grew on these media, there appeared to be linie to no interaction between them. At the lower level of dextrose supplementation ( 10 gL). few pelotons were formed in a few protoconns. Colonization did not occur at the higher level. It would be interming to determine ifthere exias a threshold level below which protocorms can be colonized on dextrose-supplemented media. Oat powder, aarch and cellulose are carbon sources frequently used in media for symbiotic germination of orchids (Rasmussen 1995). These dnsources also proved usehi for obtaining peloton formation in previously-genninated C. reginae protoconns. Starch and cellulose-containhg media were associated with the heaviest colonization and most consistent production of pelotons in the protocorms. Colonization was more variable when oat powder was added to the media Monilioid cells have been observed in the pelotons of wild-collezted C'ripediwn roots, but they are relatively uncornmon. Monilioid cells accumulate reserves (mostly glycogen) in the fûngi of the Rhizuctonia form-genus, therefore their presence in pelotons could be an indication of resource accumulation within the root or protoconn cell. The meaning of this accumulation is not clear. Although much of the transfer of nutrients probably is accomplished through the breakdown of the hyphal cells, there is some evidence that transfer may also occur through intact hyphal coils (Hadley and W~lliarnson 1971). Hyphal coils are separated from protoconn ceU cytoplasm by a perifùngal membrane (Peterson et al. 1996, Uetake and Ishizaku 1995). Could the ability of this funpus to produce large numbers of monilioid cells in the protocoms in this system indicate that transfer of sugm through live hyphae is ùnpaired? Another interpretation could be that the rate of fungal resource accumulation is greater than the utilization of carbon by the protocorms. Since monilioid cells often are formed by isolate 2209.2 in vin0 as cultures mature, monilioid cells in pelotons may also indicate a considerable tirne lag between colonization and peloton breakdown. This is supponed by the rare observations of peloton breakdown during the 26 day experiment. It is aiso possible that degradation and digestion of pelotons is not occurring under the circ~ancesof this experimem. This perhaps could explain the lack of large growth responses in colonized protocorms. However, the lack of obvious developmentai and growth ciifferences between treatments aiso means that the protocorms on media without sugar additives kept pace with those on aigar-supplemented media. While this could be the result of carbon trander through tiving fungai hyphae in pelotons, it is more probably due to the degradation of the more complex Cafbohydrates to simple sugars that were reieased into the media. Does this mean that there is no benefit to the protocorms to be colonized by fùngi? Under the conditions of this experiment, perhaps this is me, but simple sugars are not common in the natural environment. Even without direct transfer of nutrients f?om fungus to protoconn, the possession of organisms that can reluise useable sugars from complex substrates is very much like possessing the enzymes to do x, and could be very advantageous to a wiid plant. The lack of a growth response for myconhkal seedlings could also be due to the shon duration of the expenment. In a lenghier experiment, a greater difference between mycorrtiizal and non-mycorrhizd treatments may have been observed. However, mycorrhllal associations may also incur 'costs' to the protocoms in terrns of defense mechanisms. phytoalexin production and/or other factors. and may balance the carbon gain tiom the breakdown of fungal pelotons. Finaily, the naturai carbon sources of the fun& and their mode of nutrition (mutualistic with non-orchid plants, parasitic, saprophytic) is at present noi known. If these hngi typicaily enter into symbioses with other plants, either as mutualias. cornmensals or parasitedpathogens their unial nutritional sources rnay be lacking under these experimental conditions. fhey may therefore not be able to provide specific compounds necessary for the growth of the seedlings that may be provided by other symbioses. EpIorhiza and Ceratorhiza spp. fonn symptomless colonizations of the roots of non-orchid plants (Zelmer et ai. 1996). As well, a Rhizuc~oniaisolate capable of mycorrhUal symbiosis with orchid seedlings fomd a close (sometimes internal) association with the hyphae of arbuscular rnycomtiitar Lngi (Williams 1985). Large differences were not seen between the two supplementation levels of the complex carbohydrates added to the base medium in this experiment. This result could be explained by the fùngal isolate's ability to utilize these substrates. The rate at which these substrates can be attacked by fungal enzymes is probably constant regardless of their concentrations in the media. The differences might bmme apparent only as the carbon sources in the media approach depletion. Conclusions: Experiment 1 The ability of fiinsa1 isolate 2209.2 to colonize and fonn pelotons in asymbiotically gemiinated protocoms of C. regimie is substrate-dependent. In combination with this fungal isolate, starch and cellulose-amended media produced greatest numben of pelotons and most consistent colonizations. The effis of the media were largely independent of the concentrations of the carbon source used. Media supplemented with dextrose and inositol did not prornote parasitism of the protocoms, but also supponed littie to no colonization. Experiment 2 Experiment 2 was undertaken to determine if three Epulorhircr isolates from wild C'ripdium species would respond sdarly in their ability to colonize asymbiotically genninated C. regme protocorms on a variety of substrates. C'ditmreginae seedlings were combined wit h each of three E@Iorhiza isolates and ten different substrate cornbinations. Methods: Expenment 2 As in the previous experiment, carbohydrates and complex additives were used to amend a Harvais base medium (Harvais 1982) typically used for the growth of C. regme seediings iri vitro. However, in this case the potato extract, growth regulators, dextrose and casamino acid and yeast extract (CASYE)were excluded corn the base medium. 500 mg/L ammonium nitrate was used as a nitrogen source. 10 different media were prepared by adding log& of each of 5 complex carbohydrate sources. with or without CASYE (0.05 g/L yeast extract and 0.25 gR. casamino acids): starch, wheat bran, whole dark rye flour, chopped dned local Sphagm~m moss and Buckwheat flour. Al1 media were adjusted to pH 5.5 afler autoclaving. 10 mL aliquots of each medium were dispenseci into stenle 20 mL giass shell vials. 4 replicate viais for each abstrate and fungus combination were prepared. The fungi used in this experiment were three Eplorhira isolates, al1 isolated fkom the mots of wild Cypripedium species. They were 2209.2(as in Experiment l), 2227.1 from Cpr@ediurn cuIceohs Emrnet Lake Alvar, Bruce Peninniia, ON and 2220-1.1 fiom C'pediumreginue fiom Wellington County site. ON The C'diumprotocoms were of the sarne provenance as those in Experiment 1, however, they were approxhately 5 months old. Most protocoms had developed root primordia, but these had not yet elongated. Planting of the protoconns was similar to Experiment 1, except that 20 dram glass vials with screw-cap lids were used instead of Petri plates as the culture vessels. Five protocoms were removed at random fiom the germination plates and placed in each of 3 replicate vials per mediumlfun gus combination, for a total of 90 vials. The method of inoculating the vials with the fungal isolates was sirnilar to the previous experiment. Each fungus was combined with the protocoms on 10 different media. The inoculated biais were incubated as for experiment 1. Assessments At 1 1 days &er planting. 1 randornly xlected protoconn nom each via1 was aseptically removed to observe early colonization. The sarne fixation, clearing?aaining, mounting and assessrnent protocols as for the previous experiment were employed. All other protocoms (4 per via]) were harvested, fixed. cleared and aained at 80 days post-planting. Some of the srnail protocorms were lost during the clearing process. the ceUs disassociating to the degree that the protoconn could not be nnsed and stained. Results: Experiment 2 Il days Al1 thefungal isolates were able to colonize the orchid protocoms and fom pelotons. however, none of them were able to colonize the protocoms on al1 the substrates. The three fimgal isolaies differed in their ability to form pelotons on the substrates used. Degraded pelotons were already present in a few protocomis. Splwgnm (Fig. 4.4). starch and buckwheat, in declining order. produced the greatest colonization. The addition of CASEseems to have had little iduence on colonization ability of the hingi. 80 Days Al1 three fungal isolates were able to colonize the orchid protocorms and fonn pelotons (Fig. 4.8). Mycorrhizal colonization was more fiequent with isolates 2227.1 and 2209.2 than with 2220- 1.1. Colonization of at least one ce11 with a peloton per protocom occurred on al1 substrates, but was most frsquent on Sphagmm medium, and least muent on rye medium. Al1 fungi were able to form pelotons on al1 media with the exception of isolate 2220-1.1 which did not form pelotons in protocorms on wheatbran CASYE medium. Colonization of al1 protocomis in al1 repiicates ocairrd only on Sphognirm medium with isolate 2209.2 and on Buckwheat CASYE medium with isolate 2220- 1.1. Fungal isolate 2209.2 was associated with the highest abundance of pelotons per protoconn (Appendk 4.1 ). By contrast, colonization with 2220- 1.1 (Fig. 4.5) produced colo~tionswith low numben of pelotons (Appendix 4.1). Starch and Sphagmrm media supponed the greatest abundance of pelotons per protocorm. Peloton abundance was also tiigh for Sphrgrtum CASYE medium. 'Digested' pelotons were present in protocom associated with al! three isofates of Fun@ but were moa commonly observeci with isolate 2209.2 (Fig. 4.9). Digestion of pelotons occurred on al1 media except starch CASYE.Sphagmm medium supponed the greatest number of protocom with digesteci pelotons. Monilioid cells within pelotons were moa commonly produced by isolate 2209.2 (Fig. 4. 6), which fomed them on dl media except rye medium (Fig. 4.10). The other two hngal isdates both formed rnonîiioid ceils, but on fewer media None of the three isolates foimed monilioid cetls on rye medium. Monilioid cells were formed by both 2227.1 and 2220-1.1 on buckwheat and buckwheat CASYE. In addition, 2220- 1.1 produceâ monilioid cells on Sphapmm medium; 2227.1 formed them on starch and wheatbran media. Recolonization of protocom cells after the digestion of previous pelotons was rare. and was observed only in protocoms colonized by isolates 2209.2 on Sphagmm medium and 2220- 1.1 on buckwheat medium. Mortality was observed in protocorms in combination with each of the three fungal isolates. Fig. 4.1 1 illustrates the nwnber of live protocoms in each of the treatments prior to clearing and staining. Dead protocoms turned brown and soft, while Iive protocoms were white to cream in colour and turgid. Mortality of protoconns was highest with isolate 2220-1.1. There was no mortality of seedlings supponed by aarch medium, but death of protocom occurred on ail other media. Mortality was highest on Sphagmm, Sphogmm CASYE. wheatbran and wheatbran CASYE. 'Parasitism' of protocoms by the hngi was observed for al1 isolates, and on al1 media except starch and starch CASYE media (Fig. 4.12). The incidence of parasitism was highest for isolate 2220-1.1. Isolate 2209.2 was parasitic only on SphpmCASYE medium. While some of the protocorms scored as parasitized displayed defensive phenolic production, others did not. It was therefore not evident in every case if the mdj4ng hyphae seen in the cleared protocorms had causeci the protocorni's demise, or if the hgi simply exploiteci the resources of protocorrns that had died nom other causes. The fungal isolates differed in the media associated with the highest monaiity. While death of protocoms was seen in association with ail isolates on Sphgnum and Sphagmm CASYE, this was the only medium on which mortality occurred for isolate 2209.2, Isolate 2227.1 was also associated with protocorm death on buckwheat and buckwheat CASYE media, while isolate 2220-1.1 was not. However, isolate 2220-1.1 was associated with protocom death on the rye, rye CASYE, wheatbran, wheatbran CASYE and starch media. There were no obvious developmental or sire diferences between the suMMng protocorms of the various substratelfungus combinations after the 80 day experimental period. Discussion: Experiment 2 Ail three fun@ used in this experiment were able, to different degrees on the various media to colonize and form pelotons in protocorms of C. regme. This is unexpeaed in light of the unsuccessfùi attempts by previous researchen to find mycorrhizal symbionts for C'pripdi~~m seedlings (Jorgensen 1 994. Rasmussen 1995). Separating colonization âom germination through the use of asymbiotically- ~emiinatedprotocorms could have a large role to play in the successful colonizations of this experîment. Little is known of the factors that promoie germination of C. regimw. but they may not include fungal ingress. Stimulation of gennination by fungi in the absence of colonization and peloton formation has been reported (ûussault et al. 1998. Light 1993). Further, C'ripdiurn seeds hcubated in packets in their natural environment (Zelmer et al. 1996) fonned pelotons in the ceiis of their unenlarged proembryos before germination was evident. Perhaps the complexity of germination and early growth of C'ripeditm species has been underestimateci. This expenment was not conducted to determine the requirements of the fungi for different substrate components. Rather, the various complex additives to the medium were used to determine if the three isolates would be similar in their interactions with the protocorms on the different media. It is evident hom the results of this experiment that the fungal isolates. although al1 tentatively identified as EpuIorhiza sp.. were different in their responses to medium composition. This could reflect isolate-specific differences in the nutritional requirements of the fungi. Nitrogen to carbon ratios (Beyrle et al. 1995), nitrogen forni. heterotrophy for vitamins (Hadley and Ong 1978) and presence of easily- degradcd sugan Peardmore and Pegg 198 1) have ben implicated in the ability of orchid mycorrhizal fungi to fom mycorrhizal associations. The differences in responses between the fun@ in this study suggest that successhl symbiotic germination will require not only an optimal pairing of fungus and orchid seeà, but also of fungus and medium. Given these results, the failure of many root isolates to effect seed germination (Rasmussen 1995) and suppori seedling growth is not surprising. The culture medium used in vitro is likely to be very different in its propenies from the soil environment in which the root symbiosis hnctioned. Given the variability of the syrnbiotic responses of the fun@, the occasional favourable match between isolate, protocorm and medium couid be expected if many isolates are tested on a given medium. Sphagmcm was chosen for inclusion in the test media because it was the naturai substrate for seedlings at one of the C. re-e collection sites. Sphagmm medium promoted colonization by al1 the fungal isolates, and colonization of every protocorm with isolate 2209.2. The frequency of protocorms with digesied and recolonized pelotons was also highest on Sphgmm medium. Initiatly, Sphapm appeared to be an excellent addition to symbiotic medium. However, at 80 days, monality of protocorms was also hi& for this medium in combination with all isolates. For isolate 2209.2. this was the only medium on which protoconn death was recorded. Modifications to the Sphagrncm medium composition mi& result in the retention of early colonization with a broad range of hngi. and the elimination of the high mortality associated with this medium. Although symbiosis on the difierent media rather than growth, was the focus of this expenment, the lack of obvious differences in growth between treatments is notewonhy. By visual assessment. growth occurred on dl media, but large differences in growth and development arnong the su~vingprotocoms of the various treatments were not evident at the end of 80 days. This was unexpected, because many protocorms were known to be mycorrhizal. and peloton breakdown and even recolonization were recorded for some of the protoconns. As for the first experiment, there are many possible causes for this outcome, and more experiments will be required to shed iight on the meanings of these results. A possible explanation is variability. Orchid seedling variability on aga medium is high (penonal observation). Seedlings from the same capsule, sown simultaneously on the same asymbiotic medium oRen differ greatly in their rate of growth and development. Several months &er germination, many seedlings remain at the earliest protoconn stages, while others have advanced to organized seedlings I or more orders of magnitude larger, with shoots and roots. This high degree of inherent variability can confound the redts of experiments where site is assesseci, especially where sample sizes are low. It is likely that differences between the treatments based on size were undetectable with this experirnental design. E&dorhi=4 spp.. while sometimes isolated tiom Cypjwdium roots, may not be effective fùngi for supporting the growth of Cediumseedlings, or they may play a role in other, non-nutritional symbioses. Mordiopsîs solmi also foms pelotons in the roots and seedlings of some orchids, but appears at times to be parasitic on the orchids (Markovina and McGee 2000). The validity of using peloton formation as a criterion for recognizing orchid mycorrhizal symbiosis has been questioned (Markovina and McGee 2000). but mycorrhizal descriptions originaily were bas& on structure and not on function (Harley and Smith 1983). It is possible that the seedlings rely on other hngi for carbohydrates at this developmental stage. such as the clamp connection-bearing basidiomycetes seen colonizing field-incubated seeds of C'ripdkm (Zelmer et al. 1 996). Concli : Experiment 2 Under the conditions of this experiment, ail thfùngi were able to colonize C. regnue protocorms and fonn pelotons. There were trends in the colonization of the protocorms that are ascribed to medium composition or properties. However, the ability to colonize protocorms, the featum of that colonization and frequency of protocorm monality were different for each of the fiingal isolates on the various media. The establishment of orchid mycorrhizal symbiosis between C. regime protocorms and these fiingai isolates are therefore both isolate and medium dependent. Figum 4.1 to 4.6. Fungai isolate, Cypnpediurn qinue protocom and runy l colonizatioiis in protocoras cteareû in KOB and staincd with trypan Mue. Fig. 4.1. Fungal idate 2209.2 (Ep~lorhizasp.). 3 mo., potato dextrose agar. 0.5~. Fig. 4.2. C. regmae protocom at approximately 4 mo. post seeding in axenic culture on Harvais medium. 25x. Fig. 4.3. Fungal isolate 2209.2 foming pelotons in the cells of a C regiwe protocom on starch medium ( 1O@). 200x. Fig. 4.4. Fungal isolate 2227.1 in C. rrguuie protocorm cells on Sphagnm medium. 1 1 . days afier contact. 1 80x. Fig. 4.5. Early peloton formation in C. reginae protocom cells with isolate 2220- 1.1 on Spkpcrn CASYE medium. 200x. Fig. 4.6. Monilioid cells within the pelotons of a C regiirwr protocom colonized by 2209.2 on Rye CASE medium. 200x. Figure 4.7. Experiment 1. Protocorms forming pelotons with isoiate 2209.2 on 7 media types. Bars represent numben of protocorms (of a possible 10 per medium) forming pelotons with fimgal isolate 2209.2 on a variety of media. Not included on this graph are the dextrose (20g/L), hositol (lOg/L) and inositol(20g/L), because pelotons did not form in the protocorms on these media Fig. 4.8. Expenment 2,80 days post inocuhtion. Cornparison of numben of protocorms forming pelotons with tùngal hiates 2209.2,2227.1 and 2220-1.1 on each of 10 media types. Bars tepiesent the total numbers of protocorms formiag pelotons with al1 iso iates on eac h of the ten media Within each bar is indicated the number of protocorms formed by eac h of the three fimgal isolates on that medium. Fig. 4.9. Experiment 2,80 days post inoculition. Cornparison of numben of protocorms with digeated fungal pelotons aAer inocultion with isoiates 2209.2, 2227.1 and 2220-1.1 on each of 10 media types. Ban represent the total numbea of protocorms possessing ceus with digested pelotons with aii isolates on each of the ten media Within each bar is indicated the number of protoconns accounted for by each of the three fungal isoiates on that medium. Fig. 4.10. Experiment 2,80 days pstinocultion. Cornparison of numbe~of protocorms with monilioid celb within lungal pelatons afier inoculation with isolrtes 2209.2,2227.1 and 2220-1.1 on each of 10 media types. Bars represent the total numbers of protoconns in which pelotons incorporating monilio id hyphae were O bserved (al iso lates) on each of the ten media. Wthin eac h bar is indicated the number of protocorms accounted for by each of the three fungal isolates on that medium Fig. 4.11. Esperiment 2,80 days past inocuhtion. Comparison of numben of üve protocorms following inocuhtion with funpl isohtea 2309.2,2227.1 and 2220-1.1 on eacb of 10 meâia types. Bars represent the total numbea of iive protocomis inoculated with ail isolates on each of the ten media Within each bar is indicated the number of live protoconns accounted for by each of the three fungai isolates on that medium. Fig. 4.12. Experiment 2,M days post inocuhtioo. Cornparison of numbers of pamsitized protocorms foUorving inocuhtioo witb isoiates 222209.2,2227.1 and 2220- 1.1 on each of 10 media types. Bars represent the total numbers of parasitized protocorms inoculated by al1 isolates on each of eight media Within each bar is indicated the number of parasitized protocorms accounted for by each of the three fungai isolates on that medium. Starch and Starch CASYE media are omïtted because no parasitism occurred on these media. Chapter 5. Effect of inoculation with root-associated organisms on sterile-grown Cypripedim reginae seedlings Introduction In nature, orchids utilize symbiotic fungi to provide carbon and perhaps other nutnents during germination andfor early development (Peterson et al. 1998). Under cultivation. growers have found asymbiotic methods of germination and growth more convenient for large-scale production of seedlings (Arditti 19%). The growth of orchids on asymbiotic media must take place under sterile conditions because the sugar-nch media on which the seedlings are grown also provide an ideal substraie for the growth of molds and bacteria, which cm overrun the media and prevent germination or kill seedlings. When sterile asymbiotically grown seedlings reach an appropriate size, they are removed to the diverse biotic communities of the potting media and air in greenhouses and growth rwms. Simultaneous changes in temperature, light. humidity, nutrient availability, medium characteristics and air movement are expenenced by the seedlings. hiring a pend of adjustment, seedlings are susceptible to pathogenic organisms, including bacteria and fùngi. Loss of seediiigs can be very high &er the removal fiom a aerile growing environment (unflasicing). The introduction of naturallysccumng root-associated organisms holds promise for improving growth and reducing monality when sterile orchid seedlings are unflasked. A common practice arnong hobbyia orchid growers to promote vigorous seedlings is the addition of some of the potting mix of a mature plant to transfèr important organisrns to a new hreused for pohg up nerile seecüïngs (personal communications). Arditti, Michaud and Oliva 1982 (in Anderson 1990) recommend the addition of some soi1 mix from a pot containing a mature plant of the same or related species to the pots before planting sterile seedlings. The organisms responsible for the apparently improved survival and gowth observed by the growers have not been identified. Several types of organisms appear to be good candidates for the promotion of orchid seedling growth either due to known associations with orchids or because of their use as growth promoters in other agriculiural or honicultural systems. Soi1 or root-associated bacteria (Glick 1995. Holl and Chanway 1992. ONeill et al. 1992. Weller 1988). mycorrhital fungi (Smith and Read 1997, Peterson and Farquhar 1994) and cyanobanena (Toledo et al. 1995s Obreht et al. 1993. Bergman et al. 1992) have positive effects on the growth and survival of plants. The mechanisms by which they aid plants are varied, ranging from production of plant growth hormones (Giick 1995) and mycorrhiral associations (Peterson and Farquhar 1994. Smith and Read 1997) to fixation and transfer to the plant of atmospheric nitrogen (Bergman et al. 1992, Obreht et ai. 1993. Toledo et al. 1995a). Various organisrns may also be involved in the protection of the plant againn pathogens (Weller 1988). Additionally. interactions between root-associated bacteria and mycorrhiral fungi have been documented to affect plant growth and suMval (Azcon 1993, Garbaye 1994, Perotto and Bonfante 1997, Requena et al. 1997. Schelkle and Peterson 1996). The roots of wild Cjpripedium species are ofien heavily colonized by mycorrhizai fungi (see Chapter 2). While the growth-promoting abilities of some mycorrhizd fun@ on orchid protocorms have been weU documented (Peterson et al. 1998). no mycorrhizal fùngal symbiont has yet been found to promote the growth of C'ipedium protocorms or seedlings IN vitro. However. there is evidence that the combination of fungi and bacteria may be stimulatory to seed germination in Cmdim(Light 1993) and other orchid species (Wilkinson et al. 1989). As well, afgae and nitrogen-fixing cyanobactena, are frequently reponed from within the dead. multiple root epidermis (velamen) of several genera (Pridgeon 1987). including close relatives of Cypriipdium (see Chapter 6). While most species of Cypripedirtm do not have a velamen, cyanobactena and algae have been isoiated from the root surfaces of wild C'ripudium plants (see Chapter 3). Objectives The objective of this research was to detemine if the inoculation of sterile, newly udasked C'ripdium regmue seedings wit h selected root-associated organisms (bacteria, cyanobacteria, hngi). singly or in combination, wouid affect growth and survival parameters (e-g.weight, root length, number of roots. shoot height, su~valand phosphorus content of the leaves) of the seedlings. The nuIl hypothesis for this experiment is therefore that seedling growth and suMval (as assessed by the stated parameters) are not aff'ed by inoculation with the selected root-associated organisms. Methods:selection of root-associated organisrns The rationaie and mitera for the selection ofeach of the organisms used in this experiment are provided below. &gunism seledion: Cypripediim species For this experiment aerile seedlings of C'ripedium regime Walt. were utilized. This species was ~ktedbecause of its relative ease of germination in vitroTits commercial potential as a horticultuml species, and because a local source of mature wild plants was available for seed collection and isolation of root-associated organkms. Root samples fkom this species were rapidly processedi helping to ensure the viability of the root- associated-organisms isolated from them (see Chapter 3). Organism selection: fungus The fiuigus used in this experiment was select& 6om a large number of fungai isolates (see Chapter 3) obtained fiom the roots oJC. regmue, C. acactle, C. parv~jlomun,and (: arietittiim. Of these, five isolates were identified as members of the fom-genus Rhizociloniu. Aithough three of these isolates fomed pelotons in C. regtnae protocorms hi vitro (see Chapter 4), untested isolate 23 12, the most recently isolated strain of the common mycorrhiral fungus Eplorhiza repns (8emard) Moore (formerly Rhizuc~or~ia repens Bernard) from C. regrnue, was chosen for the experiment. This was due to concenu that these fun@ could lose vitality and the ability to fonn symbioses after lengthy culturing on agar media. Tui~sneIllo(one of the sexual States of Epulorhiia) isolates can be nutritionaüy exacting and difficult to preserve in vin0 (Hadley and Ong 1978). Organism selecîion: bacteria Eighty bacterial strains were isolated from the interior and surFaces of wild C'ripediurn roots (see Chapter 3). The strains were tested for their ability to grow on nitrogen -6ee media and for the promotion ofgrowth of lemice seeâlings. While none of the strains grew after repeated subcultures on nitrogen free medium, several strains promoted su~valand growth of lettuce seedlings; of these the two fadest growing bactena were chosen for this experiment. Both strains were isolated Rom C. acaule. and were Gram negative, rod-shaped cells which produce an abundant, cohesive rnatrix and form crearn to pale yeliow colonies on PDA. When observed under the microscope, they appeared to be motile, but fomal tests of bacterial motility were not perforrned. it is likely that they belong to the genus Pseudomoms. Owanism selection: eyonobactmkm Algae and cyanobaaeria were isolated from the surface of wild Cypripedium roots (see Chapter 3). From among these isolates, Nostoc hm~figa,a cyanobactenum isolated fiom C regi~we,was chosen befause it produceci heterocysts on low nitrogen media. This implied the ability to fix atmospheric nitrogen, and therefore the potential to provide fixed nitrogen to the orchid seedlings. Methods: Experirnental design The effects of these organisms and their combinations on the Cypripeditim reginae seedlings were tested in an interaction experiment. Thineen treatments of 50 plants each were used. The treatments are show in Table 5.1. Each of the four organisms xlected for the experiment was inoculated onto sterile-raiseci Cypri'ipdiiunr reginae seedlings either alone or in combinations with one or two of the other organisms. All combinations were used, with one exception. In no treatment was both bacterial strains employed. A control group of plants were mock-inoculateci with aii of the amer media. A carrier- media-fiee control was induded to determine the effects of carrier media addition- For the bacterial and algal inocula, 1 mL of liquid suspension was used. Fungal inoculum was applied at the rate of 1 cube of colonized PDA per pot. Regardless of the organisms used to inoculate each treatment. ail carrier media were added to each pot (with the exception of the media-free control). For example, pots inoculated with the fungus received a '/1 cm square of agar containing the fungus. A similar square of sterile agar was added to pots in treatments that did not receive fùngal inoculum. There were three assessrnent dates (Harvests): 8 weeks ( 10 plants per treatment). 12 weeks (20 plants per treatment) and 12 months (20 plants per treatment). plants were sarnpled wit hout replacement. Methods: Preparation of cultures Seedlings of C~pripediurnregnue used in the experiment were raised from seed obtained from a local source. Mature, dry seeds were surface-sterilized using 10% household bleach (5.25% sodium hypochlorite) for 35 minutes. rinsed three times in stede, distilled water, then sown ont0 the surface of Harvais' medium (Harvais 1982) in 1OOmm Petri plates. At 3 months, the protocomis were moved to Petri plates of Harvais medium modified by the exclusion of growth regulaton. After 6 months, the seedlings were aseptically transplanteci ( 15 seedlings per tray) into Phytatrays (Sigma) of half-strength commercial orchid medium (Sigma P6668) with SOmUL potato extract added. The potato extract was prepared by boihng 200g of potato in 1 L of deionized water, then filtering the extract through several layers of cheesecloth. The seedlings were grown at room temperature in darkness until4- 10 roots and large dormant shoot buds formed. Donnancy was broken by 2 weeks of 10°C and then 3 months at 4OC in an un-illuminated low temperature incubator. Following the cold treatment, the incubator temperature was raised to lO0C for two weeks. This was followed by two weeks at room temperature in the light. The seedlings were planted at this stage. one year and 4.5 months dersowing, as they began to produce their first leaves. Most seedlings possessed slightiy etiolated shoots or unfurling green leaves at the time of planting. Seedlings of O. lg fresh weight and above were randomly assigned to t reat ment S. Fungal inoculum was raid by placing cubes (approximately 3mm3) cut from a 3- month-old potato dextrose agar (PDA) culture of isolate 23 12 on to the surface of new PDA media in Petri plates. Approximately 25 cubes were added to each of 10 plates of PDA Mer 2 weeks cubes with new hyphal tips protruding fiom the cube were pooled and used as inocuium. Inoculum of each of the two bacterial strains was prepared in IL flasks containing 500 rnL of sterilized tryptic soy broth. To the flasks was added 5 mL Eom a 3 day old tryptic soy broth culture containing one of the two bacterial arains (3 16b-1 or 3 18b-6). The flasks were maintained at room temperature on a rotary shaker for 3 days. Growth of the bacteria, evidenced by a cloudiness of the media, was noted for both cultures der the second day. Cyanobacterial inoculum was prepared by adding filaments of Nos~uchmijkga which were scraped off the surfaee of a basal salts agar medium (ASM-3) to a iiquid culture of the sarne medium. Mer 3 weeks incubation at 26C in the light ( 16hr days). this Iiquid culture was subcultured CO 500mL of ASM-3 medium. Mer three weeks incubation during which the culture was shaken vigorously 3 iimes per week, the culture was vonexed before aliquots were removed for use as experimental inoculum. Each C. regmue seedling was assigned a unique number and planted individualiy in a 3 inch pot filled with an autoclave-sterilized 3: 1 mixture of Promix" (a peat-based horticultural produn) and perlite. Each plant was placed at random into one of the treatment groups. The root-associateci organisms were added to the pots at the time of planting. Plants were placed in random order within a controlled environment growth charnber providing 14 hour, 2 l OC days and 8 hour. 15°C nights. They were watered as required with deionized water to pot capacity to prevent differences in water availability, and to mimic the very moia environment typical for wild plants of C. regime. At the end of the 1' growing period, the pots were covered with plastic trays in a cool temperature chamber (4") in the dark for 4.5 months. hiring this time, the pots were watered with distilleci water as needed, but due to the hi& hurnidity of the chamber, watering was oniy necessary twice during dormancy. Subsequent to the dormancy period, the plants were moved into a cool greenhouse, where they were maintained at 10°C nights and 12-1 8°C degree days in full midwinter sunlight. Vandaiism of two of the greenhouse glass panes caused the night temperature to approach 0°C for a single night as the plants were unfurhg their fint leaves of the season. Some darnage to the laves occurred however, the damage was randornly distributeci among treatments. Methods: Assessments of variables Random seedlings from each treatment were harvested at 8 weeks, 12 wedts and 12 rnonths. The 12 week harvest occuned just prior to a 3 month cold induced domancy period. The 12 month harvest corresponded to the end of the next growing season. Fresh weigkts As the seedlings were taken From aerile culture for planting, the individually numbered seedlings were weighed. Prior to weighing, any remaining agar culture medium clinging to the roots was gently removed with fine forceps. Fresh weights, accomplished by gently shaking the soi1 fiom the roots, were measured again at the 8 week, i 2 week and 12 mont h harvest . Shoot &y weight, leaf wea At the theof the third harvest, the leaves of each sampled plant were removed and scanned on a flatbed scanner. Mer calibration of the image, Northem Eclipse software was used to determine the total leaf amof each plant. Once scanneci, the leaves fiom each plant were placed in small paper envelopes and dned at 60°C for one week. The Ieaves were then weighed on a precision balance, ground to fine powder using a monar and penle, and used for inorganic phosphorus content. Length of longest rooi The length of the longes root on each plant, irrespective of its position on the rhizome, was mmedat the the of planting. At each of the assessrnt dates, the length of the longest root on each plant was again measwed from rhizome to root tip. No attempt was made to follow changes of individual roots. Rtwt nuwebers At the time of planting the number of live roots per plant was recorded. At subsequent hameas. only live roots were noted. Shuot height Shoot height was measured from the base of the shoot to the tip of the longest leaf. This was required because of the convolute vemation ofthe leaves. When the leaves first apear, they are rolled and venically positioned. As the leaves mature, they flatten and assume a horizontal orientation. Shoot height was measured at al1 three assessrnent dates. Survivcrl Plants were assessed as live if they possessed a live crown and bud. Plants with decayed crowns were assessed as dead even if the root tips remained live. Leaf inorganic phosphom~s Organic phosphorous concentration of the leaves at the time of the third harvest was measured using a modified Fiske and Subbarrow method (Sigma diagnostics procedure 670). Leafsamples were ground to fine powder, weighed, ashed in a muffle furnace and the ash redissolved in a known volume of acid. This solution was reacted with ammonium molybdate to form phosphomlybdate. Fiske and Subbarrow solution was added to reduce the phosphomolbdate to a phosphomolybdenum blue cornplex. which is detected at 660 nm in a spectophotometer. The htensity of the color is proportional to the phosphorous concentration. Measurements were compared to a phosphous standard to ensure accuracy . Dormant bu& At the time of the second harvest, the plants were approaching a natural dormant period. The number of well-fonned, donnant buds was noted for each sarnpled plant. MycorrhizaI mots At the first and third harvests. the oldest living root from each sarnpled plant was removed and stored in 70% ethanol. For processing, the roots were rehydrated in deionized water. and then cleared in 1% potassium hydroxide ovemight at 60°C.mer clearing, the roots were rinsed twice in deionized water, once in acidifieci water to promote staining. and placed into a 0.05% aqueous solution of trypan blue for 1 hour. The roots were removed ffom the nain. placed on glas slides and squashed gently with a coverslip to permit observation of the root cells. Plants were considered mycorrhizai if pelotons (at any stage) were present in any of the root cells. Lever of bacten*dcoloni&un Bacteria on the root surfaces were obsemed at the first and third assessment. using the root removed for mycorrhizal colonitation. As the root was collecteci. a thin paradermal section was taken dong the longitudinai axis of the root using a nerile razor blade. The epidennai arips were storeci at 4OC in srnail, capped Eppendorf tubes containing a drop of stenle water for humidity. They were viewed within 4 hours of sampling on a Biorad WC-60 confocal laser scanning microscope, equipped with a krypton-argon mixed gas laser, utilking a 568 nrn excitation wavelength .the KI/K2 filter block and photomultiplier (PMT) 1 detector (for ernission > 56Onm)using BacLight Livddead sain (Molecular Probes). The abundance of bacteria was visually assesseci. and w here possible, shapes of the dominant bacteria were noted. Mdkods: Statïsticd analysis Statistical analysis was used to test the nuIl hypothesis that the addition of the selected root-associated organisms alone or in combination (treatments). had no signifiant effea (p>0.05) on the final weight. shoot height. fresh and dry shoot weight, leaf area, number of roots, length of longest root, and shoot phosphorous concentration. Kmskal Wallis tests (non-parametric ANOVA) were employed afler transformations failed to normalize the data. Where no signifiant treatment effects were detected within a harvest, the data from al1 treatments were pooled. Kruskal Wallis tests were then used to explore the effects of the presencedabsence of each inoculated organism on the harvested plants. The statistical package SPSS (Version 10.0) for Windows was used for the analyses. Results Hamat 1 (8 weeks) SuMval of the seedlings was high across al1 truitments; only one plant was scored as dead among the 130 sampled at 8 weeks. However. most plants suffered damage to the shoot irnmediately upon unflasking. Damaged shoots rarely produced leaves, however these were replaced by lated buds that oRen produced leaves. Soon der the pots were placed in the growth room, they were colonized by many airborne organisms. The moa evident of these were the abundant algae on the surface of the mil. These were not the algae used to inoculate the plants. intact and digesteci pelotons were confinned in the cleared and stained roots of 50 of the 60 plants inoculated with the hgalisolate. This indifates very high colonization, because only a very small part of the root system was observed, and mycorrhizal colonization can be patchy. Pelotons were not observed in the roots of uninoculated plants. Fixation with 2.5% glutaraldehyde prior to clearing and staining allowed the preservation of ceil nuclei. hlthough peloton-like structures were fonned by very thin hyphae (possibly of Pe,iicillium sp.) in root cells of a few control plants, nuclei in these cells were usually missing or degraded. in contrast to the intact appearance of nuclei in cells colonized by the fùngal inoculant. Bacteria were observed on the surface of live root segments using the BacLight Live/Dead Kit (Molecular Probes) for fluorescence microscopy . Bacteria ( moaly shon rods) appeared confined to the roo t surface. ORen occupying crevices between adjacent epiderrnal cells. They were moa abundant in the youngea faons of the root hair zone. where they appeared embedded in a matrix. They were also abundant on the epidermis of the root overlying conical ce11 colonized by the Epulorhiza isolate. Bacteria colonized control plants sparsely or not at dl. Although it was possible to visudite cyanobacteria using either of the methods used for funsi and bacteria Nostoc huniifi~gacelk were observed on the surface of only one of the 130 mots examineci. Statistical analysis revealed significant difrences between the three harvea dates. The analysis for each harvest date is therefore presented separately; harvest weight indicates the weight of the plant at that harvest date (final weight). At harvest 1 (8 weeks) there was a significant difference in hamest shoot length between the treatments (p=0.042). The treatment effe* on harvest shoot length appears to be due to treatment 13. the control without added carrier media This treatment had the highest mean rank for harvest shoot length, and may indicate an inhibitory efféct of the carrier media on the seedlings. No significant differences were seen among treatments for the variables su~val,hmest weight. length of longest root or number of roots. There were ais0 no significant differences attributable to inoculation with any of the 4 organisrns using the pooled treatment data. Hunes! 2 (12 weeks) Andysis of harvest weight. length of longest root. root number. shoot length. number of winter buds and survival data showed no significant differences (pc0.05) between the thirteen treatments (data not presented). There were also no significant differences attributable to the inoculation of the plants with any one of the 4 organisms across treatments. Bacterial. cyanobacterial and fiingal colonization was not directly observed at this hawest date. Hamesi 3 (IZ months) At the third harvest. there was a significant treatment efféct for the number of roots on the plants at time of hawest (~4.037).There were no significant treatment effects detected for any of the other variables: harvest weight. length of longest root. shoot wet and dry weights, leafarea, shoot phosphorous concentration and survival. When the treatment data were pooled, there were several significant differences attributable to the inoculation of the plants with ihree of the four organisrns. Plants inoculated with the fungai isolate (Table 5.2) were significantly different than non- inocuiated plants in the number of roots present (p4.001)and the shoot phosphorous concentration (p4.000). Table 5 -3 shows the treatments ordered by mean rank for p hosphorous concentration. Without exception, treatments t hat included the fungal inoculant ranked higher for shoot phosphorous concentration than those without fungai inoculation. Mycorrhizal colonization was confirmed by a root subsample for almoa al1 inoculated plants examineci. Plants not inoculated with this fungal isolate remained fiee of fungal colonization. Inoculation with bactenum 3 16b-1 also had a significant effect on some variables (Table 5.4). Harvest weight (p=û.008), shoot wet (p4.0 14) and dry (0.023) weight and the number of roots (p-3.037)were al1 higher in treatments inoculated with this organism. By contrast, inoculation with bactenum 3 18b-6 had linle effect on the variables with the exception of shoot dry weight (p=û.û40). which tended to be lower when inoculated with this isolate (Table 5.5). lnoculation with cyanobacteria did not have a detectable efféct on any of the measured growth and suMval parameten (Table 5.6). Discussion The Cjprïpedium seedlings of this experiment were affêcted by root-associated organimis, but inoculation effects were not evident until the end of the second growing season after unilasking. Mycorrhizal fbngal inoculation had its major effects on shoot phosphorous content and root number. Acquisition of phosphorous via a mycorrizal hgushas been previously documentai for the temperate terrestrial orchid Weta repm (Alexander et al. 1984), and may be one of the major roles of mycorrhizal symbiosis in autotrophic orchids. Surprisingly, fungal inoculation did not affect the weight or survival of the seedlings. The lack of an obvious growth response in the presence of an apparently fùnctional mycorrhizal symbiosis initially is puuling. Digestion of the pelotons was confimed. and the nuclei of the celis were oflen enlarged and lobed. In these respects, the mycorrhizal symbiosis formed between T.regittae and E. repens appeared normal and similar to the colonkation observed in wild plants. Further. in vitro work with early stage seedlings (mostly protocorms) and fungal isolates in co-culture has drawn attention to the flow of carbon fiom fungus to protocorm ( Arditti 1992). Why was there no major growth response to fungal inoculation in the C. regitae seedlings of this experiment? Several explmations for the lack of growth response to mycorrhital colonkation in this experiment can be offered. If the plants are indeed receiving carbon from the breakdown of the pelotons, but no growth enhancement occurs, then perhaps this indicates a cost to the plant of being mycorrhizal. It has been nated that orchids may be parasitic on their symbiotic fun@ (for reviews see Arditti 1997, Rasmussen 1995). A more balanced symbiosis could be indicated if there are cos to both syrnbionts. Another explanation for the lack of a mycorrhizal growth response is that the symbiosis functions differently in the presence of other organisms. and on the poorer substrates used in this experiment, than it does in dual cuiture on nutntious, sterilized media in the lab. Most previous midies of mycorrhital symbiosis have been carried out under the latter conditions. The presence of a diversity of other organisms may dampen the strong responses seen in dixenic culture. The sources offungai nutrition need to be carehlly considered. There is a possibility that orchid mycorrhiral fun@ may acquire carbon directiy ffom other plants. Triple symbioses occur between some of t he mycoheterotrop hic orchids suc h as Corallorhii-a species forest trees and hngi that can be mycorrhiral with both plant groups (Taylor and Bruns 1997, Zelmer and Currah 1995). It has also been demonstrated that binucleate Rhizucto~~instrains fomiing symptomless to growth-stimulating root symbioses with pine seedlings in tree nurseries in Finland share a cornmon genotype with those forming mycorrhizas with Finnish and Canadian orchids (Sen et al. 1999). In lab studies, where the fùngi have no access to other plants, the fun@ rnay have less carbon (or other substances) to provide to the orchid seedlings. There also the possibility that inoculation effects were not detected. There is high inherent variability in orchid seedlings. Power analysis of the presmt data suggests that sample sues much larger (100-600 plants per treatment) than those used here would be required to reduce this possibility. However, responses due to fungal inoculation seen in early seedling development are not kely to be the same as the responses of older, autotrophic seedlings or mature plants to mycorrhizai symbiosis. While protoconns lack a major resource base (Richardson et al. 1992). older, autotrophic seedlings have the ability to produce and store photosynthates. Their requirement for fungai carbohydrate imputs may be minimal. whne requirements for specific macronutrients rnay be much higher. It seems doubtful that the currency of symbiotic exchange would remain as fùngal carbon after the onset of autotrophy. The C regmue dlings in the study may be interacting with their myconhizal hgiin a similar manner to orchids in the wild. Phosporous in panicular may be important to C. regme growing in one ofits common habitats: rich fen areas. While rich fens are not low in nutrîents, they oflen have low phosphorous availability (Szumigalski and Bayley 1997). If the seedlings of this experiment had been plant4 in their natural substrate, growth and suMval differences may have been noted between the non- and fùngal-inoculated plants. In general, plants with abundant phosphorous are larger and more rapidly reach maturity than those with lower phosphorous concentrations (Salisbury and Ross 1992). Two growing seasons were not sufficient to produce sexually mature plants in this study. Growth and maturity effects may ahhave been detected in these plants growing on the experimental medium aAer a longer penod of observation. Growth responses were seen in plants inoculated with bactenum 3 16b-1.Hamea weight, shoot weight and nurnbers of roots were al1 positively influenced by this bacterium, which was originally isolated from a C. acaule root. Under the conditions of this experiment, bacterial isolate 3 16b-I acted as a plant growth promoting rhizobacterium (PGPR).This bacteriurn was unable to grow on Ntrogen-free medium (see Chapter 3) so the effects on C. reginee are probably not due to the ability to fk dinitrogen. Production of growth-promoting hormones and siderophores are other common mechanisms for the enhancement of plant growth by PGPR (Glick 1995). Funher characterization and experirnentation would be necessary to determine the mechanism for the growth eRms on C. reginae seedlings. By contrasi, inoculation with bacterial isolate 3 18b-6 significantly affecteci only a single parameter - shoot dry weight. Plants in the presence of 3 18b-6 tended to have lower shoot dry weights than uninoculateci plants. The results of this experiment, although employing only two bacterial isolats, Uidicates that bacterial inoculation can affect the growth of sterile-grown orchid seedlings, even two Kasons &er udasking. This presents the possibility of screening for effiive PGPR isolates, perhaps hom the roots of established orchids. Although the bacteria in this experiment were not positively identified, they shared many charactenstics with those of the Pset~domows,a genus known to contain PGPR (Giick et al. 1995). Isolation proceedures that target this genus (Glick et al. 1995) may prove usefui in the search for organisms to support orchid seedlings in the transistion from stenle to soi1 culture. The cyanobacterial isolate used in this experiment had no effea on seedling growth and survival. The cyanobaaeria were capable of fixing dinitrogen (evidenced by their growth on nitrogen-free medium and production of heterocysts), so an effect based on increased nitrogen availability might have been expecîed. However, there was little evidence that the cyanobactena were able to establish populations either in the soi1 or in the rhizospheres of the seedlings. NOSIOCcells were seen only once, at 8 weeks &er planting. on a root of a seedling. By this time, the sudaces of the pots were heavily coloniteâ by other algae, and it could be that the inoailated cyanobacterium was out- competed by these. It is also possible that the environmental conditions of this experiment did not permit the cyanobacterium to flourish. The fate of the cyanobactenum used as inoculum is uncertain. therefore, any interpretations of the effect of cyanobactena on C. reginae seedlings from these data are questionable. It would have been interesting to determine if the rwt-associateci organisms were interacting in ways that affected the measured growth parameters and seedling survival. Unfomnately, due to the structure of the data, the assumptions of the parametric tests needed to examine this could not be met. However, both the fungal isolate and bactenurn 3 16b- I had significant effkcts of the number of roots. This might be a nch area to explore for synergistic effects. It is difficult to determine if fungal colonization within root cells is mycorrhllal or parasitic. A method used in this research may be usefbl in assessiny the viability of the colonked cells. Giutaraldehyde fixative used before the comrnon clearing and staining proceedure preserved the nuclei of the cells even afier clearing. In a few control plants. peloton-like structures were fomed by very thin hyphae (possibly of Prnicilliim sp.). Unlike the cells colonized by the fungal inoculant. the nuclei of the cells that contained other fiingi were usually missing or degraded. This method could form the basis for distinguishing parasitic fun@ fiom symbiotic ones in the cells of cleared roots. This experiment has implications for the cultivation and conservation of Cpnipedititrm species. It has demonstrated that longterm mycorrhizai symbiosis can be established in previously asymbiotically-grown seedlings. and with an isolate obtained fiom the roots of mature wild plants. While this symbiosis did not provide growth stimulation or increase suNiva1 of the seedlings, it allowed the inoculated plants to accumulate phosphorous to a greater degree than non-inoculated plants. Cornmercially, this could be important to orchid growen because a high phosphorous aatus is implicated in earlier matunty, i.e. flowering and ovedl fecundity. For those attempting to restore populations, it could mean pater viability of introduced seedlings on phosphorous-poor substrates. Although the asymbiotic method of seed germination and growth has proven usehl this research adds to a body of evidence (e.g Anderson 1992, Zelmer and Currah 1997. Zettler 1998%Zettler 1998b) that mycorrhizal symbiosis should be a consideration in restoration and conservation strategies for orchids. Furthemore. there is evidence ftom this research that bacteria can provide a mareof growth stimulation for the seedlings, and these may be screened to find those that help buffer the seedlings in the difficult transition fiom sterile conditions to soil. Conclusions The inoculation of stenle-raised Cypripedizm reginae seedlings with root-associateci organisms at the time of unflasking had effects that were detectable afler two growing seasons. Due the structure of the data, non-parametnc tests were employed for analysis. This did not permit the exploration of interaction effects between the organisms. However, root number at harvest was significantly different between treatments. and appears to have been attributable to both fungal and bacterial inoculation. Plants inoculated wit h the fungal isolate (EpIorhiza sp. ) had significantly higher shoot phosphorous concentrations and root numbers than uninocuiated plants. Mycorrhiza symbiosis was confirmed by observations of pelotons in root samples of inoculated plants. Pelotons were not seen in uninoculated plants. although a few peloton- like structures were observeci. These were determined to be pathogenic hngi by the breakdown or absence of nuclei in these celis. The effect of bacterial inoculation varied with the isolate used. Plants inocuiated with bacterial isolate 3 16b-1 had higher harvea weights, shoot wet and dry weights and numben of roots. whereas the ody signiticant effect of inoculation with isolate 3 1Sb-6 was a reduction of shoot dry weight. Inoculation of C.reghe seedlings with the cyanobacterium Nostoc hunirfgu did not have a measureable efect on any of the growth or su~valof the seedlings. The NMV~ of the cyanobacterium after inoculation is in doubt. Table 5.1 Treatments used in inoculation experimcat Bacterium Bacterium Treatmert Plants Fungus 316b-t 318M Cynb 1 50 cm cm cm cm 2 50 cm X cm cm 3 50 cm cm X cm 4 50 cm cm cm X 5 50 X cm cm cm 6 50 cm X cm X 7 50 cm cm X X 8 50 X X cm cm 9 50 X cm X cm 10 50 X cm cm X 11 50 X X cm X 12 50 X cm X X 13 50 O O O O Legend: cm, nede carrier medium added (mock inoculation). This was 1 mL sterile tryptic soy broth without bacteria, sterile ASM-3 media without Nostoc, and a 3mm cube of steriie PDA when the fungus was not added. 0, no carrier medium added. X, indicated organism added. Cynb. Cyanobacterium (Nostuc hmjbga) TaMe. 5.2. Krusk.l Wallis test mults Tor the cornparison or noci- and inoculated plants witb the fungal isolate (Epdorhiso sp.). Baivat 3 (12 months). Variable chi2 Asymp. Sign. Hamest weight 0.553 0.457 Lengh of longest root 0.995 11.3 18 Shoot wet weight 0.02 0.887 Shoot dry weight 0.292 0.589 Number of roots 1 1.063 0.001 8 Leaf area 0.255 0.613 SUMV~ 0.045 0.832 Shoot P concentration 77.054 0.000 * Asymp. Sign., Asymptotic significance. * denotes significant difference between inoculated and uninocuiated plants at paos. Table 5.3. Shoot phosphornus concentration itHiivcst 3 (12 momtbs). Mean nnk by treatmeat. Treatment Mean rank Fungus Bac 3 16b-1 Bac 3 1 8b-6 Alga 12 123.50 X cm X X Legend: cm, stede carrier medium added (mock inoculation). This was 1 mL sterile tryptic soy broth without bacteria, aerile ASM-3 media without Nom, and a 3mm cube of aerile PDA when the fungus was not added. 0, no dermedium added. X. indicated organism added. TaMc 5.4. KnisW Wallis test results for the cornparbon of non- and inoeulatcd plants with the bacterial isolate 316b-1. Barvat 3 (12 mooths). Variable chi2 Asymp. Sign. Harvest weight 7.042 0.008 Length of longest root 1.707 0.191 Shoot wet weight 0.5.987 0.014 Shoot dry weight 5.160 0.023 1 Number of roots 4.348 0.037 Leaf area 0.469 0.493 SUMV~ 3.10 0.078 Shoot P concentration 2.086 0.149 Asymp. Siga., Asyrnptotic significance. * denotes significant difference between inoculated and uninoculated plants at p Variable chi2 Aqmp. Sign. Harvest weight 1.978 0.160 Length of longest root 0.774 0.379 Shoot wet weight 2.317 0.128 Shoot dry weight 4.224 0.040 * Number of roots 0.060 0.807 Leaf area 0.422 0.516 Su~val 0.355 0.551 Shoot P concentration 0.645 0.422 Asymp. Sign., Asymptotic significance. * denotes significant difference between inoculated and uninoculated plants at p Variable chi2 Aqmp. Sign. Harvest weight 0.519 0.471 Length of longest root 0.01 1 0.9 16 Shoot wet weight 0.635 0.426 Shoot dry weight 0.017 0.896 Number of roots 1.834 O. 176 Leaf area 0.649 0.420 Su~val 0.045 0.832 Shoot P concentration 0.253 0.615 Asymp Sm., Asymptotic significance. denotes significant difference between inoculated and uninoculated plants at p Introduction Velamen, a dead, sponge-like multiple epidhs, surrounds the roots of many orchids. In the slipper orchids, Puphiopedihm and Phragmipedium (Orchidaceae, subfamily Cypripedioideae), the velamen is udlyseveral ceIl layers thick and possesses abundant root hain on the outer surface. There is controversy regarding the funaion of the velamen; the proposed roles include the condensation of water vapour (Unger 1854 in (Pridgeon 1987). capture of the first nutnent-rich run-off from substrates during rainfall (Be~nget al. 1982) and as protection against moisture loss and mechanical damage (Dycus and Knudson 1957). These roles have relevance to epiphytic orchids, but many species of Paphiopedifum and Phragmipdium are lithophytic, temestrial or humus epiphytic in contUwously moist environments (Cash 199 1). Several authors have reponed observations of organisms such as fun@ and cyanobacteria in the veiarnen of Paphiopdifum, Phragmïpediuni and other orchids (Be~nget al. 1982. Pridgeon 1987). Dressler (1993) suggesteci that these velamen inhabitants rnight be "invited guests," implyhg an additional role for the velamen as a habitat for beneficial organisms. The isolation and idmtification of velamen organisms has not been attempta making this proposed role difncult to evaiuate. Further, there have been few investigations of the mycorrhual associations of these two genera, despite the cultivation of some species for more than 100 years. The Eequency of mywrrhiral associations of PclphiopdiIum is suggested by the inclusion of pelotons in the root illustrations of Fuchs and Ziegenspeck (1926). In one of the only isolation attempts recorded in the scientific literature (Bernard 1904). a Rhizoctoniu sp. from a cultivated plant of Payhiopdiium i~tsigw(then known as opripudiuni ir~sipe)was isolated. Symbiotic germination of Puphiopediitm seedlings using fungal isolates fiom other orchid genera has also been attempted (Light 1989). Prior to the development of asymbiotic techniques for germination of Paphiopedih~and Phrugmipedium se&. these orchids were propagated by sowing the seeds directly into the mois potting medium of established ('host') plants of the same or similar species. Although the yield of seedlings is usuaily lower, this method produces mycorrhVal seedlings that withstand environmental stresses better than those that are sterile-grown (Cash 199 1 ). It is, therefore, important to determine the mycorrhizal status of plants to be used as 'hosts' for the symbiotic germination of Puphiopediium and Phrapipedium seeds (Cash 199 1). Trade in wild-collected Pcrphiopediium and Phrugmipedium species is now severely restricted under the Convention on International Trade in Endangered Species (CITES) in order to protect the remaining wild populations. Consequently. most plants in cultivation in Canada have been grown fiom seed under aseptic conditions. It is not known if these plants, now geographically separated fiom their natural root associates, can be colorûzed by mot-associated organisms and mycorrhizal tùngi. The objective of this research was to localize, isolate and identify rwt-associated organisms, especially fungi, algae and ciliates, fiom the mots of cultivated Puphiopediluun and Phmgmipedium species. Methods Collection sites Puphiopc!diI~~mand Phrupipedi~~mspecies and hybnds were sarnpled from three greenhouses: Johnston Orchids (Edmonton, AB). Bickell Orchids (Whitby. ON) and from a collection housed at the University of Guelph Dept. of Botany greenhouse (Table 6.1). At Johnston Orchids, the plants were grown in a mixture of bark, rockwool, Osmw~du fiber. couse perlite and Sphgmm moss. Roots from Johnston orchids were sarnpled by gently unpotting mature. healthy plants and rernoving approximately 3 cm segments fiom each of two roots with actively growing root tips. The fint root was cut into smailer segments, and imediately presewed in 2.5% glutaraidehyde in HEPES buffer (O. 1M). These roots. labeled with a number identdjhng the plant from which they were detached. were dehydrated in a graded ethanol series to 70% ethanol then sent to the lab in Guelph for hrther processing. Upon amival, the roots were dehydrated to 1Wh ethanol. then infiltrated with L. R. White resin by gradually increasing the ratio of resin to ethanol from 1 :3 to 1 :O. Infiltrateci root pieces were placed in flat aiuminum trays or in gelatin capsules in pure L. R White resin and polymerized at 60°C for 4 hours. Polyrnerized sections were prepared for laser scanning confocal microscopy by affwng the resin block to a microscope slide, removing the upper layers of resin with a razor blade to expose the tissue and staining with a 0.5% solution of sulforhodamiw (Melville et al. 1998). Alternatively, some sarnples were sectioned on an ~Itramicrotorneand stained with 0.5% toluidine blue O in 1.0% sodium tetraborate for light rnicroscopy. The second root piece from each plant was placed in a plastic bag to maintain moisture levels during transportation back to the University of Guelph where the pieces were used for the isolation of endophytes following the procedure described below. The second collection site (Al Bickell Orchids) was characterized by low light levels, very high humidity and a potting medium primarily composeâ of redwood bark, Sphagmm moss and styofoam packing chips. Roots were sampled as above. Additionally. a 2 cm root segment was severed and placed immediately into liquid ASM- 3 medium for the isolation of algae and ciliates. The third greenhouse, at the University of Guelph (U of G), was considerably bnghter and drier than the first two. The potting mix was cornposed mostly of fine grade fir bark but also included perlite, Sphapm moss and styrofoam chips. Sarnpling was the same as at BickeH Orchids. Isolation and cultivation of niol-asstxiiüed organism 1. Fungi Fungi were isolated by transversely sectionhg 1 cm ninace-steriiized mot pieces ( 10% household bleach 1 min, 3 changes of sterile water, 1 min each) into approximately 1 cm discs and placing the discs, 4 to a Petri plate, on each of 3 agas-based media (corn meal agar (CMA), potato dextrose agar (PDA),Harvais orchid medium (Harvais 1982)). For isolation fiom the cortex only, mots were aseptically sectioned into 1 cm disis, then dacesterilized and placed on the agar plates. Hyphal tips emerging from the root sections were removed to fiesh media This step was repeated und pure cultures of each fungus were achieved. Samples of each isolate were stained with acid fuchsin (O. 1% in equal parts lactic acid. giycerine and deionized water) and observeci by light microscopy. Fungal isolates were stored at 4OC on PDA slants. For the plants collecteci at Johnston Orchids, an attempt was made to compare the fùngal isolates of the root with intact velamen the interior of the velamen oniy, and the root cortex only. For the intact root, segments were plated as above. Cortex-only isolates were achieved by peeling off the velamen of the root prior to surface sterilization. Velarnen-oniy isolates were obtained by removing the velamen of 1 cm long root pieces, ding the velamen pieces in W-transparent plastic packages and irradiating both i~er and outer sunaces with UV radiation in a laminar flow hood for 24hr. Following this treatment, the velamen pieces were plated onto PD& tap water agar and CMFungi growlng fiom the velamen pieces were removed to fiesh media until pure cultures were obtained. Storage and identification procedures were the sarne as for the previous fungai isolates. Testing of sclecteâ ruagai isolates Tor abüity to Corn pelotons witb Puphiopedilum protocorms To test the symbiotic potential of selected fbngal isolates, three week pst-germination Puphiopdiiluni protoconns (hybrid seedlings donated by Green Valley Orchids) were used. The seeds were germinated on a commercial orchid'mediurn (Sigma P6668) at room temperature in the dark. Stages selected for the trial were a) embryos just splïtting the testa, or b) a slightiy enlarged protocorm still covered in part by the testa. Thirty fungal isolates (Epuforhiza and Moniliops~sspp.) obtained from the roots of Paphiopedihm, Phragmipedi~~mand the closel y- rela t ed genus Cypriipedit~m(Table 3 ) were each inoculated ont0 2 replicate 60mm Petri dishes, each containing 10 Paphiopudif~mseedlings. Control plates (4) were not inoculated. Medium for al1 plates was 0.1% w/v oat powder with 7 g/L agar. A 3mm disk of the inoculant strain was cut fiom five-rnonth-old PDA cultures and placed in the center of the plate. The ten seedlings were arranged in a circle around the inoculum at half the distance between the inoculum and the edge of the plate. Each plate was sealed with parafilm and al1 plates were incubated at room temperature in Iow ambient light in a large sealed plastic bag. Mer 40 days, five protocorms taken at random tom each plate were fixed and aored in 2.5% glutaraldehyde in O. 1 M HEPES buffer for 5 days. then rinsed in distilled water. The seedlings were cleared in boiling 5% KOH for approximately 30 min until transparent. Cleared protocoms were rinsed twice in distilled water. once in acidified water, then the volume of iiquid in each via1 was doubled by adding 10û% ethanol. Seedlings were aored in this solution until nained. Three drops of O. 1% trypan blue in 1 : l : 1 water:glycerin:iactic acid solution was added to each vid wiihout drai~ng.Thiny minutes later, they were removed from the naining solution. Protocorms fiom the same repiicate were mounted together on glass slides with PVA (Salmon 1954). Gentle pressure on the coverslip allowed the dispersion of the protocom cells to faditate observation of the interÎor cells. Slides were observed by light microscopy. Peloton formation in the interna1 cells of the protocorms was considered evidence of a balanced symbiosis. Parasitisrn was indicated by the presence of rambling hyphae, disorganized or dead tissue, or colonkation of the vascular system or meristems. Replicates were xored by the percent of protocorms with pelotons relative to the number of protocorms observed per replicate. This was sometimes less than 5 since many small protocomis were lost dunng clearing or were too degraded by fun@ activity to be mounted for microscopic observation. 3. Algae All roots sarnpled for this audy were terrestrial roots growing in the poaing media. Alyae associated with the Paphiopediium and Phragnripudium roots were cultureci in ASM-3 media (Gerrath et al. 1995. Gerrath et al. 2000). Vials containing root pieces were exposed to light in a 26°C growth cabinet and renewed with fiesh media as needed until observed. A Zeiss light microscope outfitted with digital image capture equiprnent and Northem Eclipse software was used to record observations of the algae, which were identified by J. F. Gerrath. 4. Bacteria Bacteria were isdated concumently with the fungal isolations, particularly on PDA media plates. Bacterial colonies fonning on the cut surfaces of orchid roots were removed using a flamed loop and areaked ont0 PDA plates. Mer 3-10 days incubation in thc dark at 22OC, colonies were selected for re-streaking on PDA bas& on colony texture and color. This step was repeated und pure cultures representing the diversity of colony types found on each plant root were obtained. Gram naining (Gray 1990) was used to facilitate initial observation. 4. Ciliates Ciliates were cultured fiom the algal isolation samples. Approximately 1 mL of the ASM-3 media into which the orchid root segments had been placed was dispensed into 60mm Petri plates or plastic storage containers. Three boiled barley caryopses and 10 mL of Evianm or Aberfoyle Springsm minera1 water were added to each dish. Mer 3-5 days, this was repeated with the fira culture acting as the inoculum. 3-5 days later, 1 rnL of the second seneration culture was added to 5 mL Bouins fixative (Baker 1946). in which the ciliates were stored until prepared for observation. Ciliates were not seen in the uninoculated control cultures. For identificatio~quantitative protargol staining (QPS) was used following the methds of Montagnes and Lynn ( 1987). Ciliates were identified by D. Lynn and D. Acosta. Results Fungi Fungi were isolated fiom nearly al1 of the 76 Priphiopedilrcm and Phragmipedirîrn plants wnpled. Many of the resulting isolates (Table 6.2) were potential orchid mycorrhizal syrnbionts (C urrah et al. 1997% Currah et al. 199%); however. a mycorrhizal association between these fun@ and the orchids from which they were isolated could not be confirrned. The most commonly isolated taxon of orchid myconhual fungus was Epulorhka. Severai species were recognized, including the ubiquitous orchid endophyte Ephrhiza repens (Fig. 6.1). E. ufburtiensis(Fig. 6.2) and a species tentatively identified as E inquilim (Currah a al. 199%) (Figs. 6.3 -6.6). Pelotons containing Epulorhh-like hyphae were often seen in the root samples (Fig. 6.7). E inquifimwas isolated only frorn the Johnston Orchids greenhouse. At least two species of Moniliopsis (M. solani (Fig. 6.8), M. boreulis (Fig. 6.9)) were isolated in this study. Microscopie examination of resin-embedded roots revealed both peloton formation (Figs. 6.10. 6.1 1) and parasitism (evidenced by intercellular hyphae and invasion of the stele) by broad, thin-walled, dolipore-septate hyphae resembling those of Moniliups~s(Fig. 6.12). Myconhizal fiingi inside the root cortex were ofien linked to hyphae in the velamen via the passage cells of the exodermis (Fig. 6.13). Al1 hyphae in the conex resernbled Moniliopsis or Epulorhiz. No conidial, ascomycetous or clamped basidiomycetous hyphae were contirmeci within the cortex. Several of the potentidly mycorrhùal isolates were inoculated onto Paphiopedihm hybrid seedlings in sterile culture to confirm a mycorrhllal role for these fingi. Lack of colonkation or parasitism of the protocorms, characterized by invasion of the stele and meristematic areas, resulted ôorn al1 inoculations with Moniliopsis isoiates (Table 6.2). Of 20 Epuforhii=4-likeisolates, 19 wmable to form pelotons in the protocomis of the test seedlings (Table 6.3). No parasitism was noted for any of these isolates. Spiral or coiled cells @g. 6.14) were noted in the velamen of several specimens from Johnaon Orchids. Transmission electron microscopy (TEM)revealed a central hypha in some of the coiled cells of the velawn. The coiled hyphae were dolipore septate with imperforate parenthosornes (Figs. 6.18,6.19) and a thick ce11 wall distinguished by its lens-shaped, electron dense inclusions (Fig. 6.20). Similar hyphae, with less elaborate walls, were seen among pelotons in the root cortex of these plants (Fig. 6.2 1). More than 350 nobmyconhizal hngi also were isolated (Table 6.3). Of these, most belonged to one of three taxa: Zygomycetes, the anamorphic genus Trichodema and the ascomycete Chaetomium. While the first two were most commonly isolated From velamen fragments or from sections of orchid root Gth an intact velamen, known potentialiy-mycorrhizal fiingi and species of Chmtomium were rarely isolated corn the velamen (Fig. 6.22). Chaetomitm species were comrnonly isolated fiom cortex-only root samples. A single isolation of the nematode-trapping fungus Arthrobonys quickly died out afler exhausting the supply of nematodes isolated with it corn the velamen of PaphiopediItim coricolor. Also of interest were isolations of the orchid pat hogens PrstuIotiopsis (Fig. 6.23) and Colktotrichun, and of De~id~osporium(Fig. 6.24)and Thoze relia. &cterict Bacterial cells embedded in mucilaginous ma& material occurred within the velamen in close contact with the exodermis and passage cells (Fig. 6.25) and ah,rarely, within the intercellular spaces of the cortex of the root. Intercellular bacteria colonization was associateci with parasitism by a MoniIiopsis-like fungus which colonized the stele. Bacterial isolata initially grew weU on the PDA isolation medium, but won became difficult to trder, even to ,more traditional bacterial culture media such as nutrient agar. nutrient broth and tryptic sou agar. Evennialiy, transferred colonies did not grow, althoug!! staining with the LIVElDEAD BacLight Kit indicated that the cells were ail1 alive and possibly motile. Most isolated bacteria were Gram stain negative and rod or short-rd shaped. They tended to produce a thick, cohesive mucilage fkom which cells were not easily separated. They could not be identified or further characterized due to their lack of growt h in vitro. Algw and cyanobacteria Algae were seen on the surfaces of the roots plated on agar fiom the Johnaon greenhouse. Some of these were removed to solid ASM -3 medium. However, isolation rates were low, possibly due to the activities of the fungi also growing on these plates. Subsequent sarnpling at the U of G and Bickell greenhouses was accomplished using liquid medium which suppressed fimgal growth. Therefore. moa of the algae isolated were from the latter two greenhouses. 18 genera of algae were cultured and identified fiom the velamen of Paphiopediium and Phragmipedium roots (Table 6.4). Additionally, a number of diatoms were noted but not identified to genus due to their low numbers in the wnples. The rnajority of algae isolated from the Bickell greenhouse plants were biuegreen algae, more correctly termed cyanobacteria. Filamentous cyanobacteria genera Nostoc (Fig. 6.26). Ambae1~7(Fig. 6-27},Scyonemu (Fig. 6.28). Lyngbya (Fig. 6.29)and Oscillatoria (Fig. 6.30) were common in the samples. The single-celled cyanobacteria Chroococcus and Sjmechoctxcus/cystrs were also present. In the U of G greenhouse samples, cyanobacteria were less cornmon. and unicellular green algae, including a Chlorella-ke isolate (Fig. 6.3 1) and species of Sce11edesm1ilc.s (Fig. 6-32), dominated. An ununial alga isolated fiom this group of samples was Scoii~liopsisreticula~a (Fig. 6-33). which has been isolated tiom the interior of lirnestone rocks from the Niagara Escarpment, Ontario (J. Gerrath, personal communication). Using the LSCM, it was possible to localize algae in the velarnen of some root samples. Algae were associated with the velarnen not just on the surface but also in the more interior layen close to the exoderrnis (Figs. 6.34.6.35). Filaments of cyanobactena resembling Lyngbya, Awboma and Nostoc often filled the lumen of velamen cells, their axes aligned with the long avis of the cell that they occupied. Filaments of a cyanobacterium resembling the genus Riwularia were noted on the velamen surface of resin embedded Phragnripedit~mprcei roots (Fig. 6.36). This cyano bacterium was not isolated fiom the roots. Diatoms. which were seen covenng the surfaces of root hairs, were also under-reprewnted in culture. Nostoc, L~yngbyuand Chiorefia-like cells were isolated fiom the roots of two other orchid plants (Angraecumdidieri and Sophroniris weiigei) growing with the Paphiopediim and Phragmipedium species in the Bic kell Greenhouse. This may indicat e a sirnilarity between velamen habitats of different orchid genera, a preference for a particular potting medium rather than an orchid genus. or a lack of specifidty on the pan of these algae and cyanobacteria. Ciliates Al1 of the ciliates obtained in this study were memben of the senus Col@ (Table. 6.5). Six species of COI@ were cultured from 12 of the 20 root samples fiom the Bickell Greenhouse (Table 6.6). Botb Puphiopdilum and Phrugmiped~~mmots produced cultures of ciliates. A stereoscope was used to determine which cultures had live ciliates: only those with visible ciliates were hed and prepared for examination. Although there is a possibility that very small ciliates were rnissed by this method, flagellates a few microns in diameter were noted so the potential loss of diversity is probably small. The remaining isolation attempts did not result in ciliate growth. It was noted that few ciliates were seen in cultures which contained abundam cyanobacteria. Cultures of ciliates were also obtained fkom the U of G greenhouse root amples. (These are now being processed for identification). Sectioned or block-embedded root material was insufficient to positively localize ciliates because it was difficult to recog~zetransverse sections (Fig. 6.37) and the LSCM stain used (Sulforhodamine G) did not aain cilia (Fig. 6.38). However, profiles or sections seen within the velamen of some orchids possibly were those of active or encysted ciliates (Fig. 6.39). It is also possible that some of the other organisms seen on prirnary isolation plates (amoebae, nematodes) were responsible for these stmctures (Fig. 6.40). Discussion As suggested by earlier researchers, many organisms inhabit the roots of Paphiopedhm and Phrugmipedium species. The velamen is panicularly rich in organisms. Bactena, algae. cyanobacteria fun@ and ciliates were ~enin or isolated fiom the mesh-like framework of the velarnen. Most of the organisms isolated are cornmon to ubiquitous in soils or aquatic systems, however, soi1 was not used in the culture of the Puphiopedihm and Phrupipedm species sampled h this study. 1s it possible that the velamen is acting as an 'internaüzed rhizosphere' for soi! organisms, as suggested by Be~nget ai. (Benzing et ai. 1982) The provision of a habitat for microorganisms could be an important adaptation for these plants, many of which grow in mois habitats in which soi1 is scarce to absent (Cash 199 1 ). The benefits of a soil-like assemblage of organisms in close proximity to the root could be diverse. Several cyanobacteria. including Nosfroc and Anah.are known to fix atmosphenc nitrogen under aerobic conditions and to participate in nitrogen-fixing symbioses with other plants (Bergman et al. 1992) and fun@ (Havelka et al. 1982). Additionally, some species of Ly~gbyu,Oscillaforiu and some unicellular cyanobacteria may be able to provide fixed nitrogen under low p02 (Gallon 1992) experienced during the flooding of the velarnen or due to the respiration of other organisms. Nitrogen could be transferred to the orchid roots by leakage from the living filaments (Gallon 1992), or through the senescence and breakdown of older filaments. In the same way, sugars From p hotosynthetic velarnen organisms may enhance bacterial colonization and t herefore waae products from protozoa and nematodes may be available to the orchid. The heavily reinforceci exodermis excludes the velamen community from the root cortex, but mycorrhizal fun@ communicate with the velamen through passage cells. Hyphae of mycorrhizal hngi in the velarnen appear to be continuous with those in the corien allowing impon of nutnents gathered in the velamen into the orchid roots. These fùngi could be acting as scavengers, foraging in the velarnen for nutrients released by velamen organisrns and transferring them to the interior of the orchid root for assimilation by the plant. There is also evidence from this research that some mycorrhizai fun@ may be more aggressive in their approach to nutrient acquisition. The dolipore-septate spiral cells of the mycorrhual fùngus seen in the velamen of several plants from Johnston Orchids are reminiscent of mycoparasitic tiingal structures. The presence of a central hypha in some of the coils may be evidence of a mycoparasitic role for these fûngi. A final benefit of the diverse comunities in the velarnen may be the reduction of pathogens in the cortex by the physical occupation of points of entry. Le. the passage cells. Similar mechanisms have been suggested for the protective abilities of ectomycorrhiral fun@ (Schelkle and Peterson 1996). Chaetomiuni spp. were the most commonly isolated hngi with the exception of known mycorrhiral species. Additionally. they paralleled the behaviour of the mycorrhizal hngi in the isolations from velamensnly, conexsnly and whole root isolations. They were more frequently isolated from the root cortex than whole root or velarnen sarnples. There was no evidence of ascomycetous hyphae in pelotons of the roots, but this is difficult to ascertain and could be easiiy missed. Another explanation for their isolation from the cortex-oniy root sarnples is the inclusion of the exodermis as the outermoa layer of the cortex. The heavily reinforced, nate UI exodermal cells have larninated walls. In at least one sarnple, fungal hyphae were seen between the wall laminations. The presence of hyphae in this area would be difficult to detect. In thin- sectioned resin-embedded material, the exodennal walls ofien shatter between the laminations. These heavy cells walls also fluoresce brightly using suiforhodamine G, obscuring detail in LSCM examinations. The role of these cellulose-degradiig hngi in the roots of Pqhiopdifum and Phragnripdium should be exarnined in greater detail. Dressler (1993) suggested that the velarnen organisms were 'inviteci yests'. 1s there evidence of an 'invitation'? in a suil-poor environment, the mesh-like framework of secondary cell wall thickenings of the velarnen would provide both a habitat and protection from creatures larger than the velamen pores. Nutrient-rich run-off is believed to be captured and retained by the velamen until absorption into the root (Be~nget al. 1987). providing a rnoist, favourable and perhaps more stable environment for the growth of velamen organisms. Additionally, the abundance of bacteria and zygomycetes in the velamen suggests that simple sugars are available in the velarnen. Leakage of nutnents by the roots of other plant species has been well documenteci (Kapulnik 1996). In the mature root s of Paphiopudihm and Phragmipedium, leakage of sugars would probabl y occur via the passage cells of the exodemis, and may be responsible for the attraction of fùngi and algae to the velarnen above the passage ceils. Although most of the organisms isolated from the roots were readily cultured. bacterial isolates from these piants gradually bmeunculturable once removed from the orchid roots. The bacteria declined in vigor with each aibculture. to the point that they could not be transfened despite the abundance of live cells evidenced by a viability stain. This could be due to many factors, including the requirement of these isolates for specific cornpounds present in the rhizosphere but absent from the culture media. It is also possible that the bacteria were microaerophilic. In heavily colonized root velamen cells. respiration of numerous organisrns could reduce p02 and allow rnicroaerophilic bacteria to become established. When fim isolated, cells located in the middle of a large colony rnay have been protected fiom atmospheric oxygen to some extent - subculturing would have removed this protection. Bacterial colonies in the velamen could serve as food sources for protozoa (Anderson 1988, Nisbet 1984) and fungi (Barton 1988). both of which could benefit the orchid roots by providing wastes. or in the case of mycorrhizal fungi, directly supplying the nutrients obtained from the bacteria to the root cortex. Several genera of bacteria are known to provide fixed nitroçen to their associated plants (Puente and Bashan 1994) and others release plant growth promoting substances (Glick 1995). This research provides strong evîdence for a hi& Frequency of rnycorrhizal colonization arnong Pqphîopedilwn and Phragmipedium species. Pelotons were noted in most of the specimens examineci. Since only a small portion of each root system was sampled. this highlights the abundance of pelotons in the rwt systems. The role of Moniliopsis species in the roots of Prrphiopedilum and Phragmîpedîim is uncluv. While colonizations of the stele by Mondiopsis was infrequently seen, pelotons fomed by the hyphae of these fun@ were rarely degraded. and intercellular colonizations of the conex were common. Old hyphal masses formed from the ce11 walis of other mycorrhizai fun@ appeared flocculant in cells containing Moniliopis. It is possible t hat Moniliopsis spp. possess the ability to utiiize (and release?) the otherwise undegradable hypal ce11 wali components as an energy source. Moniiiopsis spp. were not able to form mycorrhizal associations with seedlings of Paphio~iIum.This was in stark contrast to isolates of L'orhiztested. which were almost dl able to form pelotons with the seedlings. The source of the organisms in the velarnen for plants which were propagated in sterile culture mua be in the environment of the plants, in this case, the geenhouse, potting media or water used in growing the plants. Mycorrhizal fungi already present in the environment of the plants mua be an important source of inoculum. Ushg other orchid genera (Bagyaraj and PoweU 1983) showed that mycorrizal colonkation of initiaiiy sterile orchid seedhgs would occur in greenhouses in the presence of other orchids. but time to colonization was hypothesized to depend upon the abundance og inoculum in the greenhouses. A pater number of organisms were associated with the plants of the Johnson Orchid greenhouse than the Bickell or U of G plant collections. This is an old orchid collection devoted to species of Puphiopudiium and Phmgmipediîium, which includes plants obtained prior to the CITES restrictions on import of mature plants or divisions from their countries of origin. Epiulorhka cj. inquilina was found exclusively in the roots of plants from this collection. As this species is not known from Canadian orchids (Cunah et al. 1997a, Zelmer et al. 1996), but has been recently described hmplants in the United states (Currah et al. 1997b), it is possible that this fungus has corne into the Johnston collection on the roots of wild-collected plants. In contrast, Epulorhiza aibertiensis has been isolated and described fiom native otchids in Alberta, Canada (Cumah et al. 1997a). where Johnston Orchids is located. Local Sphgmrm moss, used in the potting mix, may have been the source of the single isolate of this species. It is not known if the organisms that are found in association with the roots in greenhouses are the sarne as those associated with these plants in nature. Most of the organisms found in the velamen are of widespread distribution. However, the fbngi and aigae were not identified to species in most cases. At the level of species, some of these organisms may not be as widespread as suggested by their genera. Little is known about the root-associated organisms of Pqhiopediium and Phragmipedium in nature. There are reports of Prrphiopedihm species (P. primulimm and others (Cash 199 1)) growing in or below algd mats on cliff faces. In view of the high diversity of mot-associated organisms revealed by this research, it is interesting to consider what organisms are not componems of this 'cornmu~ty'that are common associates of other orchids. The lack of Ceratorhiia isolates is intereaing, particularly because this is one of the rnost common fiingal genera isolated fiom the roots of a broad range of temperate terrestrial orchids (Currah et al. 1997a). Isolates of Ceratorka have been obtained fiom the roots of greenhouse plants of Phaîaempsis, including stenle-raid seedlings planted out into the U of G greenhouse (unpublished data). Also interesting is the near absence of members of the Mycelium Radicis Atrovirens (MM) group. These fun@ have been isolated fiom a broad range of non- orchidaceous hosts (Jumponen and Trappe 1998). and are cornrnon on the roots of temperate terrestrial orchids (Cunah et ai. 1988). The lack of a soi1 substrate may be a factor in the exclusion of these very common kngi. Conclusions There is a high diversity of root-associateci organisms in the velamen of greenhouse specimens of Pophiopedihm and Phragmipediirrim. The organisms isolated fiom or seen in the velamen in this snidy include algae, cyanobacteria, bacteria, fun@ and protozoans. There is a lower diversity of organisms in the root cortices. The most common isolates from the root cortices were known or suspmed mycorrhizai fungi, including Ephrhizu and Mondiopsis species, and also the ascomycete Chaetomium. Priphiopediium and Phragmipediurn species are definitely mycorrhizai at maturity under greenhouse conditions. Ta bk 6.1 Pqhiopedilnm rad Pkrugmipedi~~mcollections. Species Green house PaphioprJiilirnr aJJIICII~ Allan BickelI Orchids Paphiopdil~tmciJJirc~tm Johnston Orchids f>aphiopudiI~~maffim Johnston Orcfüds PaphiopeJiwn appIeto~~i~mni Johnston Orchids Paphiopdil~tmarmc'11iac7tm Johnston Orchids Pophiope JiI~trnbr liai~tlwn Johnston Orchids Paphiopedilum bodegomii Allan Bickell Orchids Paphiopdil~mboxaiii Johnston Orchids Paphiopedil~~mcuIIo~ttm Johnston Orchids Paphiopdihm chambrrluiniamtm Johnston Orchids Paphiopedil~tmcharle~worthii University of Guelph Paphiopedilurn charlesworthii Ailan Bickell Orchids Puphiupe Jihm charlesworthii Johnston Orchids PaphiopeJiilurn concoIor Johnston Orchids Paphiopedil~tmhryi Ailan Bickell Orchids Paphiopedilum emersmii Johnston Orchids Paphiopedilum esqu iruki Johnston Orchids Paphiopediim ml Johnston Orchids Paphiopediitïrn glarrJt~lifrtum Johnston Orchids Prcphopedilurn glm~copiyli~tm University of Guelph Paphiopediium glmicophyiIllirm AlhBickell Orchids Paphiopedilum gratrixiamm University of Guelph Paphiopedilum gmtrixiarntm Johnston Orchids PaphiopeJitim haynaldiamim Johnston Orcfüds Pqvhiopediium henrymm University of Guelph Paphiopediàl~mhenrymm Johnston Orchids Paphiopediilm hirsïïitissimtcm Johnston Orchids Paphiopedilum hybrid seedli ng University of Guelph Paphiopedilttrn insigne University of Guelph Pcrphiopediitim insigne Allan Bickell Orchids Pcrphiopedltm insigne Johnston Orchids Paphioprdlttm Iawre~~ciamim Johnston Orchids Paphiopdiiwn marpetiemium Johnston Orchids Paphiupedium micranthm Johnston Orchids Paphiopediiwn nivmm Allan BickeIl ûrchids Pqvhiopdilum nivmm Johnston Orchids Paphiopdiitm pcnishii Allan Bickell Orchids Prphiopedilttrn phiiippineme var. Iaevigatttm Sohnston Orchids Paphiopedilum primulim~m Alla Bickell Orchids Paphiopediium primuIimm vat. Jm~m Johnston Orchids Paphiopedilm richardsoniamïm Johnston Orchids Paphiop Jihm rothchi/l Specimen Mate Fungus identity * Orchid identity Paphiopdiilm glmcophyilum anhrosporic dematiaceous fungus Paphiopedifiun, gImcophyffim Aspçirgillus sp. Puphiopedilum tnicrunthurn Aspergillus niger Phragmipdium caudatum Cephalospriiirn Phragmijxdiim c~erwiakmiawm Ceratocystis sp. Pqhiopedi fum victoria-mmiae Ceratucystis anmorphs Pcphiopediium vilIosum Cerarorhi:~sp. Phalaunupsis seeedng4 Ceratorhiza sp. Phdaenopsis seedlng4 cf E'lorhix sp. Phragmipediium cm&mcni cf Eplorhita sp. Phragmip4dium warscewiczia~iiim cf Eplorhilo repens Phragmipdium smgeniiamrm cf Eplorhiza repens PqplriopediIum insigne cf Eplorhiro reprns PuphiopPdlm imip cf Epulorhiza repens Paphiopedilum insigne cf Eplorhiz repens Phrugmipditm sargertrianrim cf Epulorhz repens Pqphiopediium gmnixiar~tlm cf Eptrlorhira repens Paphiopctilum gratriximm cf EQurorhira repens Pqhiopdilm insigne cf Epu forhiru repm Pcllphiopedilum sukhakulii cf Eplorhiza repens Puphiopdiltîrn gratriximnm cf Eplorhizo repens Phrugmipedititn calhimm cf Ejmlorhiza repens Puphiopediium sukhlii cf: Epdorhiizo sp. Phrasmipdium purptrrascens CJ Egulorhiiro sp. PclphiopedrIum wilhelminiae Chaeromitm sp. Paphiopedilum richmdmm Chaeromium sp. Puphiopedilum philippine~ise Chaetomium sp. Pcphiopedilm seedl ing @rimifimmx ?) Chaeromium sp. Puphioperüum glaucop~yllum Chuetorniun sp. Pqvhiopedilum philippinense Chwromium sp. Phragmipeditm fi~~dleya~nirn Chaeromium sp. Paphiopdilum marpettemrn Chaeromium sp. Paphiopediium concolor Cheromium SQ. Pqhiopdiium armeniacm Chaetomium sp. Puphiopedilm emersonii Chaetomium sp. Paphlopedilum grairixiarwn Ckrreomium sp. Pqhiopdihm coricofor Chetomium sp. Paphiopdlurn primulimim Chaetomium sp. Ptphiopedilum niveuni Chuetomium sp. Phragmipedium callumm Chaetomium s p. Pclphiopedilum rn~~quettearn~m Chetomium sp. Pc~phiopediluun,meniucum Chetomium sp. Pqhiopediium chrmiberiairiimm Chaetomium sp. Paphiopdilum crddic~um Chaetornium sp . Paphiopedilum primtdi~tum Chtwtomiicdm sp. Paphiopdilutn ~11~1idiumrrn Chaetomium sp. Paphiopediium emersunii Chaetornium sp. Pqhiopedil um ptimulimrm Chaetnrnittrn sp. Puphiopdiltim hirs~~itissimtcm Chaetornium sp. Paphiopediium phi Iippirierise Chetomium sp. Phturginipediunr scageoiia~nm Chtomium sp. Paphiopedil um enrl Chwtomium sp. Parphiopedihm adarcmm Cheromiim sp. Paphiopedilum primulimtm Chuetomium sp. Paphiopedilum henrymm Chaetomium sp. Phragmiipdium culIuunr Chaeromiutn sp. Pqh~opedilutniawrenciamm Choeromium sp. Paphiopedilum philrppimme Chetomium sp. Paphiopediurn hirstlitissimm Chaetomium sp. Paphiopedilum sanderianum Chetomium sp. Pqphiopedilum hymidiamrrn Clurrtomium sp. Pqhiopdilum armeniacm Choeiornium sp. Pqhiopdilurn vilfosum Chetomium sp. Pqhiopediium philippineme Chaeromium sp. Paphiopdihm hirstiitissim~~rn Chetmium sp. Paphiopedilum enrl Chaeromium sp. Paphiopedilum chclmberlai~~ianicm Chwtomium sp. Paphiopediium emersonii Chaeronium sp. Pclphiop~dilumtopperi Chaetomium sp. Paphiopedilum san&riam~rn Chuetomium sp. Paphiopdiium &cmm Chaetornium sp. Paphiopdilurn gratrixiamm Chuetomium sp. Pccphopdiium im*gne Chaetornium sp. Pqdtiopedilurn Iawremiicacm Choetomim sp. Phragmipdium caudoum Chaetomium sp. Paphiopediltim philippirtense Chaetomium sp. P@iopdilum rn~~quettearnrm Chuetomium sp. Paphiopediium primuIimm Chuetomiutn sp. Paphiopediium hqymldianum Chaetomium sp. Paphiopedt hmgratnatnximm Chaetomi~~msp. Phragmipedirim callm~rn Chuetomium sp. PqhiopdiIum i~w-pte Chetomium sp. Paphiopedilm beliatulum Chaetomiutn sp. Pqhiopdiium gratrmmm Chetomium sp. Puphiopedilum gratrïxiantlm Chetornium sp. Paphiopediiunt philippittense Ctrae~omiuntsp. P~hiopudiluni&cturn Chae~omiumsp. Pcylhiopedilum victoria-mîwiae Chuetorniuni sp. Pqhiopedilum insigne Chae~ornitmsp. Phragmipedit~mcau&ttum Chaetomium sp. Pqhiopedilum sarEde~imttcm Chaetornium sp. R Fusarium sp. Pqhiopdilutn hybrid Chaetomium sp. d Trichderma sp. P@iopedilum concolor Chuetomium sp.type 1 Phrapipedium hurtwegr r hessiae Chaetomium sp. type 1 Puphiopedi fum callos~rm Chwtomium sp. type 1 Phragmi'dium purprrarce.~ Chae~omiumsp. type 1 Paphiopdilum gfanduliffem Chae~omiumsp. type 2 Paphiopedilum victoria-regiltlle Chaetomium sp . type 2 Paphiopedilum victorieregwue Chwtomium sp. type 2 Phragmi'dium sorgentiant~rn Chaetomium sp. type 2 Pqhiopedilum victoria-regmae Chrietomium sp. type 2 Pqhioprd fum micran~hum Che~orniumsp. type 2 Paphiopdilum victoria-regriae Chwtomium sp. type 2 Pqhiopedilum gldli/erum Chaetomium sp. type 2 Phragmipedium wrge~~tiamrn Chaetomium sp. type 2 Phragrnippdum demetria Chmtomiun sp. type 2 Pqhiopedifum viciorru-regnue C'tomiump. type 2 Phragmipdium demetria Chaetomium sp. type 2 Paphiopedilum anneniamm Chaetomium sp. type 3 Puphioprdltm glardtdi$erim Chaetomium sp. Phragmipdium pqurarcerr~ Chaetomium sp. Phragmipedium hrawegri x bessiae Chaetornium sp. Phragmipedium &metria Chwtomium sp. P@iopdiIt~m anneniaa~rn -2.1 Chaetomium sp. type 1 Pttphiopediim gfmdulijenirn 1*2 Chaetomium sp. type 3 Phragniipdium lindleyamm 1.2 Chrretomit~msp. type 3 Phragmirpdium bo~sse~ianurn -1.4 Chuetomiurn sp. type 3 Ptphiopdiium esquiriolei -1.5 Chaetomium sp. type 3 Phragmi'dium purpurascens -3 -2 Chaetomium sp. type 3 Phragmipedium sarge~cnlrrn -5.1 Chaetornium (very curly) Paphopedilt~mglandtll/enm -5.1 Cisaezomium sp. type 3 PqhUopdifum armeniacum irv. 3 Chiara Pqvhiopdilurn marquettemm iw.2 Chcrima sp. Pqhiopedifum hirn~itissim~m irv. 1 Chaiara sp. Puphiopediltm marquefteamm iw. l Chcriara Pqhiopediium #ne irv. 1 ChIara sp. Pc~phiopediIumgratriximnrm -3.1 Chalara & Mo~~~~:iIfium Pcrphiopdiiuni armeniacum -2.1 Cldobotryum? sp. Pqhiopedilum wilkimir~iue -3.1 Clcrdobo~nl?sp. Paphiopedlm wiIheIminiae Colletotrichtim? sp. Pqhiopediium spiceriamm x henrymum Colletotrichum? sp. Pqphiopedilum rothschifdianurn Coiletotrichim? sp . Paphiopediilrrm rothschildia~~~m Unidenti fied conidial Paphiopediirm gimcophy Iitm Unidentified conidial Paphiopediium seediing (prnniiimm x ?) Unidentified conidial Paphiopediit~mghcophyllurn Unidentified conidial Paphiope Jiium gI~~~cophy/l/lrrm Unidentified conidial Paphiopedihm druryi Unidentified conidial Phrugmipdium waliacei Unidentified conidial Paphiopdiium dnrryi Unidentified conidid Paphiopdiium primulinum Unidentified conidial Paphiopeàilim porishii Unidentified conidial Phrugmipedium pearcii srna// pim Conicioid basidiomycete? Paphiopediium haynaidia~r~~rn Cyli&a.capon sp. Phragnipdium purpurmcem Cviindruçatpon s p. Paphiopediium cafiostim Cylindroapon s p. Paphiopedikim sc~nderiamrn Cylindrocarpon sp. Paphiopediltm hyhrid CyiirsriLacarpon sp. Puphiopediium topperi Cyii&iovn sp. Paphiopedium topw Cyli&ocaqwn sp. Phragmipedium ecuadorense Cylindrucarpon sp. Paphiopediiilm @IW Cyiindkocarpon sp. Phrupipediun, emadore~m Cylindrocarpon sp. Phrugmipdium ect~adoreme Cylinrirocarpon sp. Pqphiopediium hybrid Cyiindroccarpon sp. Phragmi'dium warscewic=ia?turn Cylindrocmpoon sp. Phr~~ptipeditimc=erw~iakouia~itim CyIirtCit.0carponq. Paphiopediilm s~~khakiiiii Cylinctiocatpon sp. Pag>hiopediftmwnderimninr Den&opnum cf: loba~um PuphiopediIrim wilhe Imi~tiae Den&oprium cf: iobatum Paphiopedilium wilheiminiae Epdorhiza-like, but with Paphiopediium primuiimm rhizomorphs Unidentified isolate Paphiopedium spicerimm EpIorhiza sp. Paph;opediIum spiceriam x henrymm EpIorhizz sp. Phrapipediwn iiradieyam~m Epdorhiza sp. Paphiopediilm grutrixia~~lm E;puiorhiza sp. ? Paphiopediium gratrixiwn~m Epiorhita albertiensis Phraginipedum ecuadoreiise Epiorhizu cjrepm Phrugmipdim boisseriamm Epulorhii=acf repens Phrugmipedium demetria Epulorhka cf repem Phragmipedium dernetrîa Epirlorhiza cf inquilina Phragmipedium cau&tum Eplorhiza cj inquilim Pqhiopdilum hirsuitissimum @ulorhia cj: inquiiina Paphiopedlum hirsuitimhtum Epdorhiira cf: inquifina Puphiopedilt~mhirst~itissimm Epdorhizo cj: iriqdima Phragmiipedium cauciatfirn Eplorhiza cf: iiiquilim Phragmipdiurn cuu&tum Eprlorhiza cf: inquilim Paphiopedilum hirsuitissimum Epdorhim cJ inpilim P~hiopedrlumhirstlitisimm Ejmlorhiza CJ irpilim Phragmipdium catirlatrum Eplorhiza cf: repens Phragmipedium hartwegr x bessiae Epdorhiza inquilim Paphiopedlum chmlesworthi1 Ejmlorh inquilim Pqhiopedilum charlesworthii Epulorhia inquilina (?) Pophopedilum esquiriolei Eplorhizu inquilina p) Puphiopdilm esquiriolei Epulorhiza inpilirw 17) Paphiopedilum topperi Eplorhi=ainquifina (?) Paphiopedilum callosum Wlorhira inqtdim (3) Pqhiopedilum esquiriolei Eplorhiza inquilina (3) Puphiopedlum toppi Ejmlorhiza inquilim (3) Paphiopedilum esquiriolei Epulorhiza inquilim (7) Phtagmipdium cailuncm Egulorhirci inquilina (3) Puphiopedium niwum Epulorhim inquilim (3) Paphiopedilum vettustum Eplorhiira inquilina (3) Paph~opedilumesquiriolei Epulorhi-0 inquilina C>) Pqhiopedilum caliosum Epulorhizz inquilina (?) Pqhiopedifum topperi Eplorhiza inquilim (?) P denotes potentidly syrnbiotic fun@ 6 included for cornparison. Collecteci at University of Guelph greenhouse. Chwtomiwn types: type 1- hghtoaiolar hyphe; type 2 - curly, type 3-coiled Tabk 6.3. Imeulation of Popkiopcdil~rnseedling with Rikbcfonia zL Mates Paphioprdilum hybrid seedlings on oat agar plates 40 days poa inoculation % with total cleared pelotons isolate identity rep 1 rep 2 protocorms originally isolated fiom: 209-2 @uIorhka sp. O 25 5 Cjpripdium regime Ëplorhi=a sp. 100 Cmdiumregime Eplorhizu sp. O Cyptipedium pan,iflon~m c.J EpIorhiza 1 O0 Phrcg purpurawe~~s %matephrus pnmtus O Phrogpupr~scem Eplorhi=a cJ inguilim 100 Pwh hi~min'm+mum Ejnilorhiiro cg repn O Phrug demetnu Epulorhio c.5 inquilim 1 00 Puph esquiriolei Moniliopsis solani O Paph espiriolei EpuIorhiito CJ repens 1 O0 Phgsugentimum Epulorhira albertiensis 40 Phrag edorenw Moniliopsis sp. O Pqph col los in^ Epulorhiza sp. 1 O0 Phrug harnuegi x bessiue EpuIorhiza c.J repens O Paph charlesworthii cJ Epulorhiza 100 Paph wilhelminiae EpIorhiza cf:inpiIi110 1 00 Phgcauthturn Moniliopsîs sp . O Pqh suwtakulii Epulothiza cJ repens 100 Pqvh sukhakulii Qulorhizo CJ inguilim 100 Pqph toppen c.f: @ulorhizu 100 Puph villosurn Eplorhiza repens 100 Pqh villostim îkmtephoruspmutus O Pa@ primtrIimm Moniliopsis sp . O Paph victoria-manae cf:Epulorhiza 100 Paph gratrixianum E;pu/orhizt c.J repens 100 Paph gratrixiunum Epulorhca cJ inquilim 1 O0 Phrag dumm Eplorhiza cg repens O Piph insigne Qttlorhizo sp. 100 Pqph hynuïdiamrm iImiritephoruspenmn~s O Paph bellrmrlrrm c.j EHorhira sp. 100 Paph be1lutuIum Tibk 6.4- Algae and cyrnobicterii isolatcd fmm the roots of Pqkioperlrum rad plants with Algal genudgroup U ofG Bickell Paphs Phrags ûthers > 1 species no no no yes- A&S no no no no no yes- A&S Micrucystis no Nosrai yes-A Osci Iiatoria no Scenedesmus no Scotell~opsis no Scyronema no Sticharaccus no Sylec~occtlsScystis no Tolpthrix no U of G, number of plants at the U of G greenhouse from which the genudgroup was cultureci (of a potential 10 plants) Bicke& nurnber of plants at Al Bickeii greenhouse fiom which the genudgroup was cultureci (of a potential22 plants including &phronitis wetigei and Angrwcum didieri Paphs, algae were cultured from the rwts of Pclplhiopedilurnspecies Pbmgs, aigae were cultured from the roots of Phrapipedium species Other, aigae were cultured fiom the rwts of non-Cypripodioideae piants (Angrueam diderei and Sophronitis wetigei) Plants with >1 kind, number of plants in which more than 1 species in the algal genus was present Tabk 6.5. Ciliatd protozoaas isolatd from the mots oîP~~iopedifun, rad Alan Bickell UofG Orchids* Greenhouse** Spccits Paphs Pbnp Cui@ ctaIhs-li ke 1 O CoIpoda injiata 1 O Colpodrr mar~pai~i-li ke O 1 Coipada spp. 1 O Cotpodrr steinii 6 1 Coi@ skinii-like wlo nucleolar 1 O mas Number of plants from which each ciliate group was isoiated Paphs = Paphiopediium spp. Phrags = Phrogmipdium spp. * of 20 plants ** of 10 plants Figures 6.1 to 6.6. Epufdiui adrtes firom tbe mats ofPickiopadilrrm and Pkmg~ediummots. Seiaed with icid fucbsin in Iiçtogîyccml; ligbt micmcopy. Fig. 6.1. @aIorhi=4 repm isdated fiom Pclpho~~dilumhirsuitissimum. On potato dextrose agar (PDA), 3 months post inoculation. 277x. Fig. 6.2. E albertiensis isolated Phrugmipedirrm epadorettse. PDA medium, 3 months. 316x. Fig . 6.3.E. cj inquilim isolated fiom Paphiopedihm chprlesworthii. PD A medium. 3 months. 300x. Fig. 6.4.E. cf: inquilm monilioid cens isolated fiom Pqhiopdiium chrrriesworthii. PDA medium, 3 months. 300x. Fig. 6.5. E cf: impilill~monilioid ceUs isdated fkom Prrpihiopditum caII0s11m. PDA medium, 1 mont h after isolation.320~. Fig. 6.6. E. mguilim monilioid celis isolated Pqhio~umcaIIosum. PDA medium &et 4 years of serial tders. 26ûx. Figum 4.7 to 6.14. Fungi irditat from, rad in situ in the mots OC P4phiopcdil~m and PAic~g~~mspecies. Fig. 6.7. Cotoriization of cortical cclls of Puphiopdihm sonderiamm by an Epu'orhiiro-likc fiingus. Rcsin cm-: kser scainin$ CO(lfOcal micro~copy;stained with SulModaminc. 20h. Fig. 6.8.Moniliopsis solani isolated from Puphiopodfum esquiroloi. PDA medium 5 rnonths. Staurd with acid fùcbsin in baoglycerol. 305x. Fig. 6.9. T. pnlt4m.s anamorph noni Puphiopodilunr charlesworthii. PDA medium, 6 months. Siaùrd Hith acid hichsin in lactoglycnol. 295x. Fig. 6.10.Moniliopsis-like fiingus (m)coloaiàng root cortical celk of Puphiopedilum henrynum. D, dolipore sepa. Resh emkddsd; stained with tolui~blue O. 2OOx. Fig. 6.1 1. Apparent myconhizal colO(ljZati011 of a T. pnmhrs-üke hlll&US. Phragmipdium boisserianum. Reoin ernbedded; staLKd with toluidine blue O. H, Hypha; DP, digened peloton. 2OOx. Fig. 6.12. Paiariosm of Phmgmipdim boisemm4m(mby a T. pnnt~n~s-ükefuagus. Nehyphae withm adodmnal and pericycle œUs. Resin emkddcd; staineû witb tduidine blue O. P. Phlmarrows, hyphae. 200x. Fig. 6.13. Cotmctim betswen velamen and cortical hypbae via passage œUs. V, velinrn: C, cortex: E>C exOdermis; PC, passage cel); arrow, hypbae. Pa@iopedilum Imtren~iunum~ 1SOx. Fig. 6.14. Coiled ceIls of fuagus m vekmen of Paphiopdilum pnmulinum.. LSCM image: nmnstmcî.ced Z Series, min embedded material stained with sulforhodamine G. 2Mk. FiIres 6JS to 6.21. Coüd cdb wWin vdimm and cortex (TEM images), fungai cokmiDltio~of corta in the mots oicpPkiopa~mrpccia. Fig . 6.15. Velamen cell of Ptphiopedilum primlimcin containhg wiled fùngal cells. H, coiled hypha; CH, central hyphe: V, velamen cdl wall. TEM microscopy. 12ûûx. Fig. 6.16. Detail of laminated will in coiled hyphae.Note electron-dense Iens-sbpeâ inclusion in the ceil wall. TEM microscopy. 28,000x. Fig. 6.1 7. Laminate walled hyphae (amows) in peioton of P@iopedihm @muiimrm. Note lobed nucleus (N) with prominent nucleolus. 250~. Fig. 6.18. Imperforate parenthome (arrows) in dolipore septum of coiled hyphal cells 6om velamen. TEM microscopy. Paphiopsdiiurn pn'mufiltum. 35,700x. Fig. 6.19. hail of imperforate parenthosorne (Fîg. 6.18). TEM microscopy. Pqhiopediiunt primrriimrm. 48,000~. Fig. 6.20. Hypha with IamiRated wall pashg hoan adjacent velamen ceii. Note the reduction of the hyphal cd Wall at the point of petration. TEM microxopy. Pophopdilum prinnrlinum. 28,000~. Fig -6.21. Hyphal ceii wdof similar hyphae in the peloton of P. priniulinum. TEM microscopy. Lens-shsped inclusions (anows) are less prominent but still evident in this partiaily digested hypha. 28,000x. Fig. 6.22. Isolation of major fimgal groups hmvelamen, whole mot, and cortex samples fiom the roo ts of Paphiopedilum and Phragmipedium species (Johnston Orc hid P~W Figures é.23 to 6.2û. Fungi, bacteria aiid cynobactcrt fmm the mots of PapkiqrcdiIum and l%mgm@dum. Fig. 6.23.PestaIotiopsts sp. conidia Isolated from Pqhiopedihm vicforia-repue. Stained with trypan blue; Iight microscopy. 36ûx. Fig. 6.24. Denuhpriirni iobatum conidia (arrow) and conidiophores. Isolated from PaphiopediI~~rnwiIkiminiiw. Stained with acid fichsin in lactoglycerol; light rnicroxopy. 36ûx. Fig. 6.25. Bacterial cells (arrows) in velamen in close contact with exodemial (Ex) and passage ce11 (P) tilosome (T). Phragmipdium boisseriamm. Resin embedded, aained with toluidine blue O; light rnicroscupy. 50x. Fig. 6.26. Nostoc qp. fiom P~hiopdihmparr'shti. Live; unstained; light microscopy. 2oOzt. Fig. 6.27. Arnhem sp..from Paphiopediium insigne. Live; unstained; light microscopy. 200x. Fig. 6.28. Siytonemu sp. from Paphiopedium wermshlm. Live; unstauied; light microscopy. 2m Fiim 6.29 to 6.33. Alge rad Cyinobicterir irolrtcd frwi the mots of Pcy~khpeu&m and Inmgmipeuhm species. Livt mitend: uastaiad: ligbt microrcopy. Fig. 6.29.Lyrgbya sp. fiom Pqhiopediium stonei. 200~. Fig. 6.30. Oscillattwia sp. âom Pqhiopedutn hkgomii. 2OOx. Fig. 6.3 1. ChloreIIa-iike cclls from Paphiopedilm pishii. 2OOx. Fig. 6.32. Scenedemus (mow)fiom PqhiopPdiun, cklesworthii. 200x. Fig. 6.33. Scotiellopsis (Iive, and ceil wall or@) from Puphiopdium wordii and Pqhiopedlum spicerimm 'Sabot Mauve' JC/AOS x P. henryamim. 2600x. Fiires 6.34 to 6.4. Pqkiopdum and [email protected] onganisms. Figs 6.346.36 and 6.384.JO: Rain embeddd; sfiined witb rulforbodamint G; vhdwith Iwr sunning confoul microscopy. Fig. 37: min embeâdeâ, sec~ioned,stained with toluidiae blut O, viewed witb ligbt microsropy. Fig. 6.34. Algae and cyanobactena on surface and within velami of f'hragmipediim coricimm. Note sheaths on Nostuc-Iike cells (arrows) and mwnlarnents, iikeiy Lyngbp. 5ûx. Fig. 6.35. Algae and cyanobacteria in deeper velamen layers of Phragniipedium waiiucei. Arrow, Nostuc-lie cells. 75x. Fig. 6.36.Rivuimà on velamen surface of Phrupiiperirumpec~cei. 36x. Fig. 6.37.Cyst-like bodies (arrows) in embedded and seaioned velamen of PcllphiopedIum ~11Wtakuiii.45x. Fig. 6.38. Ciliated protoman (Coi@ sp.). Cilia are not stained by suIforhodamine G, and so are not seen in this image. 45x. Fig. 6.39. Organisms in the velamen of Pqphiopedium vemsttrm. Arrows indicate amoeba-like celis. 40x. Fig. 6.40. Encysted organisrns, possibly protozoans, in the velamen of Pclprhiopediium spiceriamm. 50x. Chapter 7. General Conclusions Introduction At the outset of this thesis and in the introductions to the chapters that followed, key areas for exploration of the mots and root-assuciated organisms of the Cypripedioideae were identifieci. These included the (re)examinations of root anatomy as it related to colonization of mycorrhizal and other organimis, locaiiition of associated organisms on and in the roots of wild and cultivateci mature plants? isolation, culture and identification of the organisms, and experimmtation aimed at characte~ngthe associations. This chapter highiights the major findings of the thesis as they relate to these key areas. Root anatomy relative to colonization by mycorrhizal and other root- associated organisms. The mature roots of C'ripedium acaule, CC.regime. C. CQndjrhrrn, C.pa7~flonim and C. arietimm all tended to develop dark mot epidennises due to deposits of pigmenteci material on outer tangentid epiddceii walls. in some species, the outer walls were dso ligded. These dark olds mots were still live, heahhy and often containeci many hgaipeiotons. EpiddceIl shape and Ne relative to the underlying aaddce11 layers was süniiar in four C'pedium species but not in C. trierinum. in this species, a large epiderrnai cell may faciiitate colonization of the smaller diameter roots. An exdermis (with Casparian band) composed of 'long' and 'short' (or passage) cells was also seen in Cypripedium roots, although the dinerentiation between the two ceii fomwas not as evident as in the Pqphiopedhn and Phragmipddium roots. Although rnycorrhual fùngi passed through the epidennis (C'dim)or velamen (Puphiopedihm,Phragmipedm) without the formation of appressoria, colonization of the cortex generally occurnd only via the short cells of the exodemis. Cortical cells in al1 species accumulateci large amounts of starch, which tended to decrease after colonization. However, colonized cells, particularly in early peloton formation, often possessed starch grains. Glutaraldehyde Ncative was employed before the usual procedures to clear and stain roots for fun@ observation. This technique preserved the integrity of the nuclei even after clearing, and so pennitted an estimation of cortical cell viability during colonkation by fùngi. The observation that non-mycorrhizal fungal colonization often accompanied degradation or dissappearance of nuclei in colonized cells cwld fom the basis for distinguising fimg al pathogms fiom mycorrhizal fiingi. Root-associated organisms of wild Cypripedium plants Al1 Cjpripediurn species examineci in this study were associated with rnycorrhizal fungi at rnatwity. Roots sampled from mid rhizome that lacked evidence of fùngal colonization were rare, and were generally shorter than colonired roots. Colonization of individual mots varieci, but overail wlonization levels for many collections were quite high. Revious reports oflow mywrrhizail coloiiuation levels may rdma tmdency for researchers to collet Young, light-coloured roots, which generally contained few pelotons. Muhiple degraded pelotons in the cells of older mots indicated that recolonidon ofroot ceUs was common. Several stages of peloton formation and breakdomi oAen coa The roots of C'ipdiumspecies were also datedwith a diversity of organisms repwenting several trophic levels and ecologid fimctions (e-g. nitrogai fixation, primary production). Potentially mycorrhizal and siterile, dark septate tirngi. bactena (especially Gram-negative rd-shaped celis foning mucoid colonies); unicelluiar and filamentous algae and cyanobactena and ciliated protomans dl were observed on andfor isolated fiom the daceand interiors of wild-collected Cypripedium roots. Moa organisms isolated from the mots were cornmon soi1 or water organisms. There was linle preliminary evidence of a unique assemblage of organisms on the roots. However, many of the hngi and bacteria isolated &om the mots were short-lived in culture. This suggests un-met requimnents for nutrients or conditions provided by the orchid rhitosphere. Bacterial isolates fiom the orchid roots affecteci the growth and adval of lettuce Kedlings nt vitro, and some appearrd to be plant growth prornoting rbuobacteria. Additionally, one bacterial isolate (of two tested) influenced the growth of a potentidly mycorrhizal fimgus. Root-associated organisms of greenhouse specimens There was a high diversity of root-associatecl organisms in the velamen of greenhouse specimens offaphiopedilum and Phrcpipedium. Environmental conditions and age of individual greenhouses may have a greater influence on the species composition of the root-associated organisrns than does the orchid spacies. Pqhiopedilum and Phrugmiipedium specimens in the same greenhouse had sirnilar organisrns associated with them. The organisms isolated fiom or seen in the velamen of Paphiopediilum and Phragmipdium specimens included algae, bacteria. cyanobacteria, hngi and protozoans. While the velamen was a rich habitat for organisrns, the diversity of organisms in the root cortices is low. The most cornmon isolates from the mot cortices were known or suspecteci mycorrhizai fiingi, including Epulorhiria and Moniliopsis species, and aiso the ascomycete Chwromium. PuphiopediI~îmand Phrapipedium species are commonly myconhizai at maturity under greenhouse conditions. Root-associated organisms can interact with stenie-raised seedlings Several root-associated îùngi isolated fiom mature Cypripeditrm, Puphiopediilm and Phragmipedium were used to inoculate sterile-raiseci protocorms of C'ripedium regnuTe or a Paphipedilum hybrid. While di of the Qwlorhiza isolates tested were abie to colonize at least a few faphiopedihm protocomis unda sorne conditions, Monilopsis isolates rarely succeeded in foming a mycorrhizril symbiosis with the protocorms. In an experiment using three fùngal idates on ten ciiffixent substrates, the abiility to colonize protoconas, the fatures of that colonization and fiequency of protocorm mortality were différent for each of the fungal isolata on the various media. The establishment of orchid mycorrhizai symbiosis between C. reginae protoconns and these fun@ isolates are therefore both isolate and medium dependent. The inoculation of sterile-hsed autotrophic Cypripdium regme seedlings with root-associateci orgMsm at the time of unfiasking had effis that were detectable after two growing KasOns. This experiment, perhaps the largest conducteci to date with Cypripdium, utilized four root-associateci organisms (l fimgal isolate, 2 bacterial isolates and a cyawbacterium) singly and in various combinations. The number of roots possessed by plants at the end of the second growing season was significantly different between treatments, and appears to have been atmbutable to both fùngal and bacterial inoculation, Pooled data showed that plants inoculated with the fungai isolate (Quiorhiirci sp.) had significantiy higher shoot phosphorous concentrations and root numbers than unindated plants. The effea of bacterial inoculation varied with the isolate used. Bacterial isolate 3 16b- I positively afkted harvest weights, shoot wet and dry weights and numbers of roots whereas the only significant effect of inoculation with isolate 3 18b-6 was a reduction of shoot dry weight. An effiof the cyanobactenum on the secdliags was not detected, but the persistence of this organism after inoculation was uncertain. Implications of this work Membm of the Cypripedioideae are colonkd by a large number of mot-associated organimis, including bacteria, cyawbacteria, aigae, ciliated protozoa~~~and ftngi. in the genus C'n'pedium. root-Bssociateû orguiisms are mostIy restricted to the surface and a single epidennal cetl layer of the mot, while in Puphiopediium and Phragmiipdium, root- associated organisms inhabit the dead, many-layered epidennis known as the veiamen. hica organisms remain extemai to the cortex of the rwt. For the orchid grower, this means that mot-associated organisms an vulnerable to cultural practices, since they lack the protection fiom the environment that root-inhabiting endophytes wodd receive. In C'dium,Paphiopedium and Phragmipedium, fw organisrns penetrate the exOdermis of the root to coloniÉc the cortex, and the grand rnajority of thox that do are mycorrhizal fùngi. From this research it is apparent that mycorrhizsl fiuigi are mmmon within the rwts of Cypripodioideae and are part of their ecology emi at rnaturity and under greenhouse conditions. Under the conditions of this research, Epulorhiro spp. were the most commody isolated mycofthizal fùngi associated with wild Cjpripedium and cultivated Pqphiopedilum and Phragmipedim plants. There is much interest among hobbyists and commercial hortidhualias in growing C'ripedium species in culture, but despite the hundreàs of seeds Dasked each year, relatively few result in seedllligs established in soü. This appears to be due to two main factors. The fint is the low germination of seeds of rnany C'n'pedium species on the available media formulations. This dlundoubtabiy improve as modifications of methods are made and as our understandimg of the &ors wntrolling germination in Cjqwipedium seeds develops. The second reason haî cultivated Cypripedium seedlings are rmly seen is the high mortality of the seedluigs when brought out of sterile culture. Again, as methods for unfiasking continue to develop, this situation wiU undoubtably irnpmve. Howewr, one of these improveînents may be the integntion of other organisms into the propagation qstem for C'ripdium. Inoculation by two of the organisms (a bacteriurn ada mycorrhital fiuigus) collectively resulted in a grrater numben of roots, highet hawea and shoot weights and higher shoot phosphorous concentrations. These effects did not lead to significant differences between inoculated and uninoailateci plants in survival over the relatively short-term period of study (relative to the lifespan of C'ripedium plants). However, as ~eenin Chapter 2, there are large starch reserves associated with each root, and thedon a higher nwnber of roots Mght lead to pater long-term su~vai of indated plants. Similarly. the phosphorous accumulations in the mycorrhiul seedlings did not affect th& short-term su~val,but phosphorous accumulation is important in the onset of maturity and flowering. Based on the rd&of this study, 1 belim that inoculation of C'pedium seedlings with wekelected rwt-associated organisms at unflaskïng could be an aid to the growth and SuMval of sterile-raid plants. The mot-associated organisms for the inoculation experiment were selected by criteria relating to taxonomie grouping (ie. EpcIwhitcl) or demonstrateci ability to inhience the seeâiings of a rapidly-grown but unnlated plant (ie. lettuce). The existence of an efiêct of inoculation using isolates selmed for these very basic traits should open the dwr to optimization of the effècts by new, more targeted seledon processes. For example, selection of bacterial nrallis using seedlings of more quickly raid orchid @es, aich as Spirmtks spp., may result in the choice of bacterial strains that show a greater growth enhancing eff~on C'pPdium than those screened using lettuce Seedlings. Siariy, hgalcultures within EQuIo1hin could be screened for ability to promote growth and phosporous accumulation in Cypripedium seedlings. As seen in Chapter 4, the medium on which the seedlings are raidcan affect the ability of mycorrhual fiingi to colonize and interact with the dlings, so the effiveness of a myconhizai hgus may not extend to media that is different from the screening medium. Cult ivated Pqhiopedilum and Phragmipedium specimens, due to the habitat provided by the root velamen, have especially rich and extensive root calonitstions. Are these the same comunities of organisms found in the wild orchids, or just substitutes for a different and important assemblage? The root-associatecl organisms of wiid plants in these two genera are as yet unknown, and the opportunities to study these plants in their natural habitats are disappearing. Propagation of Polphopedilum and Phragmipedium from seed is accomplished in sterile culture, wfüch physically separates the seedlings fiom theù natural root floni and fauna. This research has demonstrated the abiiity of root- associated organisms to influence seedlings of Cpripdium regme, which offers a liited habitat for root colonizers. Wtth their more extensive (and weli-colonized) velamen habitats, it is possible that root-associated organisrns play a larger role in the ecology of Paphiopedilum and Phragmipdium. Similar isolation and inoculation work on plants of these genera in their counaies of origin and utiliPng natural substrates would be invaluable, and could also lead to improved methods for propagation and restoration. We are jua beginning to understand the roles that root-associated organisms play in the vitality and We cycles of wiid orchids. We must ensure that meanires to conserve the slipper orchids also consene their symbionts. This wiil be difficult where the associated organisms remain unknown. Appendix 2.1. New pelotons Appendhc 2.2. Intact pelotons I Intact / pelotons \O- I m- Appendu 2.1,2.2. Compahrons of peloton stages observed in roota of four Cypr@edimapecies. Superimposed pie charts. Each ring of the chart represents the total nuiber of 1 cm segments sampled fiom that species. Colored bars indicate the percentage of segments of each species that possessed the indicated peloton stage. The sizes of the superimposeci pie chats are not informative, and serve ody to facilitate a cornparison of percentages between spies. Appendix 2.3. degraded pelotoas Appendix 2.4. Co Uapsed pe 10 tons I : Collrpsed pelotons Appendu 23,2.4. Cornparhm of peloton stages obsewd in mots of four Cypr@dium specics. Superimposed pie charts. Each ring of the chart represents the total nvrrlber of 1 cm segments sampled fiom that species. Colored ban indiate the percentage of segments of each species that possessed the indicated peloton stage. The skof the niperimposed pie charts are mt informative, and serve ody to facilitate a comparison of percentages between pies. Appendù 4.1. Numbers of protocorms in each pebton abandance char by medidfimgal Matecombbrtioa. Experimcnt 2,80 days. Each small pphrepresents a single medium, upon which were combined C. regince protocorms and one of three fungal isolates. W'ieach graph, the x axis bas five abundance catagories (fiom left to nght): no pelotons observed, a few pelotons, moderate number of pelotons, large numbers of pelo tons, almost every cell colonized. The bars indicate the total number of protocorms in each of abundaoce class with aU isolates. Stacks within each bar indicate the conhiion of each isolate to the totaL Literature Cited Albert, V. A. 1994. Cladistic relationships of the slipper orchids (Cypripedioideae: Orchidaceae) fiom congruent morphological and molecular data. Lindleyanu 9: 115-132. Alexander, C. E., 1. J . Alexander, and G. Hadley. 1984. Phosphate uptake by G@ra repens in relation to mywrrhizaî ideaion. Niw Phyofw'sf 97: 40 14 1 1. Alexandrow, W. G. 1925. Uebet Plastimot der Biattstmktur bautartjger PBamen. Zeitschrvt firr die gestllllte Bofanik 12: 290-3 79. Andenon, k B. 1990. Improved germination and growth of rare native Ontario orchid specia. Pages 65-73 in G. M. men, P. F.J. Wes, and S. D. Rice, eds. ConseMng Cmdinm C'.University of Waterloo Press, Waterloo. -. 1992. Symbiotic and asymbiotic germination and growth of Spiranthes magnicmpomm (Orchidaceae). Lindleycirr 6: 183- 186. Anderson, R 0. 1988. Comparative protozoollog: ecology. physoioogy and Ive histo~. Springer-Verlag, New York. Arditti, J. 1992. Fun&anenta&sof wchid biokgy. John Wdey and Som, Inc., Toronto. Atwood, J. T. J. 1984. The relatioaships of the slipper orchids (subfdy C'ripedioi&œ, ûrchidpceee). Sefbycmrr 7: 129-247. Azcon, R 1993. Growth and nutrition of nodulateci mycorrhizal and non-myconhizal Hedymm coromrium as a result of treatment with fracuons fiom a plant growth-promoting Rhiwbacteria &il Biofbgy md Bitxkmiisny 25: 1O3 7- 1042. Bagyaraj, D. J., and C. L. Powell. 1983. Ocairence and uitetlsity of mycorrhizai infections in cultivateci orchids in some New Zealand nurseries. New Zeui' Jm~nwlof Agrictlh~ruilResewch 26: 409-4 1 2. Barron, G. L. 1988. Microcolonies of bacteria as a nutrient source for lipcolous and other fungi. CardianJourmi of Botany 66: 2505-25 10. Beardmore, J., and G. F. Pegg. 198 1. A technique for the establishment of mycorrhizai infection in orchid tissue grown in aseptic culture. New Phyro~@s~87: 527- 53 S. Bellone, C. H., S. D. V. C. De Bellone, R. O. Pedraza, and M. k Monzon. 1997. Cell colonization and infiedon thread fomtion Ui sugar memots by Acefubucrer diarotrophicus. &il i3ioIogy cad Biochemiw 29: %5-%7. Benzing, D. H., D. W.On, and W. E. Friedman. 1982. Roots of Sobralia nactuntk (Orchidaceae): structure and fimaion of the velamin-exodennis cornplex. Amen'm JimtnaI of Bokmy 69: 608-614. Bergmah B.. A N. Rai, C. Johuisson, and E. Soderback. 1992. Cyanobactaial-plant symbioses. S'biosis 14: 61-8 1. Bernard, M. N. 1904. Recherches expetùnentales sur la orchidees. Rewe Générde de Botmique 16: 405451,458475. Beyrle. H. F., S. E. Smith, R L. Peterson, and C. M.M. Franco. 1995. Colonization of Orchis morio protocomis by a mycorrhizal fungus: effects of nitrogen nutrition and glyphosate in modifjing the responses. Cimadia Jarrml of Botmry 73: 1 128-1 140. Brundrett, M. C., D. E. Enstone, and CC.A Paerson. 1988. A berberine-aniiine blue fluorescent stairting procedure for suberin, lignin, and dose in plant tissue. Protoplclsma 146: 133-142. Buttefielci, B. G.. B. A Meylan, and R. R Exley. 198 1. Intercellular protuberances in the ground tissue of CUCOSnucifeta L. Protop141~ma107: 69-78. Cash, C. 199 1. n>e SIpper Otchids. Timber Press. Chu, C.-C., and K. W. Mudge. 1994. Effects of prechilling and liquid suspension culture on d germination of the yellow Lady's Siipper orchid (Cyprepedum calcedus var. pubescens). LindIeyrnia 9: 15 3- 159. Clements. M. A., H. Muir, and P. J. Cnbb. 1985. A prelunlliary report on the symbiotic germination of European tenestria1 orchids. Kov Bullern 4 1: 437-445. Cnib, P. 1997. i%e genus Cjpriipedum. Timber Press, Portland. Currah, R S., S. Hambleton, and E. A. Smreciu. 1988. Mycorrhizae and mycorrhizal fungi of Caf)(psobufbara. Antericm JmmI of Bo- 75: 739-752. Currah, R S., and C. D. Zelmer. 1992. A key and notes for the genera of fiuigi myconhVal with orchids and a new @es in the genus @ulorhirri. Reports of the Tonon MycoIbgic4iInninte 30: 43-59. Cunah, R S., C. D. Zelmer, S. Hambleton, and K. A Richardson 1997a. Fungi fiom orchid mycorrhitas. Pages 1 17-1 70 Ui J. Arditti and k M. Pridgeon, eds. Orchd bioIogy: reviews mdperspectives. Kluwer Academic Publishers. Currah, R S., L. W. Zettler, and T. M. Mclnnis. 1997b. Epulorhira rquilina sp. nov. fiom PIantantherro (Orchidaceae) and a key to Qwlorhka species. Mycotrmtn LM:335-342. Curtis, J. T. 1939. The relation of specificity of orchid myconhizal fÙrtgi to the problem of symbiosis. Amerkm Jmmul of Botq26: 390-399. -. 1943. Germination adsadling development in five species of C'riipedum L. A rnerican Juidr~~iO/ Botany 3 0: 199-205. DePauw, M. A., and W. R Remphrey. 1993. In vitro germination of three C'ripedium species in relation to time of seed collection, media, and cold treatment. canadiru, Jmî17~11of Bofq7 1 : 879-885. DePauw, M. A., W. R Remphrey, and C. E. Palmer. 1995. The cytokuiin preference for in vin0 germination and protocorni growth of Cjpripedim ernddum. Antials ofBotq 75: 267-275. Dong Z., M. J. Canny, M. E. McCully, M. R Roboredo, C. F. Cabadilla E. Ortego. and R Rodes. 1994. A nitrogen-fixing endophyte of sugarcane stems: a new role for the apoplast. Plmf Physiology 105: 1139-1 147. Dressler, R L. 198 1. The orchids - mtwul histq and cI;asnficatim. Harvard University Press, Cambridge, Massachusetîs. -. 1 993. Phyfogenyand clasrr~cationO/ the orchidfatmi&. Cambridge University Press, Cambridge. Dumas-Gaudot, E., S. Slezack B. Dassi, M. J. Pozo. V. Gianinazzi-Pearson, and S. Gianina-/;ti. 1996. Plant hydrolytic enzymes (chitinases and B-1.3-glucanases) in root reactions to pathogenic and symbiotic rnicroorganisms. Phund Soil 185: 21 1-221. Durkee, S. 2000. Cjpripedium acaule and Cyprip~diumregime. Orchich 2000: 864-869. hssauit, L., M. St. Arnaud, D. Barabe, and J. A. Fortin. 1998. E@kts of Epulorhia repens on C~diumacaule (ûrchidaceae) xcd germination. kond Intemtionul Confrence on Mycorrhisw . Swedish University of Agricultural Sciences, Uppsala, Sweden. Dycus, k M., and L. Knudson. 1957. The role of the velamen of the aerial mots of orchids. Botunical Gazette 1 19: 78-87. Ekelund, F., and R ROM. 1994. Notes on protozoa in agricultural soi1 with ernphasis on haerotrophic flagellates and naked arnoebae and their ecology. FNS MicrobidogV Reviews 15: 32 1-3 53. Esau, K. 1977. Amtomy of seedplantsSJohn W1ley and Sons, New York. Esnault. A.-L., G. Mesuhara. and P. A. McGee. 1994. Involvement of exodermal passage cells in myconhizal idhion of some orchids. MyfoWcai Research 98: 672- 676. Fuchs, A.. and H. Ziegenspeck. 1926. Entwicklungsgeschichte der Axen der euiheimischen Orchideen und ihre Physiologie und Biologie. Zeitschn~jkdie gesrmrte Botmik 14: 166-260. Won, J. R 1 992. Tansley Review No. 44. Reconciling the incompatible: N2 fixation and 02. NOY Phytohgis~122: 571-609. Garbaye, J. 1994. Tansley Review No. 76: Helper bactena: a new dimension to the mycorrhizal symbiosis. New Phyt~I~s~128 : 197-2 1O. Gerrath, J. F.. J. A Gerrath, and D. W.Lanon. 1995. A preliminary account of endolithic algae of lirnestone cWs of the Niagara Escarpment. Cmiadica JmmI of BO- 73 : 788-793. -th, J. F., J. A. hth,U. Matthes, and D. W. Larson. 2000. Endolithic aigae and cyanobacteria fiom clifi of the Niagara Escarpment, Ontario, Canada. Cmtodan JmmI of Botany 78:807-8 1 S. Gill, D. E. 19%. The natural population ecology of temperate terrestrials: pink lady's- slippers, C'pediumacaule. Pages 9 1- 1 O5 in C. Ailen, ed. North Americcin Native Terrestrial Or&& Propcgation ami Productim. Glick, B. R 1995. The enhancement of plant growth by fieeliving bactena C411Qdiun Jmnual of Midiology 41 : 109-1 17. Gtick, B. R., D. M. Karaturovic, and P. C. Newell. 1995. A novel procedure for rapid isolation of plant growth promoting pseudomonads. CdfmJmml of Microbiology 41: 533-536. Gray, T. R G. 1990. Soi1 Bacteria. Pages 15-3 1 in D. L. Dindal, ed. Soil Biofogy Guide. John Wiley and Sons, inc., New York. Hadley, G.,and S. H. Ong. 1978. Nutritional requirements of orchid endophytes. Nmb Phytoiogrst 8 1 : 56 1-5 69. Hadley, G., and B. Williamson. 1971. Anaiysîs of the pst-Uifection growth stimulus in orchid mycorrhira New Phytoiogist 70:445-455. Harnbleton, S., and R S. Currah. 1997. Fungd endophytes fion the mots of alpine and bord Ericaceae. Cùmdim JmmaI of Botany 75 : 1570- 158 1. Harley, J. L., and S. E. Smith. 1983. Mycorrhizf synrbiosis. Academic Press, London, United Kingdom. Harvaiq G. 1974. Notes on the biology of some native orchids of Thunder Bay, their endophytes and symbioms. CdianJourni of Botcary 52: 45 1460. -. 1982. An impmved culture medium for growing the orchid C'ripedium reginae axenically. CdianJarrml of Botq60: 2547-2555. Havelka, U. D., M. G. Boyle, and R W. F. Hardy. 1982. Biological nitrogen fixation. Pages 3 65-422 in F. J. Stevenson, ed. Nitrogvn in agricultura/ ds- ASA rnomgrqh 22. Hewitt, E. 1966. Sand and water culture methods used in the study of plant nutrition. Commorweulth Rrireau Technical Cornmunicalion 22. Hijner, J. A., and J. Arditti. 1973. Orchid mycorrhiza: vitamin production and requirements by the syrnbionts. Americm Jo~mlof Botany 60: 829-83S. HoIl, F. B.. and C. P.Chanway. 1992. Rhizosphere coloiiization and seedling growth promotion of ldgepole pine by Bacillus pui'yxa. CdianJmmal of Microbiobgy 38: 303-308. Holm T. 1904. The root-structure of North American temestrial Orchidaceae. American Jmma! of Science 1 8 : 1 97-2 12. Holrnan, R T. 1983. Studies on the fiagrances of Nonh Amencan terrestrial orchids. Pages 22-40 in E. H. Plaxton, ed. Syniposium II ami Lecttwes NdAmericm Terrestricd OrchidS. Michigan Orchid Society, Southfield, Michigan. Hutchison, L. J., and G. L. Barron. 1997. Parasitism of algae by lignicolous Basidiomycota and other hgi. CdanJmrmf of Botuîy 75: 1006 - 10 1 1. Ingham E. R,and H-Masicotte. 1994. Rotozoan communhies around conifer roots colonized by enomycorrhkai hgi.Mjcorrhiira 5: 53-61. Jensen, W. A. 1962. Botmicd his~uchemistry.W. H. Freeman and Co., San Francisco, USA. Jorgensen, B. 1. 1994. Hardy orchids: Symbiotic in vitro propagation and cultivation. Pages 166-172 in T. Kotai and R. H. Zimrneman, eds. Emimmentai effects und their co~~trolhr pkmt tisrue culture, Kyoto, Japan. Jumponen, A., and I. M. Trappe. 1998. Dark septate endophyies: a review of facultative biotrophic root-colonking fungi. Nov PhytoIogist 140: 295-3 10. Y. 19%. Plant growth promotion by rhizosphere bacteria. Pages 769-78 1 in Y. Waisel, A. Eshel, and U. Kafkafi, eds. Phrm~s - the hi& ha& Marcel Dekker, Inc., New York. H. S. 1989. Germination in the Cypriipedum/Paphiopedih alliance. The Cm~adianOrchid Jmrmi 5 : 1 1- 1 9. Stimulation of Cypripdium seedlings by two root isolates. World Orchid Congres, Glasgow. Markovina. A.-L., and P. A. McGee. 2000. Cornparison of symbiotic and asymbiotic seed germination and development in Sarcarhiius. Lindleycmci 1 5 : 68-72. Matsubam, Y.-1..Y. Uetake, and R L. Peterson. 1999. Entry and colonkation of Aqmagus oflcinolis mots by arbuscular myconhizal hngi with emphasis on changes in host microtubules. Camdm Jourmil of Bot- 77: 1 159-1 167. McCook. L. M. 1989. The genus Phragmipdium - Part 1. Amencan Orchid Society Bulletin 58: 1095-1 101. Meinde, E. P. 18%. Beitriige zur Anatomie der Luftwurzeh der Orchideen. FIwa 78: 133-203 + 2 plates. Melville, L., S. Dickson, M. L. Farquhar, S. E. Smith, and R L. Peterson. 1998. Visubtion of mycorrtlltal fungal structures in resin embedded tissues with xanthene dyes using laser scanning confd microscopy. CdimJmml of Botq 76: 174-178. Mitchdi, R B. 1990. The work of the Sainsbury Orchid Conservation Project, Royal Botanic Gardens. Kew. Pages 95-96. North American Native Terresrriai Orchid Propagation md Producim. Mohr, U., I. Lange, T. Boller, A. W~emken,and R. Vogeli-Lange. 1998. Plant defence genes are induced in the pathogenic interaction between bean mots and Fum'um soluni, but not in the symbiotic interaction with the Ybuscular mywnhizal fiingus Glomus mosseae. New Phyroogrst 138: 589-598. Moller, 0. 1968. Wachshunsrhythmus und vegetative Vennehmng von Cpripedium calceolus. Die Orchidee 19: 222-223. -. 1977. Kritische Anrnerkungen ni "Vermehmng von C'npedium aus Sarnen". Die Orchidee 28: 184-185. Montagnes, D. J. S., and D. H. Lynn. 1987. A quantitative protargol nain (QPS)for ciliates: method description and test of its quantitative nature. Màrine Microbial Food Webs 2: 83-93. Muir, H. J. 1989. Germuiation and mycorrhizai hguscompatibility in European orchids. Pages 39-56 in H W. Pritchard, ed. Mdrnmetho& in orchid conrervrtton: lk role of phy~ioiogy~ecology and management. Cambridge University Press, Cambridge. Plisba, B. 1 984. Nunirion und feeding strategies in protozm. Crwm Helm, Ltd., London. Obreht. 2.. N. W. Kerby, M. Gantar, and P. Roweli. 1993. Effécts of root-associateci N2- fixing cyanobacteria on the growth and nitrogen content of wheat (Titzcum wlpeL. ) seelings. Biology and Fertility ojSoiis 1 5 : 68-72. O'Brien, T. P.. and M. E. McCully. 198 1. The snrdy ojpkurl structure. Principies md soiected metW. Tefmaccarphi Pty . Lt d.. Melbourne, Australia. O'Neill, G. A., C. P. Chanway. P. E. Axelrood R. A. Radley, and F. B. Holl. 1992. An assessrnent of spnice growth response spifiCity after inoculation with coexistent rhizosphere bacteria. CadianJournal of Boiany 70:2347-23 53. Perotto. S., and P. Bonfmte. 1997. Bactend associations with myconhizai fungi: close and distant niends in the rhizosphere. Trends in Mzcrobioiugy 5: 49650 1. Peterson, C. A., and D. E. Enstone. 19%. Functions of passage cells in the endodermis and exodemk of roots. Physo/og'a Piuntamm 97: 592-598. Peterson, C. A-. and C. J. Perurnalla. 1WO. A survey of angiospem species to detect hypodermal Casparian bands. II. Roots with a multiserïate hypodennis or epidennis. Botanicai Jourmi of the Linnean Sociev 1O3 : 1 13 - 125. Peterson, R L., P. Bonfante. A Faccio, and Y. üetake. 19%. The interface between fungal hyphe and orchid protocorm cells. Cmdian Jo~nr>lof Botq74: 1861-1870. Petenon, R L., and M. L. Farquhar. 1994. Mycorrhiuis - integrated development between mots and fiingi. Mycologia 86: 3 11-326. Peterson, R L., and F. C. Guinel. 2000. The use of plant mutants to shidy regdation of colonization by AM fungi. Pages 147-1 71 in Y. Kapuinik and D. D. Douds, 4s. Arbusc~illormycmhirae: moIemIiw bioiw andphysioIogy. Kluwer Academic Publishers, Dordrecht. Peterson, R L., Y. Uetake, and C. D. Zelmer. 1998. Fungal symbious with orchid protocorms. Spbiosis 25 : 29-5 5. Pridgeon, A. M. 1987. The velamen and exodennis of orchid roots. Pages 139-192 in J. Arditti, ed. Orchid Biologv: reviews Mdperspectives. Cornstock Publishing Associates, Itbaca. Pridgeon, A M., P. J. Cnib, M. W. Chase, and F. N. Rasmussen, eds. 1999. Generd innoducrion, Apstioideae. Cypripedioideae. Mord University Press, Great Britain. Puente, M.-E., and Y. Bashan. 1994. The desert epiphyte Tiifduremnutu harbours the Ntrogen-fixing bacterium Ps~udomnmarstuzzeri. Camdan JmntuI of Botuny 72: 406-408. Rasmussen, H. N. 1995. Terrestriid orchidsfrom seed $0mycotrophic ph.Cambridge University Press, Great Britain. Requem, N., 1. fimemer, M. Toro, and J. M. Bara 1997. interactions between plant- growth-pmmoting mizobacteria (PGPR), arbumlar mycorrhizal hgiand Rhizobium spp. in the thizosphere of Anthylis cytisoides, a adel legume for revegetation in mediterranean semi-arid ecosystems. Nov Phyt~I~s~136: 667-677. Richardson, K. A, R L. Petenon, and R S. Currah. 1992. Seed resewes and eady symbiotic protocorrn development of Platmthera hvperborea (Orcbidaceae). CdanJi of Botq 70: 29 1-30. Riley. C. T. 1983. Hocticultural seed germination and cornmerciai potmtial. Pages 9-1 3 in E. H. Plaxton. ed. S'posium II adlectwes North Amerimn terrestria/ orchid, Southfield Michigan. Rosso, S. W. 1966. The vegetative anatomy of the Cypripedioideae (Orchidaceae). hinKd of the Lin?&?anSociety 59: 309-34 1 . Salisbury, F. B., and C. W. Ross. 1992. Plant Physolqgy. Wadsworth Publishing company, Belmont, California. Salmon, J. T. 1954. A new polyvinyl alcohoi mounting medium. The microscope 10: 66- 68. Schelkle. M., and R. L. Peterson. 1 9%. Suppression of wmmon root pathogms by hel per bacteria and ectornycorrhizai hgïin vitro. Mycowhira 6 : 48 1-485. Sen, R,A. M. Hietala, and C. D. Zelmer. 1999. Common anastomosis and internai transcribed spacer RFLP groupings in binucleate Rhiztxtoniu isolates representing root endophytes of Phsyhstrrs, Ceratorhi= spp. fkom orchid myconhizas and a phytopathogenic anastomosis group. Nov Phyrologit 144: 33 1-341. Shefferson, R. P.. B. K. Sandercock, J. Proper, and S. R Beissinger. 2001. Eaimating dormancy and survival of a rare herbaceous perennial uskg mark and recapture models. EcoIogy 82: 145-156. She* C. J. 1974. An introduction to the ecology of the Illinois Orchidaceae. IIlimis State Mt~seurnScience Papers 1 4: 1 -89. Smith, S. E., and D. J. Rad. 1997. Mycwrhiira Synbims. Harcourt Brace & Co. Smnciu, E. A, and R. S. Cunah. 1989. Symbiotic gennienninationof ds of temestrial orchids of North Arnerka and Europe. LindIeym 4: 6-1 5. Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy . JmrnaI of Uitrastmc~ureResecvch 26: 3 1-43. Steele, W. K. 1996. Large desadling production of North Amencan Cypripedium species. Pages 1 1-26 in C. AUm. ed. North American NUIIIWTemestrial Orchid Conference, Washington, DC, USA Stoutamire, W. P. 199 1. Annual growth cycle of C'ripdium di&m Muhl. root systems in an Ohio prairie. Lirdleyana 6: 235-240. Szumigalski, A. R, and S. E. Bayley. 1997. Net above ground primary production dong a pegtlmd gradient in central Alberta În relation to environmental f-ors. &scieme 4: 385-393. Taylor, D. L., and T. D. Bruns. 1997. Independent, specidi invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proeedings of the N&ml AcOLdemy of Science, llSA 94: 45 10-45 15. Toledo, G., Y. Bashan, and A Soeldner. 1995a. Cyanobacteria and black mangroves in Northwestem MoOco: colonization, and didand seasonai nitrogen fixation on anal roots. C'kmJini17yxI of Microbiology 4 1 : 999- 10 1 1. -. 1995b. in vitro colonization and incfease in nitrogen fixation of seediing rmts of black mangrove indateci by a füamentous cyawbacteria- CdmJacrmI ofMicrbioIogy 41: 1012-1020. 1 Uetake, Y., M. L. Farquhar, and R L. Peterson. 1997. Changes in rnicmtubule arrays in symbiotic orchid protocomis during fun@ colonization and seneScence. New Phytologist 13 5 : 70 1-709. Uetake. Y ..and N. Ishiraku. 1995. Cytochemical localization of adenylate cyclase activity in the symbiotic protocomis of Spiran~hessinemis. Mycollogid Research 100: 105-1 12. Uetake. Y ., and R. L. Peterson. 1 997. Changes in adin filament arrays in protocorni cells OF the orchid speci- Spirmthes sinemis, inducad by the symbiotic fungus Ceratobasidim cornigerum. Canadian JmmaI of Botany 75: 166 1- 1 669. Vralstad, T., T. Fossheim, and T.Schumacher. 2000. Pîceirhizo bicoloruta - the ectomycorrhizal expression of the Hynienossrphtrs aggregate? New PhyoIogrst 145: 549-563. Vujanovic, V., M. St. Arnaud, D. Barabe, and G.niibeault. 2000a. Viability testing of orchid seed and the promotion of colouration and germination. Amls of Bw86: 79-86. Vujanovic, V., M. St-Arnaud, O. Barabe, and G. Thiit.2ûûûb. Phiaucephlu victwïnii sp. nov., endophyte of Cypnpedium pwrfomurn. Mycdogio 92: 5 7 1- 576. Weller, D. M. 1988. Biological contr01 of soilbome plant pathogens in the rhizosphere with bacteria AnmmI Review of Phytopathol' 26: 379407. Wherry, E. T. 19 18. The reaaion of the mils supporthg the growth of certain native orchîds. Journal of the WarhingtonAcademy of Sciences 8: 589-598. Whitlow, C. R 1983. Cypripedium Culture. Pages 2541 in E. H. Plaxton, ed. Symposium II adLectures N~orhAmerican TenemafOrchidS. Michigan Orchid Society. Sout Meld, Michigan. Wilkinson. K. G., K. W.Dixon, and K. Sivasithamparam. 1989. Interaction of mil bacteria, mycorrhual fiingi and orchid seed in relation to germination of Australian orchids. New Phyroogist 1 12: 429435. Williams, P. G. 1985. Orchidaceous rhizoctonias in pot cultures of vesicular-arbuscular myconhllal fungi. Candian Jourml of Bolany 63 : 1329-1 333. Zelmer, C. D. 1994. Interactions between nonhem terrestrial orchids and fungi in nature. M.Sc. Thesis. Botany. University of Alberta, Edmonton. Zelmer, C. D.. and R S. Currah. 1995. Evidence for a tiingal liaison between Coral/orhiza ml& (ûrchidaceae) and Pinus contortu (Pinaceae). Cdim~ Jmmlof Botany 73: 862-866. -. 1997. Symbiotic germination of Spirmthes lacera (Orchidaceae) with a naturally ocairring endophyte. Lindeyuna 12: 142- 148. Zelmer, C. D., L. Cuthbertson, and R S. Cd.19%. Fungi associated with terrestrial orchid mycorrhizas, dsand protocorms. Mycoscience 37: 439448. Zettler, L. W. 1998a. ûrchid consedon in the 21s cenhiry: the value of includlig mycorrbual hngi tc. preserve endangerd species. North Americun Native Orchid J-14: 261-269. -. 1998b. Propagation of the little club-spur orchid (Plamtheta clrivolda) by symbiotic seed germination and its ecological implications. Envir011mental and lhperirnental Boimry 3 9: 189- 195.