New Phytol. (1970) 69, 1015-1023. NON-SPECIFICITY OF SYMBOTIC INFECTION IN ORCHID MYCORRHIZA By G. HADLEY Botany Department, University of Aberdeen (Received 8 April 1970) Summary Symbiosis tests carried out between orchids from several geographical localities and thirty-two Rhizoctonias isolated from orchids, non-orchid hosts and soils of worldwide distribution, showed no evidence of any species-to-species relationship between orchid and fungus. Dactylorhiza purpurella established a symbiotic relationship with most fungi tested while Coeloglossum viride, Goodyera repens and Cymbidium canaliculatum, although less frequently symbiotic, established compatible infections with many isolates. Among orchids with photo- synthetic protocorms, Hpidendrum radicans was compatible with several fungi but three other species of Epidendrum together with Spathoglottis plicata and a Laeliocattleya cultivar were symbiotic only with Tulasnella calospora and certain Ceratobasidium isolates. Tulasnella calospora may be a universal orchid symbiont. Ceratobasidium cornigerum and Thanatephorus orchidicola commonly occur as root endophytes but are doubtfully symbiotic with most orchids. 7. cucumeris is frequently symbiotic with Dactylorhiza purpurella but less commonly so with other orchids tested. INTRODUCTION ‘The specificity of one mycorrhizal fungus to one species of orchid was advocated by Bernard (1909) and maintained by Ramsbottom (1929). Subsequently Burgeff (1936) and Curtis (1939) showed that certain species of fungi were actively symbiotic with certain taxonomic or ecological groupings of orchids. The distribution of mycorrhizal fungi must be influenced by soil conditions and the concept of ecological specificity is attractive from the evolutionary aspect. Nevertheless, many fungi infecting orchid roots to establish a symbiotic association with germinating seeds of their host while nating elsewhere may do so (Harvais and Hadley, 1967a; and see Harley, 1969). ‘The reasons for this are not fully understood. Orchid endophytes are commonly strains of the form-genus Rhizoctonia. ‘They are taxonomically obscure and there has been little precise work on their identification. By inducing many orchid Rhizoctonias to fruit Warcup and ‘Talbot (1967) were able to identify species of the genera Ceratobasidium, Sebacina, Thanatephorus and Tulasnella as mycorrhizal fungi forming coils inside host cells. Within the genus Thanatephorus, T. cucumeris is an omnivorous plant pathogen most commonly known in its imperfect state (Rhizoctonia solani). Downie (1959) first reported the isolation from roots of Dactylorhiza (Orchis) purpurella of a fungus regarded as Rhizoctonia solani and she further found that R. solani isolates from other (crop) hosts were symbiotic with germinating seed of Dactylorhiza purpurella. Among the isolates of Rhizoctonia solani used by Downie and others subsequently obtained from roots of TOI51016 G. Hapiey Dactylorhiza purpurella and Coeloglossum viride by Harvais and Hadley (1967a) only one (isolate Sz) has been induced to form the perfect stage, in this case Thanatephorus orchidicola (Warcup & Talbot, 1967). One other isolate, Rst which was symbiotic with Dactylorhiza purpurella although obtained as a pathogen on tomato plants, has been fruited as Thanatephorus cucumeris (Warcup, private communication). ‘The claim that Dactylorhiza purpurella protocorms may exhibit symbiosis with many Rhisoctonia species and strains (Harvais and Hadley, 1967b) remains unsubstantiated unless the fungi in question are taxonomically described. ‘The identification of many orchid fungi by Warcup (private communication) and the improved understanding of the nutrient requirements of the orchid-fungus system (Hadley, 1969) have led to further studies with Dactylorhiza purpurella and other orchids. This paper describes symbiosis tests between Rhizoctonias of world wide distribution and a selection of temperate and tropical orchids. MATERIALS AND METHODS Culture techniques Stock cultures of fungi were maintained on potato dextrose or potato carrot agar medium and before experimental use were subcultured on to Pfeffer 0.1°% dextrose agar or, with the nutritionally exacting isolates, Dox-yeast agar medium. For each treatment one 4 mm diameter disc of agar from the growing front was inoculated to each of five replicate tubes, usually a few days after sowing the orchid seed. Orchid seed was surface sterilized by shaking for 20-30 minutes in a 5% solution of “Domestos’, washed twice in sterile water and sown on agar slopes. ‘The experimental medium contained (w/v) 0.08% CaNO3; 0.02% KH,PO,; 0.02% MgSO47H,0; 0.02% KNOs; 0.05% (NH,)2SO, (omitted for certain species); 0.01% KCI; Fe, Zn, Cu, Mn, Mo in traces; 0.05% yeast extract (Oxoid); 0.1% dextrose; 0.4 or 0.9% ball-milled cellulose (Hadley, 1969) and 1°% agar. Yeast extract was included since the nutritional requirements of some of the more exacting fungi were not fully known. Cultures were maintained at or near ambient temperatures (c. 20° C) in darkness or, with tropical species, in diurnal conditions at 20~-25° C. They were assessed after 3-6 months by sampling from at least three replicate tubes for visual observation of germina- tion and symbiosis. Where required, measurements of length and breadth of proto- corms were made as previously described (Harvais and Hadley, 1967b). Orchids Limitations due to availability of seed meant that only certain orchids could be used. ‘Those listed here exclude many which failed to germinate or gave very low percentage germination. ‘The three north temperate species are common in north-east Scotland but occur in different habitats. Dactylorhiza purpurella (‘T. and'T. A. Steph) Soé was known to accept a broad spectrum of endophytic fungi (Harvais and Hadley, 19672) while Goodyera repens Br. was regarded as specifically associated with one fungus (Rhizoctonia goodyerae- repentis Constantin), Coeloglossum viride (L.) Hartm. occurs in calcareous habitats of pH 8-9. Cymbidium canaliculatum is one of the few epiphytic orchids of Australia, occurring on trees in open forests in the drier inland areas of the north; seed was provided by Dr J. H. Warcup.1017 Orchid mycorrhiza edeepy ‘indumy epeny uoapaoqy “yBinquiayy eave “Koy TeQUEY pueloog ‘anysautpseouryy eAupepy “ndum’y eens] us9pioqy woapaaqy “yfingnayy Arey Sandu] Bens] eyensny “S vipensny *g puejsug ‘a3prquiey uoopagy “EquOMaN vAopepy Sndwin’y yjensy exsny “g ‘eUOOPY ujensny *g ‘euaSun3 vipensny *g ‘puowso wap, wyensny “g ‘euasun edvpeyy "yong 1oBung, TOLYY 'S ‘Bhowsq woapa9qy ‘wsaps0qy uyensny °g ‘supres £11945 uyensny “g ‘AoneA Adduyy PUB|OIg “YorATUOY puspioag “uainquipa eauayy ‘runway uaapiaqy “Ysinqaany vypasny “S “ung aA eyensny °s ‘AyoT WL wyeaasny "S ‘Qjor] IT edule ‘Isog toBung PuEpoog ‘auUICy urBiz0 orydex095 (L602 ty) woSomped se parejost ‘oory ‘a4e]081 3003 Syypeandund vszy0}<)20¢7 S100 UT slo !oqD2H/d suIOpSoyIOdg uoTsoy was ‘ Aopregy (ureurezeaegy “g) roqmy orei0g wapied sruti0g {1105 si004 toyjoundund Dexys0.S9q S}00r {190 a1BBeyy “a9 squyooAy 83005 UF s[too fds marnusjay.y 81003 Ur syfoo ‘ox/0fiduey stumiqy ‘a¥eIOS! 00x !Dpeundund n=nyAO}Ka20q aakydopua 00x ‘oy ofig Duyn} axdydopua 3003 {199 are “a9 siuyonsy sysoy Auvut Jo Uaouted ui5y oruaZoyed-uo suiays Jaytonio uo oruafoyped {]iog uaBoyzed oor qualnsta ‘s0or WYN, aussered Jeol {sapiojoasoyd vuviang suoIsa] wars fore0g soqna wo thozsps fomiog usdoyed 3003 foyems0 1, 1003 UT Stoo ‘amuae«00 syRIso4aI S]00! 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HapLey Seed of Epidendrum ibaguense, E. nocturnum, E. obrienianum and E. radicans, all of which were growing in cultivation in the Los Angeles area, was provided by Dr J. Arditti of the University of California and made it possible to include these four species as an intrageneric group. A cultivar from the same locality, Laeliocattleya ev. Dee Dee was used in some experiments. Spathoglottis plicata Bl., a common ground orchid in Malaya provided a representative from a fourth geographical locality. Fungi one were isolated by plating out portions of surface-sterilized roots (Harvais and Hadley, 1967a) or by the washing and teasing technique of Warcup and Talbot (1967). Many of the isolates were identified by Dr J. H. Warcup who also provided additional isolates from Australian orchids. The symbiosis tests made use of fungi which had been taxonomically identified, together with others of interest or significance because of their origin or activity. ‘Table 1 lists the fungi with information on their source and, where known, host reaction. Resuits Isolation from orchid roots It was not possible to make isolations from all the orchids of which seed was avail- able. Table 2 shows the results for those orchids from which extensive isolation was ‘Table 2. Summary of isolations from roots of eight orchids No. of Approx. _ No.of No. of No.of strains not no. of Rhizoctonia — disti strains symbiotic isolations isolates. Rhizoctonia symbiotic with any attempted obtained ‘strains’. with host _ host tested North temperate orchids Coeloglossum viride* 120 18 4 1 2 Dactylorhiza elata 96 1 1 ° 1 D. purpurella® 600. 80 8 6 2 D. purpurella 300 1 A 1 ° Goodyera repens 132 25 1 1 ° ‘Tropical orchids Arachnis cv. Maggie Oci p 13 a Not tested 2 Spathoglottis plicata 80 14 3 ° 3 Vanda ev. Miss Joaquim 24. 3 1 1 ° * Data in part from Harvais and Hadley (1967a). attempted and it is evident that many of the fungi obtained from roots do not demon- strate any symbiotic potential in tests with germinating seed of their host. ‘Tropical orchids in particular yield few active symbionts although this is not surprising in view of observations by Hadley (unpublished) that infection of their roots is often relatively sparse and consists largely of digested non-viable hyphal masses. Infection and symbiosis Studies of the initial stages of infection of Dactylorhiza purpurella protocorms (Williamson and Hadley, 1970) showed that compatible infection of epidermal hair cells was not necessarily followed by vigorous symbiosis. The success of symbiosis can be assessed only when the protocorms have attained a degree of maturity and is difficult to evaluate in numerical terms. Nevertheless vigorous symbiotic development of evenOrchid mycorrhiza TOIQ a few protocorms may be regarded as proof of the symbiotic potential of the fungus and is designated in ‘Table 3 by the symbol ‘S’, while ‘SS’ and ‘SSS’ indicate that proportion- ally more of the population is symbiotic. Table 3. Summary of the interactions between protocorms of ten orchids and thirty-two Rhizoctonia isolates Fungi Ceratobasidium cornigerum C. obsciurum Ceratobasidium sp. indet Thanatephorus orchidicola T. sterigmaticus T. cucumeris Tulasnella calospora 1, eruciata Rhizoctonia solani Rhizoctonia sp. Rer "Ther 0393 0479 38 Fr Cs4 Der S2 060 0269 Rsr Rs6 Rst2A Rsor W16 W48 W82 W87 Amo4 Pb47 RrA 0388 0206 Rsst s Rs16 Rso4 Rsroz Spr oe Rsto Coeloglossum viride Dactylorhiza purpurella ss SSS On on OnBwe OOu ne Ome R Goodyera repens Once » g ss ssP Cymbidium canaticulatum s(S) Epidendrum ibaguense 110001 mam! anv oO SSS SS SSS iS: °O E, nocturnum 1VIGOOT11OH1111011 000000011 mos! E, obrienianum Cc ' Dy & E. radicans s(sP) wi wn itieer: age & s(sP) ° ° Laeliocattleya ev. Dee Dee 1O~o COonuncoons Spathoglottis plicata , No infection, protocorms healthy. s, Compatible infection but no growth stimulus seen. If symbiosis does not develop, this condition is analogous to s*. s*, Infection followed by death of hyphae, sometimes seen as’ hypersensitivity reacti symbiosis. P, Parasitism witho ‘compatible phase, usually resu biotic phase (‘breakaway parasitism’). due to dense growth of fungus but no jon; no growth stimulus. S, Growth stimulus; mt any evidence of a compatible phase. sP, Parasitism following ting in death of protocorms. SP, SSP, Parasitism after a sym- , Inconclusive result; protocorms often moribund or dead infection seen. ‘Symbols in parentheses indicate that a small proportion of the population was in this category. Non-symbiotic protocorms may be so because they remain uninfected (represented by <0’) or because compatible infection (s) is followed by an imbalance between host and fungus. Where the host kills the hyphae by a hypersensitivity reaction or a process of digestion there is usually no further infection and development of the orchid ceases (s*).1020 G. Hapiey Alternatively the fungus becomes aggressive, gradually advancing into the host tissue and permeating cells without forming pelotons; this parasitic phase is normally lethal and, dependent on other environmental conditions (Harvais and Hadley, 1967b), may arise with or without a preceding symbiotic phase (represented as P, sP, SP, SSP, etc.). ‘The pattern of development is shown diagramatically in Fig. 1. In any population protocorms may be found in several of the categories, which are not mutually exclusive. Table 3 records the predominant category of infection in each treat- ment. Germinated seed (protocorm) “fungus Qe No infection s Death of protocorms Compatible infection (non-parasitic), i.e. of protocorm indecisive result P Parasitism of protocorms without a compatible phase sk Hypersensitivity, or S,SS infection otherwi Marked growth stimulus, sP contained and elit e ie, symbiosis Breakaway parasitism, — usually leading to death oan es, of host Total elimination of fungus possible as old protocorm Normal autotrophic SP.SSP tissue becomes moribund phase of development As for sP (above) to mature adult plant Infection pattern may be repeated in roots? Fig. 1. Possible pathways in the development of the orchid-fungus interaction. Specificity It can be seen from ‘Table 3 that there is no absolute specificity since fungi in culture with the hosts from which they were isolated are not always symbiotic and those which are may be equally active with other orchid hosts. Nor is the orchid-fungus relationship completely random; it is evident that certain orchids are more receptive than others to a variety of endophytes and again, certain fungi are more active than others in both the degree to which they stimulate symbiotic development and the range of hosts they infect. Orchids . ‘The three north temperate orchids were infected by many of the thirty-two fungi tested and D. purpurella in particular was highly successful in establishing controlled symbiosis with twenty-one isolates, while with a further six it was compatible but not symbiotic. Coeloglossum viride, which was frequently infected but established firm symbiosis with only six of the isolates, may have been unsuited to the pH (5.6) of the culture medium. Goodyera repens was symbiotic with nine isolates but was the only orchid not stimulated by Tulasnella calospora. Furthermore, although symbiotic with its root endophyte Ceratobasidium cornigerum isolate Regr, it was not so with the three other isolates of that species.Orchid mycorrhiza 1021 Among the remaining orchids Cymbidium canaliculatum and Epidendrum radicans showed a broad spectrum of compatibility not unlike that of the north temperate species. Others were selective towards Tulasnella calospora and certain Ceratobasidium isolates. Nevertheless, the absence of compatibility was not correlated with parasitism by the fungi and in general the orchids which were resistant to symbiotic infection were also resistant to being parasitized. Fungi Certain of the isolates tested are vigorous and effective symbionts with many orchids; for instance four isolates of Tulasnella calospora from widely dispersed geo- graphical localities showed an almost identical pattern of response with the ten orchids tested. This species is clearly an active symbiont and the same may be true of certain as yet unnamed Ceratobasidium species (e.g. isolates Fr and Csq). Isolate T also may be a Ceratobasidium. By contrast four isolates of C. cornigerum, all originating as orchid endophytes, gave rather heterogeneous results while two Thanatephorus orchidicola isolates obtained from orchid roots were not symbiotic to any host tested. Isolates of T. cucumeris and Rhizoctonia solani were mostly symbiotic with Dactylorhiza purpurella and infrequently so with other hosts. Exceptions were isolates W16 and W87 (both known to be aggressive on a range of hosts) which were parasitic to several of the orchids tested. Nevertheless in other work (Williamson and Hadley, 1970; Williamson, 1969) it has been found that both of these fungi may be symbiotic with D. purpurella and the patterns of response and growth stimulus by the host are exactly as with other symbionts although the proportion of symbiotic protocorms is usually small. Isolate W48, a selective parasite of crucifer stems (Flentje, Dodman and Kerr, 1963) was vigorously symbiotic with two of the north temperate orchids. It did not exhibit any trend towards parasitism and with D. purpurella measurements at the end of the experiment confirmed that, like some other isolates of Thanatephorus cucumeris, it was able to support an extensive population of well-developed protocorms. ‘The tendency for certain fungi to pass into a post-symbiotic phase of parasitism is seen in some host-fungus combinations. In prolonged experiments this trend became more pronounced, presumably as nutrients were depleted (Harvais and Hadley, 1967b). But parasitism was rarely seen with isolates of Twlasnella calospora and the unidentified Ceratobasidium sp. (isolates F and Cs4). Discussion ‘There are two main conclusions from the results. Firstly some species of fungi, in particular Tulasnella calospora, appear capable of infecting and are actively symbiotic with most of the orchids tested. Secondly, certain orchids will accept a large proportion of the fungi tested against them. Among the four isolates of 7. calospora there is a reasonable consistency in their symbiotic activity notwithstanding the fact that they originate from various world- wide localities. T. calospora remained non-aggressive throughout the experiments and may therefore be a ubiquitous orchid endophyte. It probably includes many isolates from orchids which in the past have been referred to Rhizoctonia repens Bernard. Unpublished work has shown that Tulasnella calospora requires yeast extract for normal growth on an otherwise defined medium and it seems likely that the exacting1022 G. Hapiey nutrient requirements of this and other ‘typical orchid endophytes’ (Harvais and Hadley, 1967a) are associated with their symbiotic habit. In fact two orchid fungi, T. asymmetrica Warcup and Talbot and Sebacina vermifera Oberwinkler were excluded from the sym- biosis tests due to their complex nutrient requirements and slow growth rate. Ceratobasidium species also show a fairly broad spectrum of symbiotic activity. But the four isolates of C. cornigerum used here appear to be physiologically different for they behave differently. Indeed isolate, Thrr is aggressive with some hosts. It is evident that many different fungi exist in a variety of associations with orchid roots (Warcup and Talbot, 1967). It may be that few of these are truly endotrophic and the ability to promote in a broad spectrum of hosts the growth-stimulatory response which is recognized as symbiosis may be limited to certain species of genera such as Tulasnella and Ceratobasidium. Nevertheless many other fungi although unspecific may be symbiotic with certain orchids and there is no reason to suppose that the symbiotic stimulus varies with different fungi. Little is known about the ecology of orchid fungi. C. cornigerum has been isolated from many soils by Warcup and Talbot (1966) who have also (1967) isolated Tulasnella calospora from soil. ‘The latter fungus (as Rhizoctonia repens) has been shown to compete successfully for cellulose in soil (Smith, 1966) and to break down pectin (Pérombelon and Hadley, 1965). Downie (1943) showed that R. goodyerae-repentis was more widely distributed than Goodyera repens from which it was usually isolated. Orchid fungi may therefore be in- dependent of their hosts for survival. On the other hand G. repens although always infected with the same fungus in the field is not specific to it in experiments described here. Its apparent specificity in the natural habitat could be explained by the absence of other potential endophytes owing to the special nature of the environment (a moss/pine needle litter with a pH of 3.8) in which this orchid occurs. The key to the range of host-fungus interaction may lie in the mechanism which per- mits or prevents penetration of the host cells by the fungus. Williamson and Hadley (1970) have shown that eight fungi all initiated a compatible infection of protocorms of Dactylorhiza purpurella in a similar manner and there is no evidence of a selective mechanism in the penetration phase. The results of the present work suggest that such a broad spectrum of symbiosis is characteristic not only of D. purpurella but also of other north temperate orchids and Cymbidium canaliculatum. In all of these the proto- corms are heterotrophic. Among orchids which are photosynthetic in the protocorm stage, Epidendrum radicans is symbiotic with a broad range of the fungi tested and all others are infected by only the most benign fungi. The results are not inconsistent with the hypothesis that autotrophic protocorms are compatible with a restricted group of endophytes. There is no evidence of specificity between one host and a single species or strain of fungus. Finally, in these experiments there was no evidence that any of the fungi stimulated germination of the host; infection occurs only after the initial stage of germination in all the orchids tested. ACKNOWLEDGMENTS I am extremely grateful to Dr J. H. Warcup who made most of the identifications of fungi and provided many isolates from his own collection. I also thank Dr Warcup, Dr J. Arditti and Dr J. B. Kenworthy for collecting or supplying seed of various orchids.Orchid mycorrhiza 1023 It is a pleasure to thank Miss Florence Greig and Mrs Esther Gray for much assistance with technical work. REFERENCES Bernarp, N. (1909). L’évolution dans la symbiose. Annls Sci. nat. (Bot.), Sér 9, 9, 1. Bunceer, H. (1936). Samenkeimung der Orehideen. Fischer, Jena. Curtis, J. T. (1939). ‘The relation of specificity of orchid mycorrhizal fungi to the problem of symbiosis. Am. 7. Bot., 26, 390. DowniE, D. G. (1943). Source of the symbiont of Goodyera repens. Trans. Proc. bot. Soc. Edinb., 33, 383. DowntE, D. G. (1959). The mycorrhiza of Orchis purpurella. Trans. Proc. bot. Soc. Edinb., 38, 16. Frenty, N. T., Dopman, R. L, & Kerr, A. (1963). The mechanism of host penetration by Thanatephorus cucumeris. Aust. J. biol. Sci., x6, 784. Haptey, G. 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Wirtiamson, B. (1969). Studies on infection and nuclear hypertrophy in orchid mycorrhiza, Ph.D. thesis, University of Aberdeen. : Wittiamson, B. & Haptuy, G. (1970). Penetration and infection of orchid protocorms by Thanatephorus cucumeris and other Rhizoctonia isolates. Phytopathology. (In press.) ciated with orchids. NewThis document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.