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Taxonomy, culture and mycorrhizal associations of some zygosporic Endogonaceae
Mycol. Res. 94 (2):173-178 (1990)
173
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Taxonomy, culture and mycorrhizal associations of some zygosporic Endogonaceae
J. H. WARCUP* Deparfment of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia, 5064
Taxonomy, culture and mycorrhizal associations of some zygosporic Endogonaceae. Mycological Research 94 (2):173-178 (1990). Endogone aggregata, E. tuberculosa and Sclerogone eucalypti gen. et sp. nov. have been shown to form ectomycorrhizas with Eucalyptus and other Australian ectomycorrhizal hosts, mycorrhiza complexity differing depending on fungus and host. E. flamrnicorona, however, formed ectomycorrhiza only with Pinus. E. reticulata grew among the roots of eucalypts and other plants but appeared to be non-mycorrhizal. E. aggregafa, E. tuberculosa, E. reticulata and S. eucalypti all grew on weak agar media, but E. flammicorona although
often growing extensively from sporocarps was not subcultured successhlly. Key words: Ectomycorrhiza, Endogonaceae, Sclerogone eucalypti, Agar culture of Endogone, Eucalyptus, Pinus. Zygosporic species of Endogonaceae in the genus Endogone (Gerdemann & Trappe, 1974), are generally considered to form ectomycorrhizas in contrast to chlamydosporic or azygosporic species in Glomus, Gigaspora, Acaulospora and Sclerocystis, which with few exceptions form vesiculararbuscular mycorrhizas. Few species of Endogone, however, have yet been shown to form ectomycorrhizas. Known ectomycorrhizal species include E. flammicorona Trappe & Gerdemann (Fassi & Palenzona, 1969; Trappe & Gerdemann, 1972) and E. lactiflua Berk. & Br. (Walker, 1985) both on pines, and the species tentatively designated ' E . eucalypti' from eucalypts (Warcup, 1975). Recently Berch & Fortin (1983) cultured E. pisiformis Link: Fr. on agar and showed that it was non-mycorrhizal. During research on ectomycorrhizal associations of plants in southern Australia, sporocarps of Endogonaceae were collected and attempts made to grow the fungi in agar culture and to establish pot cultures for examination of mycorrhizal associations. Ectomycorrhizas formed by aseptate fungi from the field were examined similarly. The results of these studies are recorded in this paper.
MATERIALS A N D M E T H O D S Sporocarps were collected by raking through surface soil and litter or they were recovered from pot cultures. E. flammicorona fruited regularly and often abundantly in pine plantations in the Adelaide Hills. Sporocarps of Endogonaceae under eucalypts, although never common, were found more frequently after fire than in undisturbed sites. Aseptate ~-
Present address: 6 Seafield Ave., Kingswood, S.A., 5062
ectomycorrhizas were occasionally found during examination of eucalypt seedlings from burnt areas or regeneration sites. Attempts at obtaining pot cultures were made using Eucalyptus obliqua L'Her. o r Pinus radiata D. Don as host plants. Intemal pieces of sporocarp, blocks of agar (4-5 mm2) containing fungal hyphae, or plugs of mycorrhizal roots were placed on tap roots of seedlings during transplanting in an autoclaved sand-soil mix low in available .phosphate. Plants . were grown in a growth room; details of soil mix and growing conditions are given in Warcup & McGee (1983). Mycorrhiza experiments were done under the same conditions but using a wider range of host plants and inoculum was always a plug (ca 0.1 g) of freshly harvested mycorrhizal roots. Attempts at obtaining agar cultures were made from mycorrhizal roots or from sporocarps. Mycorrhizal roots were washed thoroughly then placed in drops of sterile water in isolation plates. Tips of mycorrhizas were cut into lengths of 3-5 mm and older portions removed. Intemal pieces of sporocarp were cut out aseptically and gently teased into pieces of 1mm3 or smaller in drops of sterile water in isolation plates. Isolation plates were poured with molten but cooled Czapek-Dox+ yeast agar ($N)prepared with double glass distilled water (Warcup, 1975). Streptomycin (100 ug ml-l) and tetracycline (50 vg ml-') were used to control bacteria. The amount of growth obtained from different fungi varied markedly. Most showed initial growth from the inoculum which ceased after a few days; others continued growth for longer periods. Contamination by other fungi, especially Mortierella, was a problem, especially in isolations from mycorrhizal roots. Growth in plastic Petri dishes was poor unless they were wrapped with parafilm to minimize water loss. The crucial stage in obtaining agar cultures was usually
Ectomycorrhizal zygosporic Endogonaceae whether the initial growth could be subcultured successfully. Hyphal tips died rapidly so that subculturing required more growth free from contamination than is usual with fungi.
174 (1-2 cm2)often increased. Occasionally thin-walled intercalary hyphal swellings, to 20 pm, were formed in branch hyphae, these were not cut off by septa and did not aid survival of the fungus in agar. Limited growth on agar was also reported by Tandy (1975a) and Chu-Chou & Grace (1979).
RESULTS Endogone flammicorona Trappe & Gerdemann, Trans. Br. mycol. Soc. 5 9 : 403-407 (1972).
Sporocarps of variable size, to 3 cm, globose to subglobose and soft when young, hard, lobed or irregular at maturity. Peridium thin, white, becoming buff, often incomplete in old specimens. Gleba at first white, becoming salmonaceous, with zygospores showing as orange granules, usually exuding a milky latex when cut. Zygospores 56-86 x 54-70 ym, globose, ellipsoid or obovoid, enclosed in a tightly adhering hyphal mantle. Specimens examined: ADW16399, 16400, 16401, Kuitpo, under Pinus radiata, June, July, Sept. 1971. J. H. Warcup. Further sporocarps have been collected from Kuitpo on many subsequent occasions.
The fungus was always found in the vicinity of pines. Sporocarps were hypogeous in pine nurseries or pot cultures, but occurred in the lower layers of litter or embedded at the soil surface in pine plantations. At Kuitpo Forest sporocarps were found from June to November except in drought years. In pot culture E. flammicorona formed ectomycorrhizas with Pinus radiafa and P. pinaster Ait. with which sporocarps were formed in 5-6 months. Mycorrhizas were not formed with Eucalyptus obliqua from sporocarp inoculum or when eucalypt seedlings were grown close to young P. radiafa with actively growing mycorrhizas formed by E. flammicorona. Tandy (1975a) reported that E. sieberi (L.) Johnson did not become mycorrhizal with sporocarpic or mycorrhizal inoculum of E. jlammicorona. Previously E. flammicorona has been shown to be ectomycorrhizal with Pseudofsuga menziesii (Mirbel) Franco (Fassi, Fontana & Trappe, 1969; Chu-Chou & Grace, 1979), Ps. douglasii (Lindl.) Carr. (Fassi & Palenzona, 1969), 10 species of Pinus (Fassi & Palenzona, 1969; Fassi et al., 1969; Tandy, 1975 a) and Cedrus deodora (Roxb.) D. Don (Tandy, 1975 a). The ectomycorrhizas formed by E. flammicorona are pale yellow to brown, unbranched or branched dichotomously to give a finger-like appearance. Root hairs are usually sparse or absent from mycorrhizas. Thick-walled wide hyphae ramify along the root surface with finer hyphae occurring between epidermal cells and in places forming a thin sheath across them. The fungus forms an extensive Hartig net in the cortex and both long and short roots may be infected. The mycorrhizal roots are of similar appearance to those formed by the ectendotrophic Ascomycetes (Danielson, 1982) but the hyphae generally lack septa and are not intracellular. The mycorrhizas formed by E. bctiflua on Pinus conforfa Dougl. ex Loud. are similar (Walker, 1985). Endogone flammicorona collected from Kuitpo often grew well initially on isolation plates of Q N agar, especially on the surface of the medium. Growth in agar was slower and aerial hyphae rare. Growth usually slowed within 4 to 5 d and all growth ceased within a month. Subcultures from colonies with strong initial growth showed little growth into the fresh Q N medium although density of hyphae within the inoculum plug
Endogone reticulata Tandy, Ausf.
1. Bof.
2 3 : 849-866
(1975). Sporocarps hypogeous, pale to deep brick red, irregular, to 2 cm, formed between and sometimes incorporating soil material. Peridiurn to 40 Urn thick, often lacking in part, of compacted thin-walled hyphae 3-5 ym diam, paler than gleba. Gleba orange to pale red, of thin-walled hyphae, 4-8 pm diam with larger branched, thick-walled structural hyphae, 12-16 pm diam present. Latex absent. Zygospores 80-110 ym, brick red, spherical, ovoid, or irregular from pressure, with a 1-2 layered mantle, not aggregated. Spore wall in 2 layers, a thin dark exospore, 1-2 ym thick, and a thicker hyaline endospore, 4-10 i.lm thick. Gametangia usually parallel, wide, 24-44 pm, subequal with a wide attachment to the zygospore. Specimens examined: ADW 16986, Mt Lofty, under eucalypts, 25 July 1984, P. A. ~ c ~ e e ; 16987, ' k ~Stirling, ~ in garden, 5 Sept. 1984, P. A. McGee; ADW 16988, Eagle-on-Hill, under eucalypts, 11 Sept. 1984, P. A. McGee.
The sporocarps recorded here differed from that discussed by Tandy (1975 b) in being hypogeous not epigeous, being irregular in shape and containing incorporated organic matter. Attempts at establishing pot cultures with E. obliqua showed that the fungus grew well in the rhizosphere but rarely on the surface of roots and did not form rnycorrhizas. Host plants remained small (Table 1). The fungus formed long hyphae with areas of dense chains of hyphal swellings, 8-15 i.lm diam, on secondary hyphae. Hyphal swellings were not cut off by septa, most were densely ~ a c k e dwith protoplasm but some were without contents. Culture of the fungus on agar was attempted from pieces of sporocarp and from washed hyphae with hyphal swellings collected from among roots of E. obliqua. Growth occurred from both types of inoculum, the fungus usually growing initially as long unbranched hyphae. These hyphae were very sensitive to the presence of certain unidentified bacteria on the isolation plates. If the tip of a hypha grew near a bacterial colony, the whole hypha, possibly 2-3 cm long, collapsed and died. A few hyphae, however, were subcultured successfully and grew into large colonies that produced copious hyphal swellings. With age such colonies became dull reddish-brown, partly from colour of hyphae and partly pigment in the agar. The presence of copious hyphal swellings packed with contents did not aid survival and without subculturing such colonies died within 6 to 8 wk. A variety of media (Warcup, 1975) were tried to improve growth and survival but none was better than iN agar. Presence of autoclaved seed, millet, hemp, oat and colonies of Rhodoforula on the medium did not increase growth. Endogone reficulata resembles E. pisifomis (Berch & Fortin, 1983) in that both grow on weak agar media, ramify in the root zone of plants but d o not appear to be mycorrhizal.
J. H. Warcup Table I. Comparison of shoot gowth of six e~torn~corrhizal species with and without inoculation with Endogone aggregata, E. tuberculosa or E. reticulata
Host Eucalyptus obliqua Brunonia australis Podotheca angustifolia Poranthera microphylla Stylidium graminifolium Lobelia tenuior
t
E. agg.
E. tub.
E. ref.
Nil
700
535 175
54 80 445
Means of 3 replicates, harvested after 8 weeks. - no test.
Endogone aggregata Tandy, A w t . J. Bot. 2 3 : 849-866 (1975).
Sporocarps hypogeous, greyish-white, hard, ovoid to irregular, to 1.8 cm. Peridium greyish-white, becoming dark brown on drying, often incomplete, tough, to 150 ym thick, can bepeeled from gleba. Gleba initially white, developing aggregates of yellow zygospores with white sterile hyphae in between. Latex absent. Zygospores spherical to ovoid, sometimes irregular from pressure, 9 6 1 4 6 x 106-176 ym, with a 2layered wall, endospore 6-10 ym wide, exospore 8-14 ym, exospore appearing laminated in aniline blue. Zygospore aggregates irregular, to 1 mm diam, with many spores. Large external hyphae, 14-28 pm diam, with ribbed walls were associated with the sporocarps. Specimens examined: ADW 16990, sporocarps from Kuitpo Forest, 15 July 1984,under old planted E. maculata Hook., site bumt in 1983.
Ectomycorrhizas were established with E. obliqlla and Melaleuca uncinafa R. Br. The fungus also formed ectomycorrhizas with numerous herbs including Brunonia awtralis Sm., Podotheca angustifolia (Labill.) Less., Poranthera microphylla Brogn., Stylidiurn graminifolium Sw. ex Willd. and Lobelia tenuior R. Br. (Warcup, 1988). Marked shoot growth responses followed inoculation of E. aggregata to E. obliqua and the above herbs (Table I). Examination of the e~tom~corrhizas of E. aggregata with the hosts in Table 1 suggested that the pattern of formation of mycorrhizas in the different hosts was similar though the mycorrhizas appeared different. An exception might be B. australis which has much slower growing roots than the other species. Runner hyphae, 6 1 0 ym diam, grew along and to a lesser extent across the surface of roots. O n some roots secondary hyphae grew in the junctions of epidermal cells and from these arose a Hartig net and a fine one-cell-thick sheath across epidermal cells. There were runner hyphae in the rhizosphere as well as on the root surface. Both E. obliqua and B. awtralis had 'classical' ectomycorrhizas, the mycorrhizas being wider through tangential elongation of epidermal cells than non-mycorrhizal roots, slightly so in E. obliqua, markedly so in B. awtralis. The mycorrhizas had few root hairs, a fungal sheath, 8-10 pm thick usually enclosing the tips of mycorrhizas, and a Hartig net. In the other four herbs the sheath was usually one-cell-
thick but might be thicker in patches, might not cover all epidermal cells and rarely covered the root tip. A Hartig net occurred in all species, except possibly S. graminifolium, but not necessarily between all epidermal cells. Root hairs were present but depressed in number and length compared with those on uninfected roots. The number of external hyphae in the rhizosphere was usually greater in these species than with E. obliqua and B. australis. Mycorrhizas on Poranthera microphylla had areas of enlarged epidermal cells (Kope & Warcup, 1986). Occasional intercalary hyphal swellings, 16-22 ym diam, were noted in external hyphae around P. microphylla. Endogone aggregata grew readily from sporocarp pieces if these were relatively free from bacteria and contaminating fungi. Hyphae were often profuse on the surface of the medium. Surface growth was difficult to cut and tended to tear and die when hyphae were cut during subculturing. Good growth in the agar was desirable in making subcultures. Colonies were white especially if profuse hyphal swellings, to 40 ym, were present. Growth was improved using Fe Edta in the :N medium. E. aggregata grew in culture for some 18 months but was eventually lost, possibly through insufficient subculturing. ,Endogone tuberculosa Lloyd, Mycol. Writings (Mycol. Notes 56): 799 (1918).
Sporocarps hypogeous, irregular, 1.5-2-0 cm, grey from the coat of soil particles adhering to the peridium. Peridium white where visible, thin. Gleba pale yellow from closely packed zygospores in large irregular areas with veins of white mycelium between, drying dingy olive yellow, incorporating soil particles. Glebal hyphae thin-walled, 5-9 ym diam, closely packed, crushed in the areas with zygospores. Zygospores closely packed, variable in shape, globose to ovoid but mostly irregular from pressure, 54-96 x 50-102 pm, wall with 2 layers, endospore 1-2 pm wide, exospore 2-4 ym. Zygospores arise as buds from persistent gametangia. Latex absent but copious oil released from cut surfaces. Specimens examined: A D W 16991, Kui fpo Forest, under old planted E. maculata, 2 7 July 1984, area bumt in 1983. Endogone tuberculosa formed pot cultures readily on E. obliqua from sporocarp pieces and from hyphae in agar and on other
Ectomycorrhizal zygosporic Endogonaceae hosts from mycorrhizas. The mycorrhizas were similar to, but differed in detail, from those formed by E. aggregata. Growth responses of plants to E. fuberculosa are given in Table I. Well-defined ectomycorrhizas were formed with E. obliqua and B. ausfmlis with a fungal sheath, 6-10 pm thick, and Hartig net. The mycorrhizas, especially on E. obliqua, were woolly in appearance from many short, fine, empty hyphae projecting from the sheath. External hyphae, 6-10 um diam, were common in the rhizosphere with occasional globose to ovoid terminal hyphal swellings, 30-35 pm diam, arising on short lateral branch hyphae. The e~tom~corrhizas formed on Lobelia fenuior, Stylidium graminifolium and Podofheca angusfifolia were generally similar to those formed on these hosts by E. aggregata. O n Poranfhera microphylla, however, the fungus formed more and greater enlargement of patches of epidermal cells where the fungus was present. The enlarged epidermal cells were covered by a sheath, to 4 pm thick, with a prominent Hartig net between cells. Endogone fuberculosa also grew well from sporocarp pieces though not as vigorously as E. aggregafa. Cultures were white with most hyphae submerged in the agar. However, after some weeks, the agar cultures were lost due to contamination. Sclerogone Warcup, gen. nov.
Etym.: Greek sclero- (hard) and -gone (reproductive organs). In reference to the sclerotioid sporocarps Sporocarpia minuta, hypogaea, bmnnea, dura, non aggregata. Pertdium et gleba ex hyphis compactis intertextis aseptatis et parietibus crassis constantes. Zygasporae generatim paucae, hyalinae, suspensoris celiulae evanescentes. Sp. typ.: Sclerogone eucalypti Warcup (syn. Endogone eucalypti ined., Warcup, 1975)
Sporocarps minute, hypogeous, brown, hard, not aggregated. Peridium and gleba of compacted interwoven thick-walled hyphae. Zygospores generally few, hyaline, suspensor cells evanescent.
Gerdemann & Trappe (1974) considered that the genus Endogone Link: Fr. consisted of several diverse elements that were not closely related. They retained the zygosporic species in Endogone sensu stricfo and raised four segregate genera for species with ~ h l a m ~ d o s p o r or e s azygospores. However, even in Endogorre sensu sfricfo species differ in characters such as sporocarp construction and presence or absence of latex, and a better understanding of sporocarp formation may indicate further segregate genera. Sclerogone is considered such a segregate genus and differs from other known species of Endogone in its small, hard, sclerotioid sporocarps. E. alba (Petch) Gerdemann & Trappe also has small ovoid sporocarps, but they are aggregated and fused together in pulvinate masses. Sclerocysfis has small hard sporocarps but they contain chlamydospores. Sclerogone eucalypti Warcup, sp. nov.
(Fig. 1)
Sporocarpla minuta, pro parte maxima minus quam 0.5 rnm, hypogaea, non aggregata, brunnea, irregulariter globosa, 2-12 (-78)
Fig. 1. Sclerogone eucalypti. A, Habit sketch of a sporocarp with four zygospores; B, cells of sporocarp; C, young zygospore.
J. H. Warcup zygosporas contenta. Zygosporae hyalinae, subglobosae vel globosae, 48-68 x 52-76 vm. Sporae panes hyalinus, lucens, 2-4 vrn latus, suspensoria cellulae evanescentes. Hyphae vegetative variabiles, 3-7( (-16) vm diam, et aliquando pariete crasso ad 5 vm. Holotypus ADW 16992 (N191) ex Eucalypti obliquae, Kuitpo For., S. Australia, 7 Sept. 1973.
Sporocarps minute, mostly less than 0.5 mm, hypogeous, not aggregated, brown, irregularly globose, containing 2-12 ( - 78) zygospores. Zygospores hyaline, subglobose to globose, 48-68 x 52-76 urn. Spore walls hyaline, glistening, 2-4 urn thick, suspensor cells evanescent. Vegetative hyphae variable 3-7 (-16) ym diam, and may be thick-walled, to 5 ym.
177 rhizas included Endogone aggregafa, Sclerogone eucalypti and Glomw tubiforme Tandy (Warcup, 1985). Five pot cultures did not form sporocarps and the identity of the fungi remains unknown.
DISCUSSION
While Endogone reficulafa, like E. pisifomis (Berch & Fortin, 1983), appeared to be a saprophyte, the other species investigated all formed ectomycorrhizas which differed in complexity depending on fungus and host plant. The mycorrhizas formed by E. flammicorona on pine had little sheath and a prominent Hartig net in the cortex of roots. The Few spores were found with attached gametangia, but absence development of mycorrhizas by the other species from of subtending hyphae and spore contents of globules of nearly eucalypt forests appeared similar in that runner hyphae uniform size (Gerdemann & Trappe, 1974) are other evidence travelled along and to a lesser extent across roots, secondary of their zygosporic nature. Young zygospores had shining hyphae grew in the grooves between epidermal cells and from highly refractive walls that appeared single layered. Mature these arose a thin sheath across epidermal cells and a Hartig spores from dried material had a 2-layered wall, the outer net between cells. Both sheath and Hartig net might be about 1 um and the inner 1-3 pm wide, with a thin, external, continuous or discontinuous, the sheath might be predominon-staining (mucilaginous) layer. nantly one-cell-thick or develop into a many layered mantle. Specimens examined: Holotype, ADW 16992 (N191).isolated from E. With S. eucalypti the sheath tended to remain thin, but with E. obliqua grown in soil from burnt area, Kuitpo Forest, 7 Sept. 1973. aggregafa and E. fuberculosa complexity of sheath depended on Subsequently found ectomycorrhizal on naturally occumng eucalypt host and both species formed 'classical' ectomycorrhizas on seedlings from the same area. Further collections from mycorrhizas Eucalyptus and the perennial herb, Brunonia australis. have been made from the Grampian Mountains, Victoria and from Apart from E. flarnmicorona which was not subcultured, the several sites in Tasmania. Most collections have been made from species investigated grew satisfactorily on weak agar media seedlings from burnt sites or from heat-treated soil (Warcup, 1983). though cultures tended to die unexpectedly especially if kept Sclerogone eucalypti has a wide host range and pot cultures without frequent subculturing. All species formed hyphal usually containing sporocarps were formed with E. pbliqua, swellings to a greater or lesser extent on agar and around E. regnans, and E. sieberi. Mycorrhizas were also formed on roots in soil. Swellings did not become cut off by septa and Lepfospemzum sp., Melaleuca uncinata, Angophora cosfafa it is probable that they are sites of temporary accumulation of (Gaertn.) Dmce, Poranthera microphylla and Brunonia australis. cytoplasm and nutrients rather than resting structures. Both Mycorrhizas on eucalypts often had little reduction in the on agar and in soil empty swellings were often present besides number of root hairs present and occasionally a sheath was those packed with contents. Berch & Fortin (1982, 1983) formed around the base of hairs (Warcup, 1975). Runner reported hyphal swellings in E. incrassata Thaxter and E. hyphae travelled along roots with secondary hyphae growing pisifomis and they occur in vigorous colonies of Glomus in the grooves between epidermal cells. Across many tubiforme so they appear common in the Endogonaceae. Endogone flammicorona was only found in association epidermal cells a fine sheath, usually one-cell-thick but with and only formed ectomycorrhizas with pines, introduced occasionally thicker, was formed: where the sheath was plants in Australia. Whether the species from eucalypt forests prominent a Hartig net was present. Sclerogone eucalypti grew well on a variety of weak media will form mycorrhizas with pines was not investigated, but (Warcup, 1975). Intercalary hyphal swellings occurred on they showed no specificity with a range of Australian hosts secondary hyphae, and occasionally on recently isolated and formed ectomycorrhizas on all hosts tested with marked strains small sporocarps formed on agar. S. eucalypti showed growth responses in an autoclaved soil mix low in available marked stimulation of growth in the presence of maize or phosphate. Malajczuk, Molina & Trappe (1982) reported that cotton meal oil (0.2 g 100 ml-') if added to i N agar but not eucalypts and pines formed ectomycorrhizas in common with several broad-host-range fungi, but that fungi which are to water agar. known to associate exclusively with members of the Pinaceae, e.g. Suillus and Rhizopogon, did not form mycorrhizas with Taxa from mycowhizas any Eucalyptus species. The data here suggest that E. During examination of seedlings from burnt sites in South flammicorona belongs to the latter group. Australia and eucalypt regeneration coupes in Tasmania, I wish to thank Dr C. Palzer, Forestry Commission, numerous seedlings of Eucalyptus or Acacia were found with mycorrhizas formed by ectomycorrhizal Endogonaceae. In Tasmania for gift of seed of Eucalyptus and providing seedlings attempts to form pot cultures from these mycorrhizas about and soil from Tasmania, and Dr P. A. McGee for sporocarps half gave contaminating ascomycete or basidiomycte ecto- of Endogone reticulafa. mycorrhizas, but several pot cultures of Endogonaceae were established. Ectomycorrhizal species obtained from mycor-
Ectomycorrhizal zygosporic Endogonaceae
178
specificity and mycorrhizal compatibility with Pinus radiata. New Phytologist 91,467-482. BERCH, S. M. & FORTIN, J. A. (1982). Germination of zygospores TANDY, P. A. (1975a). Studies of sporocarpic Endogonaceae in Australia. Ph.D. Thesis, University of Adelaide. of Endogone incrassata. Mycologia 74,861-864. BERCH, S. M. & FORTIN, J. A. (1983). Endogone pisifarmis: axenic TANDY, P. A. (1975 b). Sporocarpic species of Endogonaceae in Australia. Australian journal of Botany 23, 849-866. culture and associations with Sphagnum, Pinus sylvestris, Allium TRAPPE, J. M. & GERDEMANN, J. W. (1972).Endogoneflammicorona cepa and Allium porrum. Canadian journal of Botany 61,899-905. sp. nov., a distinctive segregate from Endogone lactiflua. Transactions CHU-CHOU, M. & GRACE, L. J. (1979). Endogone flammicorona as a of the British Mycological Society 59,403-407. mycorrhizal symbiont of Douglas fir in New Zealand. New Zealand WALKER, C.(1985). Endogone lactiflua forming ectomycorrhizas with loumal of Forest Science 9,344-347. Pinus contorts. Transactions of the British Mycological Society 84, DANIELSON, R. (1982). Taxonomic affinities and criteria for 353-355. identification of the common ectendomycorrhizal symbiont of WARCUP, J. H. (1975). A culturable Endogone associated with pines. Canadian ]ournal of Botany 60, 7-18. eucalypts. In Endomycorrhizas (ed. F. E. Sanders, B. Mosse & P. B. FASSI, B. & PALENZONA, M. (1969). Sintesi micorrhizica tra Pinus Tinker), pp. 53-63. London, U.K.: Academic Press. strobus, Pseudotsuga douglasii ed "Endogone factiflua". Alfionia 15, WARCUP, J. H. (1983). Effect of fire and sun-baking on the soil 105-114. microflora and seedling growth in forest soils. In Soils: A n FASSI, B., FONTANA, A. & TRAPPE J. M. (1969). Ectomycorrhizae Australian Viewpoint, Division of Soils, CSIRO, pp. 735-740. formed by Endogone lactiflua with species of Pinus and Pseudotsuga. CSIRO: Melboume/Academic Press: London, U.K. Mycologia 61, 412-414. GERDEMANN, J. W. & TRAPPE, J. M. (1974). The Endogonaceae WARCUP, J. H. (1985). Ectomycorrhiza formation by Glomus tubiforme. New Phytologist 99,267-272. of the Pacific Northwest. Mycologia Memoir 5, 1-76. KOPE, H. H. & WARCUP, J. H. (1986). Synthesized ectomycorrhizal WARCUP, J.H. (1988). Mycorrhizal associations and seedling development in Australian Lobelioideae (Carnpanulaceae). associations of some Australian herbs and shrubs. New Phytologist Australian ]oumal of Botany 36, 461-472. 104,591-599. MALAJCZUK, N., MOLINA, R. & TRAPPE, J. M. (1982). Ecto- WARCUP, J. H. & McGEE, P. A. (1983).The rnycorrhizal associations of some Australian Asteraceae. New Phytologist 95,667-672. mycorrhiza formation in Eucalyptus 1. Pure culture synthesis, host
REFERENCES
(Received for publication 28 February 1989)
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Taxonomy, culture and mycorrhizal associations of some zygosporic Endogonaceae
J. H. WARCUP* Deparfment of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia, 5064
Taxonomy, culture and mycorrhizal associations of some zygosporic Endogonaceae. Mycological Research 94 (2):173-178 (1990). Endogone aggregata, E. tuberculosa and Sclerogone eucalypti gen. et sp. nov. have been shown to form ectomycorrhizas with Eucalyptus and other Australian ectomycorrhizal hosts, mycorrhiza complexity differing depending on fungus and host. E. flamrnicorona, however, formed ectomycorrhiza only with Pinus. E. reticulata grew among the roots of eucalypts and other plants but appeared to be non-mycorrhizal. E. aggregafa, E. tuberculosa, E. reticulata and S. eucalypti all grew on weak agar media, but E. flammicorona although
often growing extensively from sporocarps was not subcultured successhlly. Key words: Ectomycorrhiza, Endogonaceae, Sclerogone eucalypti, Agar culture of Endogone, Eucalyptus, Pinus. Zygosporic species of Endogonaceae in the genus Endogone (Gerdemann & Trappe, 1974), are generally considered to form ectomycorrhizas in contrast to chlamydosporic or azygosporic species in Glomus, Gigaspora, Acaulospora and Sclerocystis, which with few exceptions form vesiculararbuscular mycorrhizas. Few species of Endogone, however, have yet been shown to form ectomycorrhizas. Known ectomycorrhizal species include E. flammicorona Trappe & Gerdemann (Fassi & Palenzona, 1969; Trappe & Gerdemann, 1972) and E. lactiflua Berk. & Br. (Walker, 1985) both on pines, and the species tentatively designated ' E . eucalypti' from eucalypts (Warcup, 1975). Recently Berch & Fortin (1983) cultured E. pisiformis Link: Fr. on agar and showed that it was non-mycorrhizal. During research on ectomycorrhizal associations of plants in southern Australia, sporocarps of Endogonaceae were collected and attempts made to grow the fungi in agar culture and to establish pot cultures for examination of mycorrhizal associations. Ectomycorrhizas formed by aseptate fungi from the field were examined similarly. The results of these studies are recorded in this paper.
MATERIALS A N D M E T H O D S Sporocarps were collected by raking through surface soil and litter or they were recovered from pot cultures. E. flammicorona fruited regularly and often abundantly in pine plantations in the Adelaide Hills. Sporocarps of Endogonaceae under eucalypts, although never common, were found more frequently after fire than in undisturbed sites. Aseptate ~-
Present address: 6 Seafield Ave., Kingswood, S.A., 5062
ectomycorrhizas were occasionally found during examination of eucalypt seedlings from burnt areas or regeneration sites. Attempts at obtaining pot cultures were made using Eucalyptus obliqua L'Her. o r Pinus radiata D. Don as host plants. Intemal pieces of sporocarp, blocks of agar (4-5 mm2) containing fungal hyphae, or plugs of mycorrhizal roots were placed on tap roots of seedlings during transplanting in an autoclaved sand-soil mix low in available .phosphate. Plants . were grown in a growth room; details of soil mix and growing conditions are given in Warcup & McGee (1983). Mycorrhiza experiments were done under the same conditions but using a wider range of host plants and inoculum was always a plug (ca 0.1 g) of freshly harvested mycorrhizal roots. Attempts at obtaining agar cultures were made from mycorrhizal roots or from sporocarps. Mycorrhizal roots were washed thoroughly then placed in drops of sterile water in isolation plates. Tips of mycorrhizas were cut into lengths of 3-5 mm and older portions removed. Intemal pieces of sporocarp were cut out aseptically and gently teased into pieces of 1mm3 or smaller in drops of sterile water in isolation plates. Isolation plates were poured with molten but cooled Czapek-Dox+ yeast agar ($N)prepared with double glass distilled water (Warcup, 1975). Streptomycin (100 ug ml-l) and tetracycline (50 vg ml-') were used to control bacteria. The amount of growth obtained from different fungi varied markedly. Most showed initial growth from the inoculum which ceased after a few days; others continued growth for longer periods. Contamination by other fungi, especially Mortierella, was a problem, especially in isolations from mycorrhizal roots. Growth in plastic Petri dishes was poor unless they were wrapped with parafilm to minimize water loss. The crucial stage in obtaining agar cultures was usually
Ectomycorrhizal zygosporic Endogonaceae whether the initial growth could be subcultured successfully. Hyphal tips died rapidly so that subculturing required more growth free from contamination than is usual with fungi.
174 (1-2 cm2)often increased. Occasionally thin-walled intercalary hyphal swellings, to 20 pm, were formed in branch hyphae, these were not cut off by septa and did not aid survival of the fungus in agar. Limited growth on agar was also reported by Tandy (1975a) and Chu-Chou & Grace (1979).
RESULTS Endogone flammicorona Trappe & Gerdemann, Trans. Br. mycol. Soc. 5 9 : 403-407 (1972).
Sporocarps of variable size, to 3 cm, globose to subglobose and soft when young, hard, lobed or irregular at maturity. Peridium thin, white, becoming buff, often incomplete in old specimens. Gleba at first white, becoming salmonaceous, with zygospores showing as orange granules, usually exuding a milky latex when cut. Zygospores 56-86 x 54-70 ym, globose, ellipsoid or obovoid, enclosed in a tightly adhering hyphal mantle. Specimens examined: ADW16399, 16400, 16401, Kuitpo, under Pinus radiata, June, July, Sept. 1971. J. H. Warcup. Further sporocarps have been collected from Kuitpo on many subsequent occasions.
The fungus was always found in the vicinity of pines. Sporocarps were hypogeous in pine nurseries or pot cultures, but occurred in the lower layers of litter or embedded at the soil surface in pine plantations. At Kuitpo Forest sporocarps were found from June to November except in drought years. In pot culture E. flammicorona formed ectomycorrhizas with Pinus radiafa and P. pinaster Ait. with which sporocarps were formed in 5-6 months. Mycorrhizas were not formed with Eucalyptus obliqua from sporocarp inoculum or when eucalypt seedlings were grown close to young P. radiafa with actively growing mycorrhizas formed by E. flammicorona. Tandy (1975a) reported that E. sieberi (L.) Johnson did not become mycorrhizal with sporocarpic or mycorrhizal inoculum of E. jlammicorona. Previously E. flammicorona has been shown to be ectomycorrhizal with Pseudofsuga menziesii (Mirbel) Franco (Fassi, Fontana & Trappe, 1969; Chu-Chou & Grace, 1979), Ps. douglasii (Lindl.) Carr. (Fassi & Palenzona, 1969), 10 species of Pinus (Fassi & Palenzona, 1969; Fassi et al., 1969; Tandy, 1975 a) and Cedrus deodora (Roxb.) D. Don (Tandy, 1975 a). The ectomycorrhizas formed by E. flammicorona are pale yellow to brown, unbranched or branched dichotomously to give a finger-like appearance. Root hairs are usually sparse or absent from mycorrhizas. Thick-walled wide hyphae ramify along the root surface with finer hyphae occurring between epidermal cells and in places forming a thin sheath across them. The fungus forms an extensive Hartig net in the cortex and both long and short roots may be infected. The mycorrhizal roots are of similar appearance to those formed by the ectendotrophic Ascomycetes (Danielson, 1982) but the hyphae generally lack septa and are not intracellular. The mycorrhizas formed by E. bctiflua on Pinus conforfa Dougl. ex Loud. are similar (Walker, 1985). Endogone flammicorona collected from Kuitpo often grew well initially on isolation plates of Q N agar, especially on the surface of the medium. Growth in agar was slower and aerial hyphae rare. Growth usually slowed within 4 to 5 d and all growth ceased within a month. Subcultures from colonies with strong initial growth showed little growth into the fresh Q N medium although density of hyphae within the inoculum plug
Endogone reticulata Tandy, Ausf.
1. Bof.
2 3 : 849-866
(1975). Sporocarps hypogeous, pale to deep brick red, irregular, to 2 cm, formed between and sometimes incorporating soil material. Peridiurn to 40 Urn thick, often lacking in part, of compacted thin-walled hyphae 3-5 ym diam, paler than gleba. Gleba orange to pale red, of thin-walled hyphae, 4-8 pm diam with larger branched, thick-walled structural hyphae, 12-16 pm diam present. Latex absent. Zygospores 80-110 ym, brick red, spherical, ovoid, or irregular from pressure, with a 1-2 layered mantle, not aggregated. Spore wall in 2 layers, a thin dark exospore, 1-2 ym thick, and a thicker hyaline endospore, 4-10 i.lm thick. Gametangia usually parallel, wide, 24-44 pm, subequal with a wide attachment to the zygospore. Specimens examined: ADW 16986, Mt Lofty, under eucalypts, 25 July 1984, P. A. ~ c ~ e e ; 16987, ' k ~Stirling, ~ in garden, 5 Sept. 1984, P. A. McGee; ADW 16988, Eagle-on-Hill, under eucalypts, 11 Sept. 1984, P. A. McGee.
The sporocarps recorded here differed from that discussed by Tandy (1975 b) in being hypogeous not epigeous, being irregular in shape and containing incorporated organic matter. Attempts at establishing pot cultures with E. obliqua showed that the fungus grew well in the rhizosphere but rarely on the surface of roots and did not form rnycorrhizas. Host plants remained small (Table 1). The fungus formed long hyphae with areas of dense chains of hyphal swellings, 8-15 i.lm diam, on secondary hyphae. Hyphal swellings were not cut off by septa, most were densely ~ a c k e dwith protoplasm but some were without contents. Culture of the fungus on agar was attempted from pieces of sporocarp and from washed hyphae with hyphal swellings collected from among roots of E. obliqua. Growth occurred from both types of inoculum, the fungus usually growing initially as long unbranched hyphae. These hyphae were very sensitive to the presence of certain unidentified bacteria on the isolation plates. If the tip of a hypha grew near a bacterial colony, the whole hypha, possibly 2-3 cm long, collapsed and died. A few hyphae, however, were subcultured successfully and grew into large colonies that produced copious hyphal swellings. With age such colonies became dull reddish-brown, partly from colour of hyphae and partly pigment in the agar. The presence of copious hyphal swellings packed with contents did not aid survival and without subculturing such colonies died within 6 to 8 wk. A variety of media (Warcup, 1975) were tried to improve growth and survival but none was better than iN agar. Presence of autoclaved seed, millet, hemp, oat and colonies of Rhodoforula on the medium did not increase growth. Endogone reficulata resembles E. pisifomis (Berch & Fortin, 1983) in that both grow on weak agar media, ramify in the root zone of plants but d o not appear to be mycorrhizal.
J. H. Warcup Table I. Comparison of shoot gowth of six e~torn~corrhizal species with and without inoculation with Endogone aggregata, E. tuberculosa or E. reticulata
Host Eucalyptus obliqua Brunonia australis Podotheca angustifolia Poranthera microphylla Stylidium graminifolium Lobelia tenuior
t
E. agg.
E. tub.
E. ref.
Nil
700
535 175
54 80 445
Means of 3 replicates, harvested after 8 weeks. - no test.
Endogone aggregata Tandy, A w t . J. Bot. 2 3 : 849-866 (1975).
Sporocarps hypogeous, greyish-white, hard, ovoid to irregular, to 1.8 cm. Peridium greyish-white, becoming dark brown on drying, often incomplete, tough, to 150 ym thick, can bepeeled from gleba. Gleba initially white, developing aggregates of yellow zygospores with white sterile hyphae in between. Latex absent. Zygospores spherical to ovoid, sometimes irregular from pressure, 9 6 1 4 6 x 106-176 ym, with a 2layered wall, endospore 6-10 ym wide, exospore 8-14 ym, exospore appearing laminated in aniline blue. Zygospore aggregates irregular, to 1 mm diam, with many spores. Large external hyphae, 14-28 pm diam, with ribbed walls were associated with the sporocarps. Specimens examined: ADW 16990, sporocarps from Kuitpo Forest, 15 July 1984,under old planted E. maculata Hook., site bumt in 1983.
Ectomycorrhizas were established with E. obliqlla and Melaleuca uncinafa R. Br. The fungus also formed ectomycorrhizas with numerous herbs including Brunonia awtralis Sm., Podotheca angustifolia (Labill.) Less., Poranthera microphylla Brogn., Stylidiurn graminifolium Sw. ex Willd. and Lobelia tenuior R. Br. (Warcup, 1988). Marked shoot growth responses followed inoculation of E. aggregata to E. obliqua and the above herbs (Table I). Examination of the e~tom~corrhizas of E. aggregata with the hosts in Table 1 suggested that the pattern of formation of mycorrhizas in the different hosts was similar though the mycorrhizas appeared different. An exception might be B. australis which has much slower growing roots than the other species. Runner hyphae, 6 1 0 ym diam, grew along and to a lesser extent across the surface of roots. O n some roots secondary hyphae grew in the junctions of epidermal cells and from these arose a Hartig net and a fine one-cell-thick sheath across epidermal cells. There were runner hyphae in the rhizosphere as well as on the root surface. Both E. obliqua and B. awtralis had 'classical' ectomycorrhizas, the mycorrhizas being wider through tangential elongation of epidermal cells than non-mycorrhizal roots, slightly so in E. obliqua, markedly so in B. awtralis. The mycorrhizas had few root hairs, a fungal sheath, 8-10 pm thick usually enclosing the tips of mycorrhizas, and a Hartig net. In the other four herbs the sheath was usually one-cell-
thick but might be thicker in patches, might not cover all epidermal cells and rarely covered the root tip. A Hartig net occurred in all species, except possibly S. graminifolium, but not necessarily between all epidermal cells. Root hairs were present but depressed in number and length compared with those on uninfected roots. The number of external hyphae in the rhizosphere was usually greater in these species than with E. obliqua and B. australis. Mycorrhizas on Poranthera microphylla had areas of enlarged epidermal cells (Kope & Warcup, 1986). Occasional intercalary hyphal swellings, 16-22 ym diam, were noted in external hyphae around P. microphylla. Endogone aggregata grew readily from sporocarp pieces if these were relatively free from bacteria and contaminating fungi. Hyphae were often profuse on the surface of the medium. Surface growth was difficult to cut and tended to tear and die when hyphae were cut during subculturing. Good growth in the agar was desirable in making subcultures. Colonies were white especially if profuse hyphal swellings, to 40 ym, were present. Growth was improved using Fe Edta in the :N medium. E. aggregata grew in culture for some 18 months but was eventually lost, possibly through insufficient subculturing. ,Endogone tuberculosa Lloyd, Mycol. Writings (Mycol. Notes 56): 799 (1918).
Sporocarps hypogeous, irregular, 1.5-2-0 cm, grey from the coat of soil particles adhering to the peridium. Peridium white where visible, thin. Gleba pale yellow from closely packed zygospores in large irregular areas with veins of white mycelium between, drying dingy olive yellow, incorporating soil particles. Glebal hyphae thin-walled, 5-9 ym diam, closely packed, crushed in the areas with zygospores. Zygospores closely packed, variable in shape, globose to ovoid but mostly irregular from pressure, 54-96 x 50-102 pm, wall with 2 layers, endospore 1-2 pm wide, exospore 2-4 ym. Zygospores arise as buds from persistent gametangia. Latex absent but copious oil released from cut surfaces. Specimens examined: A D W 16991, Kui fpo Forest, under old planted E. maculata, 2 7 July 1984, area bumt in 1983. Endogone tuberculosa formed pot cultures readily on E. obliqua from sporocarp pieces and from hyphae in agar and on other
Ectomycorrhizal zygosporic Endogonaceae hosts from mycorrhizas. The mycorrhizas were similar to, but differed in detail, from those formed by E. aggregata. Growth responses of plants to E. fuberculosa are given in Table I. Well-defined ectomycorrhizas were formed with E. obliqua and B. ausfmlis with a fungal sheath, 6-10 pm thick, and Hartig net. The mycorrhizas, especially on E. obliqua, were woolly in appearance from many short, fine, empty hyphae projecting from the sheath. External hyphae, 6-10 um diam, were common in the rhizosphere with occasional globose to ovoid terminal hyphal swellings, 30-35 pm diam, arising on short lateral branch hyphae. The e~tom~corrhizas formed on Lobelia fenuior, Stylidium graminifolium and Podofheca angusfifolia were generally similar to those formed on these hosts by E. aggregata. O n Poranfhera microphylla, however, the fungus formed more and greater enlargement of patches of epidermal cells where the fungus was present. The enlarged epidermal cells were covered by a sheath, to 4 pm thick, with a prominent Hartig net between cells. Endogone fuberculosa also grew well from sporocarp pieces though not as vigorously as E. aggregafa. Cultures were white with most hyphae submerged in the agar. However, after some weeks, the agar cultures were lost due to contamination. Sclerogone Warcup, gen. nov.
Etym.: Greek sclero- (hard) and -gone (reproductive organs). In reference to the sclerotioid sporocarps Sporocarpia minuta, hypogaea, bmnnea, dura, non aggregata. Pertdium et gleba ex hyphis compactis intertextis aseptatis et parietibus crassis constantes. Zygasporae generatim paucae, hyalinae, suspensoris celiulae evanescentes. Sp. typ.: Sclerogone eucalypti Warcup (syn. Endogone eucalypti ined., Warcup, 1975)
Sporocarps minute, hypogeous, brown, hard, not aggregated. Peridium and gleba of compacted interwoven thick-walled hyphae. Zygospores generally few, hyaline, suspensor cells evanescent.
Gerdemann & Trappe (1974) considered that the genus Endogone Link: Fr. consisted of several diverse elements that were not closely related. They retained the zygosporic species in Endogone sensu stricfo and raised four segregate genera for species with ~ h l a m ~ d o s p o r or e s azygospores. However, even in Endogorre sensu sfricfo species differ in characters such as sporocarp construction and presence or absence of latex, and a better understanding of sporocarp formation may indicate further segregate genera. Sclerogone is considered such a segregate genus and differs from other known species of Endogone in its small, hard, sclerotioid sporocarps. E. alba (Petch) Gerdemann & Trappe also has small ovoid sporocarps, but they are aggregated and fused together in pulvinate masses. Sclerocysfis has small hard sporocarps but they contain chlamydospores. Sclerogone eucalypti Warcup, sp. nov.
(Fig. 1)
Sporocarpla minuta, pro parte maxima minus quam 0.5 rnm, hypogaea, non aggregata, brunnea, irregulariter globosa, 2-12 (-78)
Fig. 1. Sclerogone eucalypti. A, Habit sketch of a sporocarp with four zygospores; B, cells of sporocarp; C, young zygospore.
J. H. Warcup zygosporas contenta. Zygosporae hyalinae, subglobosae vel globosae, 48-68 x 52-76 vm. Sporae panes hyalinus, lucens, 2-4 vrn latus, suspensoria cellulae evanescentes. Hyphae vegetative variabiles, 3-7( (-16) vm diam, et aliquando pariete crasso ad 5 vm. Holotypus ADW 16992 (N191) ex Eucalypti obliquae, Kuitpo For., S. Australia, 7 Sept. 1973.
Sporocarps minute, mostly less than 0.5 mm, hypogeous, not aggregated, brown, irregularly globose, containing 2-12 ( - 78) zygospores. Zygospores hyaline, subglobose to globose, 48-68 x 52-76 urn. Spore walls hyaline, glistening, 2-4 urn thick, suspensor cells evanescent. Vegetative hyphae variable 3-7 (-16) ym diam, and may be thick-walled, to 5 ym.
177 rhizas included Endogone aggregafa, Sclerogone eucalypti and Glomw tubiforme Tandy (Warcup, 1985). Five pot cultures did not form sporocarps and the identity of the fungi remains unknown.
DISCUSSION
While Endogone reficulafa, like E. pisifomis (Berch & Fortin, 1983), appeared to be a saprophyte, the other species investigated all formed ectomycorrhizas which differed in complexity depending on fungus and host plant. The mycorrhizas formed by E. flammicorona on pine had little sheath and a prominent Hartig net in the cortex of roots. The Few spores were found with attached gametangia, but absence development of mycorrhizas by the other species from of subtending hyphae and spore contents of globules of nearly eucalypt forests appeared similar in that runner hyphae uniform size (Gerdemann & Trappe, 1974) are other evidence travelled along and to a lesser extent across roots, secondary of their zygosporic nature. Young zygospores had shining hyphae grew in the grooves between epidermal cells and from highly refractive walls that appeared single layered. Mature these arose a thin sheath across epidermal cells and a Hartig spores from dried material had a 2-layered wall, the outer net between cells. Both sheath and Hartig net might be about 1 um and the inner 1-3 pm wide, with a thin, external, continuous or discontinuous, the sheath might be predominon-staining (mucilaginous) layer. nantly one-cell-thick or develop into a many layered mantle. Specimens examined: Holotype, ADW 16992 (N191).isolated from E. With S. eucalypti the sheath tended to remain thin, but with E. obliqua grown in soil from burnt area, Kuitpo Forest, 7 Sept. 1973. aggregafa and E. fuberculosa complexity of sheath depended on Subsequently found ectomycorrhizal on naturally occumng eucalypt host and both species formed 'classical' ectomycorrhizas on seedlings from the same area. Further collections from mycorrhizas Eucalyptus and the perennial herb, Brunonia australis. have been made from the Grampian Mountains, Victoria and from Apart from E. flarnmicorona which was not subcultured, the several sites in Tasmania. Most collections have been made from species investigated grew satisfactorily on weak agar media seedlings from burnt sites or from heat-treated soil (Warcup, 1983). though cultures tended to die unexpectedly especially if kept Sclerogone eucalypti has a wide host range and pot cultures without frequent subculturing. All species formed hyphal usually containing sporocarps were formed with E. pbliqua, swellings to a greater or lesser extent on agar and around E. regnans, and E. sieberi. Mycorrhizas were also formed on roots in soil. Swellings did not become cut off by septa and Lepfospemzum sp., Melaleuca uncinata, Angophora cosfafa it is probable that they are sites of temporary accumulation of (Gaertn.) Dmce, Poranthera microphylla and Brunonia australis. cytoplasm and nutrients rather than resting structures. Both Mycorrhizas on eucalypts often had little reduction in the on agar and in soil empty swellings were often present besides number of root hairs present and occasionally a sheath was those packed with contents. Berch & Fortin (1982, 1983) formed around the base of hairs (Warcup, 1975). Runner reported hyphal swellings in E. incrassata Thaxter and E. hyphae travelled along roots with secondary hyphae growing pisifomis and they occur in vigorous colonies of Glomus in the grooves between epidermal cells. Across many tubiforme so they appear common in the Endogonaceae. Endogone flammicorona was only found in association epidermal cells a fine sheath, usually one-cell-thick but with and only formed ectomycorrhizas with pines, introduced occasionally thicker, was formed: where the sheath was plants in Australia. Whether the species from eucalypt forests prominent a Hartig net was present. Sclerogone eucalypti grew well on a variety of weak media will form mycorrhizas with pines was not investigated, but (Warcup, 1975). Intercalary hyphal swellings occurred on they showed no specificity with a range of Australian hosts secondary hyphae, and occasionally on recently isolated and formed ectomycorrhizas on all hosts tested with marked strains small sporocarps formed on agar. S. eucalypti showed growth responses in an autoclaved soil mix low in available marked stimulation of growth in the presence of maize or phosphate. Malajczuk, Molina & Trappe (1982) reported that cotton meal oil (0.2 g 100 ml-') if added to i N agar but not eucalypts and pines formed ectomycorrhizas in common with several broad-host-range fungi, but that fungi which are to water agar. known to associate exclusively with members of the Pinaceae, e.g. Suillus and Rhizopogon, did not form mycorrhizas with Taxa from mycowhizas any Eucalyptus species. The data here suggest that E. During examination of seedlings from burnt sites in South flammicorona belongs to the latter group. Australia and eucalypt regeneration coupes in Tasmania, I wish to thank Dr C. Palzer, Forestry Commission, numerous seedlings of Eucalyptus or Acacia were found with mycorrhizas formed by ectomycorrhizal Endogonaceae. In Tasmania for gift of seed of Eucalyptus and providing seedlings attempts to form pot cultures from these mycorrhizas about and soil from Tasmania, and Dr P. A. McGee for sporocarps half gave contaminating ascomycete or basidiomycte ecto- of Endogone reticulafa. mycorrhizas, but several pot cultures of Endogonaceae were established. Ectomycorrhizal species obtained from mycor-
Ectomycorrhizal zygosporic Endogonaceae
178
specificity and mycorrhizal compatibility with Pinus radiata. New Phytologist 91,467-482. BERCH, S. M. & FORTIN, J. A. (1982). Germination of zygospores TANDY, P. A. (1975a). Studies of sporocarpic Endogonaceae in Australia. Ph.D. Thesis, University of Adelaide. of Endogone incrassata. Mycologia 74,861-864. BERCH, S. M. & FORTIN, J. A. (1983). Endogone pisifarmis: axenic TANDY, P. A. (1975 b). Sporocarpic species of Endogonaceae in Australia. Australian journal of Botany 23, 849-866. culture and associations with Sphagnum, Pinus sylvestris, Allium TRAPPE, J. M. & GERDEMANN, J. W. (1972).Endogoneflammicorona cepa and Allium porrum. Canadian journal of Botany 61,899-905. sp. nov., a distinctive segregate from Endogone lactiflua. Transactions CHU-CHOU, M. & GRACE, L. J. (1979). Endogone flammicorona as a of the British Mycological Society 59,403-407. mycorrhizal symbiont of Douglas fir in New Zealand. New Zealand WALKER, C.(1985). Endogone lactiflua forming ectomycorrhizas with loumal of Forest Science 9,344-347. Pinus contorts. Transactions of the British Mycological Society 84, DANIELSON, R. (1982). Taxonomic affinities and criteria for 353-355. identification of the common ectendomycorrhizal symbiont of WARCUP, J. H. (1975). A culturable Endogone associated with pines. Canadian ]ournal of Botany 60, 7-18. eucalypts. In Endomycorrhizas (ed. F. E. Sanders, B. Mosse & P. B. FASSI, B. & PALENZONA, M. (1969). Sintesi micorrhizica tra Pinus Tinker), pp. 53-63. London, U.K.: Academic Press. strobus, Pseudotsuga douglasii ed "Endogone factiflua". Alfionia 15, WARCUP, J. H. (1983). Effect of fire and sun-baking on the soil 105-114. microflora and seedling growth in forest soils. In Soils: A n FASSI, B., FONTANA, A. & TRAPPE J. M. (1969). Ectomycorrhizae Australian Viewpoint, Division of Soils, CSIRO, pp. 735-740. formed by Endogone lactiflua with species of Pinus and Pseudotsuga. CSIRO: Melboume/Academic Press: London, U.K. Mycologia 61, 412-414. GERDEMANN, J. W. & TRAPPE, J. M. (1974). The Endogonaceae WARCUP, J. H. (1985). Ectomycorrhiza formation by Glomus tubiforme. New Phytologist 99,267-272. of the Pacific Northwest. Mycologia Memoir 5, 1-76. KOPE, H. H. & WARCUP, J. H. (1986). Synthesized ectomycorrhizal WARCUP, J.H. (1988). Mycorrhizal associations and seedling development in Australian Lobelioideae (Carnpanulaceae). associations of some Australian herbs and shrubs. New Phytologist Australian ]oumal of Botany 36, 461-472. 104,591-599. MALAJCZUK, N., MOLINA, R. & TRAPPE, J. M. (1982). Ecto- WARCUP, J. H. & McGEE, P. A. (1983).The rnycorrhizal associations of some Australian Asteraceae. New Phytologist 95,667-672. mycorrhiza formation in Eucalyptus 1. Pure culture synthesis, host
REFERENCES
(Received for publication 28 February 1989)
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