Australasian Mycologist

Volume 24(1) ISSN 1441-5526 AUSTRALASIAN MYCOLOGICAL SOCIETY Office Holders

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Volumes 1-14 (2) published as the Australian Mycological Newsletter (ISSN 1322 1396); volumes 14 (3)- 17(4) published as the Australasian Mycological Newsletter (ISSN 1329-4377).

AUSTRALASIAN MYCOLOGIST

Editorial Board: Managing editors: Cheryl Grgurinovic* & Jack Simpson

Editorial Committee: Kevin Hyde Wieland Meyer John Walker

The Australasian Mycologist is published three times a year by the Australasian Mycological Society.

This issue was published on 14 June 2005. © Australasian Mycological Society, Inc. 2005.

The opinions expressed in the Australasian Mycologist are not necessarily those of the editors or publisher, and they do not accept responsibility for any information or advice contained herein. *correspondence and manuscripts relating to the Australasian Mycologist should be sent to: Dr Cheryl Grgurinovic, 15 Sprent Street, Narrabundah, A.C.T. 2604, Australia.

Australasian Mycological Society (ABN 86 738 494 713).

Cover image: Inocybe violaceocaulis © P.B. Matheny. CXViiRSITY 3lr rJYi:.i.DI^.C£OyS FUIISI IN A cm??mG SOIL

Z. Commandeur \ P.M. Letcher 2 and P.A. McGee 1*

? School of Biological Sciences A12, University of Sydney 2006, Australia, email: peterm@bio. usyd. edu. au 2 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL USA 35487.

* Corresponding author.

Abstract

This pilot investigation found that cropping had an insignificant impact on the diversity of chytridiaceous fungi at an experimental site in semi-arid northern NSW, Australia. A total of 14 different chytrids were observed on baits placed with soil. Ten different fungi were found in soil from each of a vegetated adjacent block, and a cotton/wheat rotation, eight fungi were found under each of a cotton/cotton and cotton/vetch rotations, and six under a fallow (no plants) treatment. Six fungi were common to each treatment. One fungus was only found at the adjacent site, three others were only found under the rotations. The data indicate that diversity of chytridiaceous fungi and plants were unrelated at the experimental site.

Z. Commandeur, P.M. Letcher & P.A. McGee (2005). Diversity of chytridiaceous fungi in a cropping soil. Australasian Mycologist 24 (1): 1-6.

Introduction

Chytridiaceous fungi, herein called chytrids (Barr periodically dry, mineral deficient environment. Thus 2001), have been collected from various the factors that determine diversity of chytrids in environments including sphagnum bogs (Sparrow & cultivated soils might be unrelated to the diversity of Lange 1977), beaches and dunes (Booth 1971a, the plants growing in the soil. 1971b), halomorphic soils (Booth 1969), and drought affected soils (Willoughby 1984, Willoughby & Rigg Chytrids form zoospores which require free water for 1983). Chytrids are probably ubiquitous in soil dispersal. Chytrids may rapidly pass from stage to (Longcore 2001). stage of their life cycle, with the total time as short as 48 hours (Ward 1939). In ecological terms, such The nutrition of chytrids is not well understood fungi may be thought of as ruderal (though see (Powell 1993). Experiments in axenic culture (Barr Andrews 1992) in that the size of populations may 2001), and use of diverse baits to trap different increase rapidly when nutrients are available. If soil species, indicate that a diversity of substrates may be chytrids follow this pattern, then their populations used by chytrids. Thus we might predict that a large may markedly fluctuate in soil used for cropping. array of different carbon-based substrates in soil Chytrids might be relatively abundant during periods would support a high diversity of chytrids. For of 'recovery' from disturbance such as flooding and instance, diverse plant populations might sustain cultivation, and depleted during extended periods of diverse populations of chytrids as happens for other plant growth when other fungi are more competitive. fungi (Apinis 1958, Christensen & Whittingham 1965, Wicklow et al. 1974) while relatively high Cultivation of soil is associated with loss of carbon, concentrations of organic matter might support and changes in plant exudates and organic matter abundant populations of chytrids (Lee 2000). The (Ayanaba et al. 1976, Ellert & Gregorich 1996, diversity of chytrids under heath vegetation in the Srivastava & Singh 1989). Cropping reduces the Sydney basin was less than under nearby dry diversity of plants and plant remains. Decreases sclerophyll, wet sclerophyll and rainforest vegetation in carbon stores in cropping soil are attributed to (Letcher, McGee & Powell 2004), possibly due to the inputs of carbon being reduced, and rates of decay of plant litter being enhanced. The effect of cropping on (total 25) in each of the rotations and the adjacent site chytrids is unknown. Disturbance, especially on 11 March 2003, 2 May 2003 and 17 June 2003. cultivation, often increases the concentration of Samples were collected from the fields prior to biologically available nutrients in soil which may irrigation to reduce the effect of flooding on chytrids. lead to a temporary increase in abundance of chytrids No samples were taken from the cotton/vetch rotation (Lozupone & Klein 2002). However, a reduction of in March. The river site was only sampled in March chytrid abundance and diversity is predicted for soils to provide a check on diversity in undisturbed soils, in which cropping is long-standing. and to indicate whether the common species are widespread in the district. Soil samples were returned The present study investigated the diversity of to the laboratory immediately, and stored at 4°C until chytrids in an agricultural soil. The objective was to processed. determine whether chytrid diversity is directly associated with plant diversity. If the prediction is Ten g of each soil sample was placed in a separate supported then the diversity of chytrids will be Petri dish and 40 ml of sterile deionized water added. highest in soils where many annual and perennial The soil was briefly stirred. Baits of sterile pine plants are growing together, followed by cropped pollen, keratin (snake skin), chitin (prawn soils with simple rotations, and least diverse in long exoskeleton) and cellulose (onion leaf epidermis) term fallow where few plants grow. were floated on the surface of the water and the Petri dish lid replaced. Baits were examined microscopically after six and 12 days (Barr 1969). Materials and Methods Identifying any particular species requires multiple Soil samples were collected from replicated plots of observations over a period of several days to weeks the experimental field at the Australian Cotton in order to observe all the characters needed to Research Institute, Narrabri, NSW, Australia identify each fungus (Letcher & Powell 2002b). For (ACRI:149°47'E 30°20'S). Rotations in place for the present study, chytrids were grouped based on more than three years include cotton/cotton (cotton in morphology, and then, where possible, chytrids were summer, fallow in winter), cotton/wheat (cotton in isolated to pure culture on YpSs (yeast, peptone and summer, wheat in winter), cotton/vetch (cotton in soluble starch), PYG (yeast, peptone and glucose), summer, vetch in winter), and fallow (without PmTG (peptone, tryptone and glucose) or cellulose plants). Soil was also collected from plots set out on medium, and their cultural characteristics used to an uncultivated adjacent site located less than 100 m tentatively identify the fungi (Karling 1977, Sparrow from the experimental field. The adjacent site had a 1960). cover of annual and perennial grasses and herbs, and a few shrubs. Soil samples were also collected on one Diversity of chytrids was calculated for each field occasion from a normally moist site located beside site using the Gleason index (Burnett 2003) and again the Namoi River in Narrabri (river site), about 10 km using the Simpson index (Simpson 1949). Abundance southeast of the ACRI. is notoriously difficult to estimate. Here, we used the proportion of pollen grains colonised by chytrids, or Cotton (Gossypium hirsutum L.) was sown on visually estimated as the proportion of cellulose, 3 October 2002 in the cotton beds of the rotation chitin or keratin substrate occupied by sporangia to experiment. All crop rotations were irrigated and the indicate abundance. adjacent and river sites were not irrigated. Cotton and adjacent plots were defoliated on 31 March 2003 and The data on diversity in this pilot study were both again on 7 April 2003. Cotton lint was harvested skewed and kurtosed. The samples over time were from 7 to 15 May 2003. The cotton beds were then from within one field indicating a lack of cultivated using vertical harrows. Vetch (Vicia sativa independence. Thus the nonparametric Mann- L., cv. Namoi) and wheat (Triticum aestivum L., cv. Whitney was used to compare diversity under each Yallaroi) were sown on 23 May 2003 into the type of vegetative cover using data for each harvest cotton/vetch and cotton/wheat rotations respectively. as the sample (Zar 1996). The Sign test was used to compare presence and absence of fungi under each Four or five soil samples (approximately 100 grams type of vegetative cover (Freund & Simon 1992). each) were collected on 16 December 2002 at each of three depths (surface, 5 cm and 10 cm) from each of five replicate plots of all of the rotations and the Results adjacent site to determine if soil depth affected the presence of chytrids. Subsequently, five soil samples Of the 14 chytrids identified at the ACRI (Fig. 1), were collected at less than 10 cm depth from all plots a total of ten different chytrids was found in the soil Table 1. Frequency of collection, and Simpson's Index and Gleason Index of chytrids at ACRI. Co/Co - cotton/cotton rotation, Co/W - cotton/wheat rotations, Co/V - cotton/vetch rotation, fallow (Fal) and adjacent (Adj) site, with the (n) number of samples for each site. * Unable to collect samples March 2003, # single collection. Fungus Co/Co Co/W Co/V* Fal Adj Total Namoi number of River# sites (90L (87) (62) (90) J?0L_„ (15) Rhizophlyctis 3 4 3 3 4 5 1 rosea Catenophlyctis sp. 4 4 3 4 3 5 1 Unknown C 3 4 2 3 3 5 0 Rhizophydium 0 0 2 0 1 2 1 subglobosum Rhizophydium 4 4 3 4 4 5 0 sphaerotheca Rhizophydium 4 4 3 4 4 5 1 pollinis-pini Unknown G 0 0 0 0 3 1 0 Unknown H 1 3 0 0 0 2 | 0 Unknown I 0 0 1 0 0 1 1 Rhizophydium sp. 0 1 0 0 0 1 1 parasite 1 Rhizophydium sp. 0 1 0 0 0 1 0 parasite 2 Catenophlyctis sp. 1 0 0 0 1 2 0 Allomyces sp. 1 1 1 2 4 2 5 1 Allomyces sp. 2 0 1 0 0 1 2 6 Others 3 Total 8 10 8 6 10 10 Hs 0.54 0.65 0.57 0.45 0.74 f | 3.32 HG 1.11 1.79 1.45 1.11 1.77

collected from the adjacent site and the cotton/wheat similar trend. The Simpson indices (Hs) (Table 1) rotation, eight from cotton/vetch rotation, and indicate the highest chytrid diversity is found at the the cotton/cotton rotation and six from the fallow soil adjacent site at the ACRI, followed by the (Table 1). Six chytrids were present at all sites. One cotton/wheat rotation, cotton/vetch rotation, chytrid was only found at the adjacent site (Table 1). cotton/cotton rotation, and the fallow rotation as the While most chytrids were seen in the first sampling, least diverse. The Simpson index is suitable for large species' richness increased over time in soil from the sample sizes (N > 80) (Burnett 2003) and so is cotton/wheat rotation (five to ten), and in the fallow not useful for measuring diversity at the river site (N plots (five and six, Table 2). Two chytrids are = 15). The Gleason index (HG) is a simpler measure parasites on other chytrids and both were uncommon. of diversity, less sensitive to sample size (Burnett

Because the richness of chytrids was similar at each 2003). The HG of chytrid diversity at the river site depth in the first collection of samples, a single was well above that of any of the ACRI sites, where sample was collected subsequently. Ten chytrids the fallow and the cotton/cotton rotation were the were obtained from the river site, of which four were least diverse sites (Table 1). common at the ACRI, and three were not found at ACRI (Table 1). Analysis of data using the Mann- The abundance of the six common fungi at ACRI Whitney test indicates that species richness did not (Table 3) is presented because insufficient data are differ significantly between sites at ACRI (P > 0.05). available for the remainder. Only Catenophlyctis sp. The presence of chytrids found associated with each was abundant. Abundance of all other chytrids was type of vegetative cover did not differ significantly low at all times (Table 3). Rhizophydium pollinis-pini between sites or between the river and ACRI (Sign was present at all sites at ACRI and found on 16% test, P > 0.05). of pollen from the river site in March. Abundance of Rhizophlyctis rosea was low and infrequent at The two indices used to indicate diversity showed a all sites at ACRI, and relatively high at the river site Table 2. Number (cumulative number) of chytrids isolated from soil under each rotation, and the adjacent site at ACRI, and the river site at four times. * = no sample collected. Mixed Cotton Cotton Cotton Fallow River Plants Cotton Wheat Vetch December 2002 9 6 5 5 * March 2003 ,6(10) 4(7) 9(9) * 5(5) 10 May 2003 5(10) 6(8) 6(9) 7 ("8) A© * June 2003 6(10) 5(8) 8(10) .5(8) 6(6) *

Table 3. Mean (± SD) abundance of common chytrid morphotypes in soil from the rotations at the ACRI. Co/Co Co/W Co/V Fallow Adjacent Morphotype Bait Unknown C Pollen 2±2 2.4 ±0.8 2.2 ± 1 2.8 ±2.5 0.5 ± 0.4 Rhizophydium Pollen 2.2 ±2 1.6 ± 1.1 1.5 ± 1 1.5 ±0.8 0.9 ± 0.3 sphaerotheca Rhizophydium Pollen 3.9 ±4.8 1.6 ±0.8 1.5 ± 1.1 0.9 ±0.3 1.5 ±0.7 pollinis-pini Rhizophlyctis Onion 0.8 ± 1 2.8 ± 4.2 2.3 ±2.8 0.5 ± 0.6 3.9 ±3.1 rosea Catenophlyctis Keratin, 12.3 ±2.9 4.4 ± 4.2 13.7 ±6.1 18.8 ± 10 1.9 ± 1.9 sp. chitin Allomyces sp. Keratin, 0.8 ± 1.5 0.8 ± 1.5 0.8 ± 1.2 0.5 ±0 1 ± 1.4 chitin

where it colonized 22% of the onion skin. adjacent than the fallow site, similar numbers were found in rotations with severely reduced plant diversity. Interestingly, two parasitic chytrids were Discussion found in cultivated soils used in the cotton/wheat rotation. If the parasites are removed from A total of six to ten chytrid morphotypes were found calculations, the cotton/wheat rotation has eight in the soils collected on four occasions from plots in chytrids, the same as the other two rotations. Six five sites at the ACRI over a period of seven months. chytrids were common to all sites at the ACRI. One In addition, limited sampling from the river site chytrid was relatively abundant at the rotation sites resulted in ten morphotypes, the same as the adjacent and absent in the adjacent site. The remaining site. Though diversity indices indicated differences, chytrids were rare. Thus ten chytrids were isolated these differences are not statistically significant and from each relatively undisturbed site, eight from our hypothesis of a decline in diversity with reduced cropped soils, and six from the fallow site. While the plant diversity is rejected. Cropping appears to have trend is towards lower diversity of chytrids with no statistically significant impact in diversity of fewer plants, six chytrids were found at all sites. chytrids at ACRI. The inclusion of the fallow site provides a simple test Using a similar sampling process, 14 different of the importance of plant materials for growth and chytrids were observed in open heath, and 23 survival of chytrids. The fallow site has no crop under dry sclerophyll vegetation of the Sydney Basin cover, though it may have had the occasional weed of eastern Australia (Letcher et al. 2004). While the over the last three years. Some insects may have shed number of chytrids in the heath appears to be similar exoskeletons, and fungi no doubt continue to slowly to the adjacent and river sites, the dry sclerophyll grow during periods when the soil is moist. In other vegetation had a significantly greater diversity. words, limited carbon continues to be deposited on Letcher et al. (2004) observed 14 out of 34 chytrids and in the soil. However, these substrates would be on pollen, which is similar to the ratio found at the rare and possibly localised at the soil surface. Six ACRI, indicating that our observations are common morphotypes were found at all sites at the comparable. Overall, limited chytrid diversity was ACRI. observed at the ACRI and in heath vegetation. Thus it might be argued that a factor other than plant The substrates used by these chytrids in the field diversity limits overall diversity of chytrids. remain unclear. The fallow site has extremely low concentrations of organic carbon and the carbon is in While more chytrids were found in soil from the a form that is thought to be complex and resistant to degradation (Hulagalle 2000). The data support the the river site are moist for much of the year. The possibility that some widespread chytrids may rotation soils are irrigated and their moisture content degrade complex carbon substrates in nature or use a is kept at levels to maximise plant growth. The diversity of widespread substrates for growth and adjacent site is not irrigated. The climate is naturally development. hot and dry. A similar diversity of chytrids in soil from the river, adjacent and the cotton/wheat rotation Alternatively, the fungi may be present in soil as sites does not support a hypothesis of available resistant structures that germinate in baited solutions. moisture being related to chytrid diversity, but this If the fungi are present as resistant structures in soil, lack of relationship may be due to other climatic and the structures germinate during the baiting factors such as heat (Gleason et al. 2004) and procedure, then reasons for remaining dormant associated desiccation. require elucidation. The ACRI is located in the hot, dry, semi-arid part of In this investigation four different baits resulted in northern NSW, Australia, where heat and lack of different arrays of chytrids being observed. water may have an overriding importance in Presumably different substrates are important for determining chytrid diversity. The dissemination of specific taxa of chytrids in nature. The limited chytrids in nature is unclear, though data on survival diversity of chytrids observed may be due to use of of some chytrids indicate that they may be inappropriate baits for the chytrids present at the transported with dust (Gleason et al. 2004). Thus ACRI. settled areas may have chytrids of exotic origin adapted to the current conditions in addition to Similarly, we observed one abundant chytrid. The indigenous species adapted to the original conditions. technique we used is only indicative. The coverage of With this in mind it is interesting to note that one baits may indicate an increased proportion of chytrid was only found at the adjacent site, and then zoospores settling on the bait, or the rapid release of in three of the four collections. A further three fungi zoospores from few sporangia, rather than the were only found at the river site. These data support relative number of sporangia present in soil. the view that some chytrids have specific conditions Catenophlyctis sp. survives high temperatures when regulating their survival, and that any one location dry (Gleason, Letcher & McGee 2004) and has other may have a limited diversity under hot, dry, semi-arid cultural characteristics that appear important for environments. These conditions remain to be growth at Narrabri (Gleason, Letcher & McGee elucidated. unpublished). Thus the relative abundance of the chytrid may be ecologically important. A limited diversity of chytrids was found at the ACRI. Though plant and fungal diversity appear to The primary source of organic energy in soil arises be only weakly correlated, the case for plants from photosynthesis. Thus many heterotrophic reducing the size of populations of some chytrids is organisms, including fungi, are closely linked to also supported. Indeed, chytrids were found where plants. Catenophlyctis sp. was slightly more abundant there have been no plants for the last three years, in soil from the fallow than the vegetated plots indicating that some chytrids may use complex indicating a negative relationship between the organic substrates. While it is clear that chytrids chytrid and plants. Amongst many factors, the size of are present in soil from hot dry semi-arid regions, the populations of chytrids is likely to be related to the mechanisms regulating their presence and function presence and availability of food, and competition are, as yet, unclear. with other microbes for that food. In addition, plants or plant remains may release to the environment compounds that are toxic to some microbes. Thus the Acknowledgements relation between plants and chytrids may be complex. Our thanks go to Dr David Nehl for allowing us These hypotheses can be tested in controlled access to his rotation experiments at the ACRI. experimental conditions. Completion of this study was supported by NSF- PEET Grant #DEB-9978094. Possible explanations for low overall diversity are unclear. Letcher & Powell (2001, 2002a) observed more chytrids under mosses where presumably water References is sequestered for extended periods and is more Andrews, J.H. (1992) Fungal Life-History Strategies, evenly distributed through the soil profile. Thus in G.C. Carroll & D.T. Wicklow (eds), The Fungal moist sites might yield greater diversity of chytrids, Community. 2nd edn, pp. 119-145. Marcel Dekker, including those susceptible to desiccation. Soils from New York. Apinis, A.E. (1958). Distribution of microfungi in Letcher, P.M., McGee, P.A. & Powell, M.J. (2004). soil profiles of certain alluvial grasslands. Angew. Zoosporic fungi from soils of New South Wales. Pflanzensoziol. 15, 83—90. Australasian Mycol. 22, 99-115. Ayanaba, A., Tuckwell, S.B. & Jenkinson, D.S. Letcher, P.M. & Powell, M.J. (2001). Distribution of (1976). 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P.B. Matheny 1 and N.L. Bougher 2

1 Biology Department, Clark University, 950 Main St., Worcester, Massachusetts 01610 USA. CS/RO Forestry and Forest Products, PO Box 5 Wembley, WA 6913, Australia. Current address: Western Australian Herbarium, Department of Conservation and Land Management, Locked Bag 104, Bentley Delivery Centre, WA 6983, Australia.

ABSTRACT

A violet species of Inocybe, for which the name I. geophylla var. lilacina has often been misapplied, is described as new from urban bushlands and Eucalyptus forests and plantations in southern Western Australia. The name I. violaceocaulis is proposed to accommodate this species that is characterised by the violet to lilac colour, elliptic basidiospores, the presence of pleurocystidia, infrequent caulocystidioid cells, presence of a veil, and host association with myrtaceous plants. The taxonomy and systematic position of I. violaceocaulis within Inocybe are discussed.

P.B. Matheny & N.L. Bougher (2005). A new violet species of Inocybe (Agaricales) from urban and rural landscapes in Western Australia. Australasian Mycologist 24 (1): 7—12.

•

INTRODUCTION MATERIALS AND METHODS

The taxonomy of Inocybe species in Australia is Colour notes of field collections were documented poorly known (Grgurinovic 1997, Miller & Hilton with Munsell Color Charts (1954), Ridgway (1912) 1987). Recently, however, re-examination of and Kornerup & Wanscher (1967). Sections of fresh J.B. Cleland's type material of Inocybe from South and dried material were placed in or rehydrated with Australia, studies of herbarium material at PERTH 3% KOH and examined under a compound light and CSIRO in Perth, and first-hand collections of microscope. Italicised measurements refer to mean Inocybe species from Western Australia have shown dimensions following Matheny, Aime & Henkel that numerous taxa are undescribed, and that (2003). The ratio of spore length to width is European names have been misapplied to Australian presented as the q value. Line drawings were done species (Watling 1985). One of the most conspicuous with the aid of a drawing tube. Herbarium of these undescribed taxa in southern Western abbreviations follow Holmgren, Holmgren & Barnett Australia is a violet-coloured representative of (1990). For explanation of anatomical characters, e.g. Inocybe with smooth basidiospores and cauloparacystidia, see Kuyper (1986). pleurocystidia. This species occurs in urban bushlands in and around Perth, sand dune systems, and in native karri {Eucalyptus diversicolor) forests TAXONOMY in south-west Western Australia. Many herbarium collections at PERTH that correspond to this species Inocybe violaceocaulis Matheny & Bougher were labelled 'Inocybe geophylla var. lilacina (Peck) sp. nov. (Figs 1-3). Gillet' or referred to this north temperate name. However, members of the I. geophylla (Fr. : Fr.) Etymology: in reference to the violet-coloured stipe. Kumm. group differ in several morphological details as well as phylogenetic history. As a result, I. Pileus primo violaceus deinde 'Cinnamon-Brown' violaceocaulis is proposed for this new violet- velumbrinus, 1.5-3.5 cm latus, nec squarrosus, coloured Inocybe from Western Australia. siccus, caro immutabilis, odor spermaticus. Lamellae b

Figure 1. Microscopic characters of Inocybe violaceocaulis (holotype). Fig. 1a. Basidiospores. Fig. 1b. Basidia. Fig. 1c. Pleurocystidia. Fig. 1d. Cheilocystidia. Fig. 1f. Caulocystidioid cells. The scale bar is equal to 10 urn for basidiospores and 25 urn for other cells. adnexae vel subadnatae, primo violaceotinctae vel age to obtusely conical or campanulate to plano­ pallidus-griseus deinde brunneae, pallidae-fimbriatae. convex, with a large but low obtuse umbo; margin Stipes 2-A cm x 4-9 mm, cortina praeditus, basis non decurved, undulate with age; surface dry but marginato-bulbosus, solidus, fibrillosus, 'Light somewhat lubricous when moist, smooth at the centre Wistaria Violet' vel 'Plumbago Gray', basis but may becoming diffracted-scaly with age, silky- cremeotinctus. Basidiosporae laeves, ellipticae, 7.5- fibrillose to fibrillose towards the margin, the fibrils 9.5 x 5-5.5 um. Cauloparacystidia nulla. diverging around the centre with age, the margin Pleurocystidia 52-63 x 11-15 um, fusiforma. In occasionally split; ground colour brown or sylvis eucalyptinis vel urbanis. Holotypus hie umbrinous—'Cinnamon Brown', 'Dresden Brown', designatus in PERTH (E7030 / PBM 2164), isotypus or 'Snuff Brown', generally a lighter shade or in WTU. 'Tawny-Olive' towards the margin (10YR 4/3-4/4- 5/3-5/4), with a lilac superficial layer at least Pileus 1.5-3.5 cm, conical in youth, expanding with when young, which wears away with age to reveal the Figure 3. Basidiomata of Inocybe violaceocaulis (E8055) collected under introduced Eucalyptus maculata and Lophostemon (Tristania) confertus in Perth, Western Australia. umbrinous ground colour; context pallid, unchanging under E. diversicolor F. Muell., Allocasuarina where bruised, up to 5 mm thick under the disc; decussata (Benth.) L.A.S. Johnson, and Agonis odour spermatic; taste not remarkable. Lamellae flexuosa (Willd.) Sweet, also in moist old sand dunes narrowly adnate, seceding, close, up to 40 reaching under Agonis flexuosa; Perth to Denmark, Western the stipe, with a few tiers of lamellulae, light gray Australia; May through August and October. (10YR 6/1), at times with a weak lilac tinge, when young becoming yellowish brown (10YR 5/4), the Material examined: AUSTRALIA. Western edges white-fimbriate, ventricose, up to 5 mm wide. Australia: E0747, in bush near NE carpark of Stipe 2-4 cm x 4-6 mm at the apex, enlarged to CSIRO, Perth, 21 June 1990, leg. N. Bougher; swollen at the base, at times rounded-bulbous, there E0801, on red earth soil in 1986 plantings of up to 9 mm diam.; basal mycelium white but sparse Eucalyptus globulus, Carpenters block off 7 Day Rd, and not conspicuous; cortina fugacious, pale violet; near Manjimup, leg. N. Malajczuk and G. Hardy, surface fibrillose, nowhere agglutinated, pruinose at 11 October 1991; E0898 (KS 648/93), in deep litter extreme apex or not at all, lilac or grayish lilac— in Karri forest under E. diversicolor, Corymbia 'Light Wistaria Violet' to 'Plumbago Gray' (15- calophylla, Allocasuarina decussata, and Agonis 16B4) throughout except for the pallid to cream- flexuosa, Mt Shadforth Res., adjoining NW corner lot tinged base; dried specimens retaining their 406, 22 May 1993, leg. K. and A. Syme; E3737 violaceous tinges; cortext coloured like the surface, (ZT2760), in bush under C. callophylla and central and lower context pallid. Stipe (and pileus) E. marginata, Kings Park, Perth, 26 June 1985, leg. surface orange with 15% KOH. E. Horak; E5916, north side of Herdsman lake under introduced Eucalyptus, on edge of mowed grass next Basidiospores 1.5-8.6-9.5 (-10.0) x 5.0-5.2-5.5 u.m, to park, Perth, 12 July 1997, leg. N. & M. Bougher; Q = 1.36-7.65-1.90 (n = 42/3), smooth, mostly E7013 (PBM 2164) (holotype, PERTH; isotype, elliptic but on occasion subreniform to WTU), on lawn under E. gomphocephala in front of subamygdaliform, at times with a suprahilar CSIRO reception bldg, Perth, 6 Aug. 2001, leg. depression, the apices rounded, yellowish brown in P.B. Matheny; E7014 (PBM 2165), same locality as KOH with thickened walls, the apiculus distinct, in E7013, 7 Aug. 2001, leg. P.B. Matheny; E7016 deposit dark brown (6F8) (Fig. la). Basidia 25-32 x (PBM 2167), on lawn under E. gomphocephala, Bold 7-8 um, 4-sterigmate, clavate, hyaline (Fig. lb). Park east of Perry Lakes Stadium, Perth, 7 Aug. Pleurocystidia 52-63 x 11-15 um, fusiform, usually 2001, leg. P.B. Matheny; E7017 (PBM 2168), Bold without a distinct neck, thin-walled to only slightly Park but different location than above, 7 Aug. 2001, thick-walled, the walls 0.5-1.5 (im thick, hyaline; leg. P.B. Matheny; E7019 (PBM 2170), Bold Park apices obtuse, sparsely crystalliferous; with a basal along Brookdale Ave, under E. gomphocephala, pedicel (Fig. lc). Cheilocystidia similar to 8 Aug. 2001, leg. P.B. Matheny; E7025 (PBM 2176), pleurocystidia (Fig. Id), mostly thin-walled; Bold Park, east of Perry Lakes Stadium under paracystidia clavate, thin-walled, hyaline (Fig. le). E. gomphocephala, 16 Aug. 2001, leg. P.B. Matheny; Caulocystidioid cells infrequent, restricted to extreme E7026 (PBM 2177), same locality as E7025, 16 Aug. apex, 55-78 x 13-16 um, fusiform to subcylindric, 2001, leg. P.B. Matheny; E7045 (PBM 2198), on thin-walled, hyaline, the apices mostly bare (Fig. If); ground in Karri forest under E. diversicolor along cauloparacystidia not observed; superficial hyphae trail around the Karri Valley Resort, Karri Valley, interwoven, hyaline, at times faintly violet in mass in 19 Aug. 2001, leg. P.B. Matheny; E7053 (PBM KOH but pigments cytoplasmic and not incrusted, 2205), in lawn on ground under E. marginata, the hyphae cylindric, 4-11 um diam. Pileipellis a Subiaco entry into Kings Park, Perth, 27 Aug. 2001, cutis of smooth, cylindric hyphae, 5-12 um diam., leg. P.B. and S.D. Matheny; E8053 (CSIRO), parallel to somewhat interwoven, no clear velipellis Cunningham Terrace Park, Daglish, Perth, hyphae on mature pilei, light or faint cinnamon in 31°57'05.2"S, 115°48'30.2"E, among mulch and mass; tramal hyphae hyaline, refractive hyphae not woodchips under introduced E. marginata and observed. Clamps present. E. gomphocephala, 9 July 2004, leg. N.L. Bougher and R. Hart; E8055 (CSIRO) corner of Morden Road Habitat: in small clusters or singly on grassy lawns in and Ednah Street, Wembley Downs, Perth, parks and landscaped areas under Eucalyptus 31°55'15.0"S, 115°46'36.4"E, in grassy lawn under gomphocephala DC, E. marginata Sm., and planted E. maculata and Lophostemon {Tristania) Corymbia calophylla (Lindl.) K.D. Hill & confertus, 16 July 2004, leg. N.L. Bougher; PERTH L.A.S. Johnson, under introduced Eucalyptus 05302331 (KS 333/91), in moist old sand dunes in maculata Hook, and Lophostemon (Tristania) moss below Agonis flexuosa, near Limpoo River, confertus (R.Br.) Peter G.Wilson & J.T. Waterh., in 5 Juniperina Creek area, 14 Aug. 1991, leg. K. Syme; year-old E. globulus Labill. plantation, in Karri forest PERTH 00755702, in sandy soil, Cannington, no date, leg. R. Hilton; OKM 24599 (VPI), on the ground under Agonis at Lurie Hall, UWA campus, distinct from it. The I. geophylla group contains the Perth, 22 May 1991, leg. O.K. and H. Miller, and following north temperate species: I. pudica Kiihn., L. and M. Bailey. I. lilacina (Peck) Kauffman, I. agglutinata Peck, I.fuscodisca (Peck) Massee and I. armenica Commentary: the name I geophylla var. lilacina Huijsman, most of which lack violet or lilac (Peck) Gillet has been misapplied for many years to pigmentation (Matheny 2005). For a consideration of this conspicuous violet-coloured Inocybe from other European lilac-coloured taxa with Western Australia. Like members of the I. geophylla pleurocystidia, see Esteve-Raventos & Villarreal group the spores are elliptic and the cystidia at most (2001), Kuyper (1986), and Stangl & Veselsky slightly thick-walled. However, members of the (1982). I. geophylla group, which occur in north temperate regions, are characterised by numerous caulocystidia at the stipe apex, and agglutinated fibrils at the base Acknowledgements of the stipe and on the pileal disc. Inocybe We thank Mr Graham H. Bell of the State Herbarium violaceocaulis (Figs 2 and 3) is distinguished by of South Australia (AD) for the loan of J.B. Cleland's infrequent caulocystidioid cells at the extreme apex type collections of Inocybe and the curator and staff of the stipe, absence of agglutinated fibrils, and at PERTH for loans of Inocybe material. Orson K. association with myrtaceous plants in Australia. Miller, Jr kindly shared with us his Inocybe collections from Australia. Travel and stay in Perth Several smooth-spored species of Inocybe with violet were funded to the first author by an anonymous colouration from Indomalaya and Australasia have donor and facilitated by staff at CSIRO Forestry and been documented by Horak (1980). Inocybe ionides Forest Products in Perth, Western Australia. Work by Corner & E. Horak, described from Sabah, appears the second author was undertaken as part of the Perth similar but lacks a cortina and has no pleurocystidia. Urban Bushland Fungi project funded by Inocybe subgeophylla Hennings is not conspicuously Lotterywest. The accession, AY732208, was lilac and occurs with fagaceous hosts in Indonesia. produced in the lab of David Hibbett and supported Inocybe violeipes E. Horak and 1. violaceovelata by the Assembling the Fungal Tree of Life project E. Horak, described from Papua New Guinea, are (DEB 0228657) funded by the National Science squamulose and have stipes with belts of veil tissue. Foundation. We thank two anonymous referees for The latter does not have elliptic spores. No violet- their constructive comments and suggestions. coloured species with smooth basidiospores are reported from New Zealand (Horak 1977). An examination of Cleland's type collections of Inocybe References at the State Herbarium of South Australia (AD) Esteve-Raventos, F. & Villarreal, M. (2001). Inocybe indicates that Cleland did not previously describe lavandulochlora, una nuova specie della sezione I. violaceocaulis. This species is also not described in Lilacinae R. Heim. Rivista di Micologia 3, 215— Grgurinovic (1997). 223. Grgurinovic, C.A. (1997). Larger fungi of South Combined rpbl, rpbl, and nuclear large subunit Australia. The Botanic Gardens of Adelaide and (25 S) ribosomal RNA nucleotide sequences strongly State Herbarium & The Flora and Fauna of South suggest that I. violaceocaulis (E7045 and E7013, Australia Handbooks Committee, Adelaide. GenBank accession numbers AY380404 and Holmgren, P.K., Holmgren, N.H. & Barnett, L.C. AY732208, respectively) evolved in a major clade (1990). Index herbariorum part I. Herbaria of the with other smooth-spored species of Inocybe that world. 8th edn. Regnum Vegetabile 120. New possess pleurocystidia (Matheny 2005). Within this York Botanical Garden, New York. strongly supported group, I violaceocaulis is nested Horak, E. (1977). Fungi Agaricini Novaezelandiae in a clade with the following north temperate species: VI. Inocybe (Fr.) Fr. and Astrosporina Schroeter. I. pusio P. Karst., I. sindonia (Fr.) P. Karst, New Zealand Journal of Botany 15, 713-747. I. queletii Maire & Konr. and I. flocculosa (Berk.) Horak, E. (1980). Inocybe (Agaricales) in Sacc. Of these, only I. pusio shares violet Indomalaya and Australasia. Persoonia 11,1-37. pigmentation of the basidiocarps. Matheny (2005) Kornerup, A. & Wanscher, J.H. (1967). Methuen indicates that I. violaceocaulis is sister to a clade handbook of colour. 2nd edn. Methuen & Co. Ltd, containing I. queletii and I. flocculosa. This set of London. species, in addition to the lilac tinged I. pusio, is Kuyper, T.W. (1986). A revision of the genus sister to a clade containing I. geophylla and related Inocybe in Europe. I. Subgenus Inosperma and the taxa. None of the taxa enumerated above evolved in smooth-spored species of subgenus Inocybe. the I. geophylla group and thus, are phylogenetically Persoonia (Suppl.) 3, 1-247. Matheny, P.B. (2005). Improving phylogenetic Munsell Soil Color Charts (1954). Munsell Color inference of mushrooms with RPB1 and RPB2 Company, Inc., Baltimore. nucleotide sequences (Inocybe; Agaricales). Ridgway, R. (1912). Color standards and color Molecular Phylogenetics and Evolution 35, 1—20. nomenclature. Published by the author, Washington Matheny, P.B., Aime, M.C. & Henkel, T.W. (2003). DC. New species of Inocybe from Dicymbe forests of Stangl, J. & Veselsky, J. (1982). Risspilze der section Guyana. Mycological Research 107,495-505. Lilacinae Heim. Ceskd Mykologie 36, 85-99. Miller, O.K., Jr & Hilton, R.N. (1987). New and Watling, R. (1985). Impressions of Australian interesting agarics from Western Australia. mushrooms. Victorian Naturalist 103, 116-122. Sydowia 39, 126-137. PHAEOPHLEOSPORA EPICOCCOIDES 1L£ ,F LI/EASE Of

J. A. Simpson \ Y. Xiao 2 and Bi, H.-Q.1

1 Forest Resources Research, NSW Department of Primary Industries, P.O. Box 100, Beecroft 2119, Australia. Current address: 75 Sprent Street, Narrabundcth, ACT2604 2 Forestry Academy of Sichuan Province, 18 Xing Hui Xi Lu Rd, Chengdu 610081, China.

J.A. Simpson, Y. Xiao & H.-Q. Bi (2005). Phcteophleospora epicoccoides leaf disease of Eucalyptus in China. Australasian Mycologist 24 (1): 13-14.

While examining stands of Eucalyptus grandis and both Corymbia and Eucalyptus (Sankaran et al. 1995, E. grandis x E. urophylla hybrids being grown in Walker et al. 1992). By contrast, P. delegatensis is plantations for wood production in Sichuan Province, known only from E. delegatensis and E. obliqua China, we observed that the leaves were often rather (Crous 1998), while P. lilianiae is known only from chlorotic. When examined closely it was observed Corymbia exima (Walker et al. 1992). These latter that on the chlorotic leaves there were abundant two species of Phaeophleospora are known only small aggregations of black-coloured spores on the from Australia. The most damaging species from an abaxial surface. Selected specimens were pressed and economic perspective is the tropical P. destructans returned to Australia for further study. which is known from E. camaldulensis, E. grandis, E. urophylla and hybrids of those species in East Microscopic examination showed the spore masses Timor, Indonesia, Thailand and Vietnam (Old et al. were of holoblastic, rough-walled conidia, extruded 2003, Wingfield et al. 1996). The teleomorph, where from immersed, subepidermal or substomatal, known, is a species of Mycosphaerella Johanson unilocular pycnidia, with ornamented, dark (Crous & Wingfield 1997). pigmented, percurrent conidiogenous cells but no conidiophores. This combination of characters Collections examined: China, Sichuan, Jia Jiang, indicated a species of Phaeophleospora Rangel Eucalyptus grandis, five year old trees, J.A. Simpson (Crous et al. 1997). The Phaeophleospora conidia (C059), 10.vii.2004 DAR 76880. Jia Jiang, present on the eucalypt leaves collected in China are E. grandis x E. urophylla, two years old, 2-5 euseptate, 33-58 x 3.2-5.2 um, narrowly J.A. Simpson (C061), 10.vii.2004 DAR 76881. obclavate, with apex obtuse, straight or curved, brown, thick-walled, verruculose. The fungus was Most species of Mycosphaerella, and related identified as P. epicoccoides, and is a new record for anamorphs, that infect eucalypt leaves cause so- China. called mycosphaerella leaf blotch disease (MLB) (Crous & Wingfield 1997) with necrosis of infected Five species of Phaeophleospora are known to occur tissues prior to spore production i.e. these fungi are on eucalypts: necrotrophs (Thrower 1966). Species of P. delegatensis (R.F. Park & Keane) Crous; Phaeophleospora are unusual in that the species are P. destructans (M.J. Wingfield & Crous) facultative biotrophs, spreading intercellularly Crous, F.A. Ferreira & B. Sutton; through the leaf mesophyll, but often not causing leaf P. epicoccoides (Cooke & Massee) Crous, necrosis prior to sporulation. Infection usually results F.A. Ferreira & B. Sutton; in premature defoliation, but spore production can P. eucalypti (Cooke & Massee) Crous, occur on infected leaves for a long period of time. F.A. Ferreira & B. Sutton; and Hood et al. (2002) made similar observations with P. lilianiae (J. Walker, B. Sutton & I. Pascoe) P. eucalypti on E. nitens in New Zealand. There is Crous, F.A. Ferreira & B. Sutton. massive spore production from chlorotic green leaves. Conidia are dispersed by water splash and by Phaeophleospora epicoccoides and P. eucalypti have wind. been recorded from many parts of the world, including Australia. They have broad host ranges in Often considered of minor importance, P. epicoccoides has been associated with serious defoliation in Crous, P.W. & Wingfield, M.J. (1997). New species plantations in South Africa (Knipscheer et al. 1990) of Mycosphaerella occurring on Eucalyptus leaves and New South Wales (Simpson unpublished), and in in Indonesia and Africa. Canadian Journal of nurseries in Australia (Walker 1962). Management of Botany 75,781-790. the pathogen is difficult. Old et al. (2003) suggested Crous, P.W., Ferreira, F.A. & Sutton, B. (1997). A the only viable management strategy for plantations comparison of the fungal genera Phaeophleospora is to select and use resistant or tolerant germplasm. and Kirramyces (Coelomycetes). South African This approach has been successful in Indonesia (Old Journal of Botany 63, 111-115. et al. 2003). In Sichuan, the eucalypt trees are being Hood, I.A., Chapman, S.J., Garner, J.F. & Molony, grown for fibreboard production or veneer, on a six K. (2002). Seasonal development of septoria leaf or seven year rotation, from seed gathered locally blight in young Eucalyptus nitens plantations in from superior trees in existing plantations. In this New Zealand. Australian Forestry 65, 153-163. situation of intensively managed, very short rotation Knipscheer, N.S., Wingfield, M.J., Swart, W.J. crops, management by tree selection and tree (1990). Phaeoseptoria leaf spot of Eucalyptus in breeding should be achievable. More than 130 South Africa. South African Journal of Forestry species of eucalyptus have been introduced to 154, 56-59. Sichuan in the past 100 years (Li & Hu 2003). There Li, X. & Hu, T. (2003). Genetic resources of has been no comprehensive study of the pathogens eucalypts and their development in Sichuan, in present in these plantings that are the most northerly J.W. Turnbull (ed.), Eucalypts in Asia. ACIAR in China. The phenology of eucalypts is complex Proceedings 111, 123-125. with repeated new flushes of foliage, and leaves of Old, K.M., Wingfield, M.J. & Yuan, Z.Q. (2003). A different morphology depending upon the age of the Manual of Diseases of Eucalypts in South-East tree (Hood et al. 2004). The impact of Asia. Center for International Forestry Research, P. epicoccoides on growth of eucalypt plantations in Bogor Barat, Indonesia. Sichuan is not known. We would expect that Sankaran, K.V., Sutton, B., Minter, C.F. & Minter, fungicides would provide cost effective control of the D.W. (1995). A checklist of fungi recorded from pathogen in eucalypt propagation nurseries but not in Eucalyptus. Mycological Papers 170, 1—376. commercial plantations. Thrower, L.B. (1966). Terminology for plant parasites. Phytopathologische Zeitschrit 56, 258- 259. Acknowledgements Walker, J. (1962). Notes on plant parasitic fungi. 1. The Australian Centre for International Agricultural Proceedingss of the Linnaean Society of New South Research supported this study as part of ACIAR Wales 87, 162-176. Project FST/2001/086. The assistance of Mme Hu, Walker, J., Sutton, B.C. & Pascoe, I.G. (1992). Sichuan Academy of Forestry, Chengdu, China, is Phaeoseptoria eucalypti and similar fungi on gratefully acknowledged. The collections from China Eucalyptus, with a description of Kirramyces gen. were admitted to Australia under AQIS permit nov. (Coelomycetes). Mycological Research 96, 200410709. 911-924. Wingfield, M.J., Crous, P.W. & Boden, D. (1996). Kirramyces destructans sp. nov., a serious References pathogen of Eucalyptus in Indonesia. South African Crous, P.W. (1998). Mycosphaerella spp. and their Journal of Botany 62, 325—327. anamorphs. Mycologia Memoir 21, v + 1-170. THE FUNGAL ENDOPHYTES OF DIPODIUM VARIEGATUM (ORCHIDACEAE)

J.J. Bougoure 1 and J.D.W. Dearnaley23

1 School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Australia. 2 Department of Biological and Physical Sciences, The University of Southern Queensland, Toowoomba 4350, Australia. 3 Author for correspondence: Fax +617 4631 1530; Email: [email protected]

Abstract

We have attempted to grow fungi from pelotons isolated from roots of the myco-heterotrophic orchid species Dipodium variegation on 3 different growth media. As well, we have analysed the fungal DNA within roots of the orchid using ITS-PCR analysis, cloning and molecular sequencing.

Fungi failed to grow out from pelotons isolated from plant roots. ITS rDNA sequences were successfully amplified and cloned from roots of three orchid plants. Comparison of these sequences with ITS rDNA in GenBank revealed that the fungal community of D. variegatum roots consists of Russula spp. and non-mycorrhizal soil Deuteromycetes.

J.J. Bougoure & J.D.W. Dearnaley (2005). The fungal endophytes of Dipodium variegatum (Orchidaceae). Australasian Mycologist 24 (1): 15-19.

Introduction

Myco-heterotrophs are plant species that obtain a rrhizal fungi, associated with myco-heterotrophic carbon source from an associated fungus. Myco- species, are representatives of the homobasidio- heterotrophic plants exist as both monocots and mycetes (Table 1). dicots, with most being monocotyledonous. The largest group of such plants is the Orchidales, 280 Research into the fungal endophytes of Australian species of which are entirely myco-heterotrophic terrestrial orchids has mostly focussed on the (Leake 1994). Although myco-heterotrophy is autotrophic orchid species (e.g. Huynh et al. 2004, generally linked with achlorophyllous plants, a Milligan & Williams 1988, Perkins & McGee 1995, number of species exist that obtain carbon both Perkins et al. 1995, Pope & Carter 2001, Warcup autotrophically and heterotrophically. For example, 1971, 1973, 1981) while the myco-heterotrophic most members of the Family Orchidaceae, which are species have largely escaped attention. Warcup generally autotrophic when mature, pass through a (1985, 1991) successfully isolated Rhizoctonia-like heterotrophic phase during their development, often fungal endophytes from two species of the rare associated with the limited food reserves of the subterranean orchid Rhizanthella. He named the generally minute seeds (Smith & Read 1997). endophyte of R. gardneri, Thanatephorus gardneri sp. nov. after succeeding in producing its teleomorph Fungi are generally quite easy to isolate from (Warcup 1988). pelotons (hyphal coils) in orchid roots and are amenable to growth in pure culture. Although a The orchid genus Dipodium or 'hyacinth orchids', diversity of orchid species have been sampled across contains a number of leafless myco-heterotrophic different geographical areas of the world, the fungal species that appear to rely heavily on carbon endophytes are largely similar (Rasmussen 2002, provided by endophytic fungi (Warcup 1990). The Smith & Read 1997). The fungi are all identity of these fungal endophytes is unknown. basidiomycetes with the majority belonging to the Warcup (1981, 1991) succeeded in isolating a fine, form genus Rhizoctonia, within the white, undamped and slow growing endophyte heterobasidiomycetes (Table 1). A few orchid myco- from roots of D. punctatum J.E. Sm. but was unable to Table 1. A list of fungal genera capable of forming orchid mycorrhizas. Anamorphic and teleomorphic genera are included for the Heterobasidiomycetes. Adapted from Rasmussen (2002) and the recent systematic overview of Basidiomycetes according to Jens H. Petersen, http://www.mycokey.com/systematics.html.

Heterobasidiomycetes Homobasidiomycetes Genera Order Order Genera Anamorph Teleomorph Ceratorhiza Ceratobasidiwn Gloeocystidiales Russula Ceratobasidiales Oliveonia Moniliopsis Thanatephorus Hymenochaetales Erythromyces Thelephora, Tulasnellales Epulorhiza Tulasnella Thelephorales Tomentella Auriculariales Sebacina Agaricales Armillaria, Mycena

Figure 1. Cross section of root of D. variegatum showing large numbers of brown-coloured fungal structures. Scale bar = 100 um. assign the fungus a taxonomic identity other than plants at Toohey State Forest, Brisbane, QLD, 'not a rhizoctonia'. Warcup (1981) isolated a Australia (27°33'S, 153°02'E) and two plants from Tulasnella-Uke fungal endophyte from the epiphytic Duggan Park, Toowoomba, QLD, Australia D. pandanum F.M. Bail. In this study we have used (27°35'S, 151°59'E). One cm long root portions two approaches to identify the fungal endophyte of were cut and rinsed under tap water. These portions the myco-heterotrophic orchid Dipodium variegatum were then soaked in 100% bleach for 30 seconds and R. Br., a widespread woodland species in South-east rinsed with sterile dFLO three times. Thin, transverse Queensland (Stanley & Ross 1989). We have isolated sections were then cut from the root portions and single pelotons from roots of the orchid using these were then crushed to release individual microtechniques and attempted to grow these on a pelotons from cortical cells (some uncrushed sections number of different media. As well, we have used were kept and photographed using a Nikon E600 PCR-amplification of rDNA ITS regions, cloning and upright photomicroscope (Nikon Corporation, sequence comparison of fungi from colonised Tokyo, Japan)). While viewing under a compound D. variegatum roots. The results of these microscope, healthy (non-collapsed) individual investigations are presented here. pelotons were removed with a 2.5 ul volume micropipette to malt extract agar (MEA) or one Materials and Methods sixth strength neutral dox yeast (NDY6) agar plates containing 15 mg 1" streptomycin and 15 mg Acquisition of orchid and fungal material 1" tetracycline or potato dextrose (PD) agar 1 Roots were collected from two Dipodium variegatum plates containing 100 mg 1" streptomycin and 50 mg 1"' tetracycline. At least six individual pelotons were successfully removed from root cortical cells these removed from each plant. Culture plates were sealed failed to grow on ME, NDY-6 and PD agar. and incubated in the dark at 20°C for at least three weeks. Molecular analysis of Dipodium endophytes ITS-PCR amplification produced a number of bands Molecular analysis of Dipodium endophytes between 600 and 800 bp for each DNA sample (data Roots were collected from three flowering Dipodium not shown). Full length ITS sequences were obtained variegatum plants at Duggan Park, Toowoomba. for two clones from each of the three sampled DNA was extracted from these roots using a DNeasy D. variegatum plants and these have been deposited Plant mini kit (Qiagen, Doncaster, Vic, Australia) in GenBank with the accession codes listed in Table following the manufacturer's instructions. 2. Both clones from Plant 1 showed high identities (90% over 393 bp) to Russula occidentalis Singer The fungal ITS region of each sample was amplified (Table 2). The first clone from Plant 2 had a high in 50 pi reaction volumes, each containing 38 pi identity to Trichoderma hamatum (Bonorden) Bainier (99% over 607-611 bp) while the second sterile distilled H20, 5 pi 10X buffer (50 mM KC1, 10 mM Tris-HCl, 0.1% Triton X-100; Invitrogen clone had 89-88% identity (over 337-372 bp) to two Australia, Mt Waverley, Vic, Australia), 2.5 pi species of Russula (Table 2). The first clone from Plant 3 had high identity to a Verticillium sp. (95% 50 mM MgCl2 (Invitrogen Australia), 1 pi 10 mM dNTP (Invitrogen Australia), 1 pi of each of the over 585 bp) and an uncultured fungus (97% over fungal specific ITS IF primer (Gardes & Bruns 1993) 367 bp), while the second clone had affinity (96 & and ITS4 (White et al. 1990), 0.5 pi of Taq DNA 95% over 166-168 bp) to two homobasidiomycete polymerase (Invitrogen Australia) and 1 pi of fungi, Halocyphina villosa Kohlm. et Kohlm and Merisimodes fasciculata (Schwein.) Earle (Table 2). extracted genomic DNA. PCR amplifications were performed in a Thermo Hybaid PCR Express thermocycler (Integrated Sciences, Willoughby, NSW, Australia) with 35 cycles of 95°C for 1 min., Discussion 50°C for 1 min. and 72°C for 1 min., with a final Sequence comparison of the cloned fungal ITS rDNA incubation at 72°C for 10 min. These reactions were samples with GenBank revealed that the fungal performed in duplicate and a negative control was community of D. variegatum roots consists of a included without DNA. The resulting amplification number of species. The non-mycorrhizal products were electrophoresed in 2% (w/v) agarose Deuteromycetes, Trichoderma hamatum and gels with ethidium bromide, and visualized under UV Verticillium sp. are likely to be contaminants on the light. orchid roots as they are common soil inhabiting fungi (Alexopoulous, Mims & Blackwell 1996). As ITS-PCR products were purified using a DNA Halocyphina villosa is a marine fungus (Hibbert & purification kit (Roche Applied Science, Castle Hill, Binder 2001) and Merismodes spp. have never been NSW, Australia) prior to cloning with the pGEM-T recorded as forming orchid mycorrhizas (see Easy vector system (Promega, Annandale, NSW, Rasmussen 2002) the results strongly suggest that the Australia), both conducted as per the manufacturer's primary peloton forming fungi in D. variegatum instructions. Sequencing reactions were performed in roots are members of the homobasidiomycete genus 10 pi volumes containing 400 ng of purified plasmid Russula. This has good correlation with molecular DNA, 6.4 pmoles of T7 promotor primer at the studies of species of North American myco- Brisbane laboratory of the Australian Genome heterotrophic orchids which have also shown to be Research Facility (AGRF). ITS sequences were colonised by Russula spp. (Taylor & Bruns 1999). analysed using BLAST searches While the sample size here is small, these findings (http://www.ncbi.nlm.nih.gov/). also compare favourably with other studies of myco- heterotrophic orchids which have shown quite specific fungal associations (Selosse et al. 2002, Results Taylor & Bruns 1997, 1999). Indeed, as more molecular analyses of autotrophic orchid endophytes Fungal isolation are being conducted it appears that the lack of Fungal colonisation was obvious within the roots of specificity usually attributed to these orchid types the Dipodium plants with the cortex containing large (e.g. Warcup 1981, Zelmer et al. 1986) may not be numbers of brown-coloured structures (Fig. 1). the case (see Bougoure et al. 2005, McCormick et al. At higher magnification these structures contained 2004). both collapsed fungal material and intact hyphae (Fig. 2). Although single fungal pelotons were Table 2. Closest two matches from BLAST searches of fungal sequences amplified from the three D. variegatum plants. Included are the two closest GenBank matches and accession codes, sequence identity and overlap of each match. Plant & GenBank Closest species match & accession code Sequence Sequence clone Accession identity (%) overlap (bp) no. Code Plant 1 AY702070 Russula occidentalis AY228349.1 90 393 clone 1 Russula occidentalis AY534206.1 90 393 Plant 1 AY702071 Russula occidentalis AY228349.1 90 393 clone 2 Russula occidentalis AY534206.1 90 393 Plant 2 AY702072 Trichoderma hamatum AY154937.1 99 607 clone 1 Trichoderma hamatum Z48816.1 99 611 Plant 2 AY702073 Russula Solaris AF418627.1 89 337 clone 2 Russula lepida AY061686.1 88 372 Plant 3 AY702074 Verticillium sp. AY 172097.1 95 585 clone 1 Unculturedfungus AF504849.1 97 367 Plant 3 AY702075 Halocyphina villosa AY571042.1 96 166 clone 2 Merismodes fasciculata AY571052.1 95 168

The endophytes of a number of European and North ous species of terrestrial orchids (including members American myco-heterotrophic orchids have been of the Russulaceae in the former study). The latter recently identified via molecular biology techniques. authors suggest that some orchid endophytes may Taylor & Brans (1997, 1999) and McKendrick et al. have antibiotic sensitivity or have an obligate (2000a) found that Cephalanthera austinae and dependency on their plant hosts. Corallorhiza trifida were colonised by members of the Thelephoraceae, whereas Corallorhiza maculata In conclusion, in the absence of pure culture and C. mertensiana were colonised by the synthesis of an association under experimental Russulaceae. Selosse et al. (2002) recently showed conditions, we have identified the primary peloton- that Neottia nidus-avis was colonised by sebacinoid forming fungal endophytes of D. variegatum as fungi, while Taylor et al. (2003) showed that the members of the Russulaceae using molecular-based primary fungal partner of Hexalectris spicata was techniques. It would be interesting to examine the also a member of the Sebacinaceae. Together with fungal endophytes of other Dipodium species to the present study, these analyses show that both determine if these were similarly colonised by these homobasidiomycetes and heterobasidiomycetes can homobasidiomycetes. be endophytes of myco-heterotrophic orchids.

Research into myco-heterotrophic orchids and their Acknowledgements fungal partners suggests that they are often involved We thank the University of Southern Queensland and in tripartite interactions with surrounding tree and the Centre for Biomedical Research for their shrub species, thus providing the orchid with a financial support, the Queensland Parks and Wildlife greater and more reliable nutrient source Service for granting us a permit to collect orchids and (McKendrick et al. 2000a, b, Selosse et al. 2002, Mr Damian Bougoure, Mr Austen Chen and Mrs Warcup 1985). The orchids used in this study all Adele Jones for advice and technical assistance. occurred close to species of Eucalyptus and it is possible that the orchids are linked to these tree species via ectomycorrhizal associations. Support for References this comes from the fact that Russula species, which Alexopoulos C.J., Mims C.W. & Blackwell M. are well known ectomycorrhizal fungi in Australia (1996). Introductory Mycology. John Wiley & (Brundrett et al. 1996), have been implicated in Sons, New York, USA. tripartite relationships between trees and myco- Bougoure J.J., Bougoure D.S., Cairney J.W.G. & heterotrophic orchids (Taylor & Brans 1999). Dearnaley, J.D.W. (2005). ITS-RFLP and sequence analysis of endophytes from Acianthus, Caladenia The lack of success in growing extracted pelotons and Pterostylis (Orchidaceae) in south eastern may be owing to the absence of specific cultural Queensland, Australia. Mycological Research 109, requirements in the media used or that there were 452-460. problems with the extraction methodology. Taylor & Brundrett, M., Bougher, N., Dell, B., Grove T. & Brans (1997) and Zelmer et al. (1996) have also Malajczuk, N. (1996) Working with mycorrhizas in encountered difficulties in isolating fungi from vari­ forestry and agriculture. The Australian Centre for International Agricultural Research. Canberra, achlorophyllous orchid Neottia nidus-avis (L.) Australia. L.C.M. Rich, and neighboring tree ectomycorrhiza. Gardes, M. & Bruns, T.D. (1993). ITS primers with Molecular Ecology 11, 1831-1844. enhanced specificity for basidiomycetes— Smith, S.E. & Read, D.J. (1997). Mycorrhizal application to the identification of mycorrhizae and symbiosis. Academic Press, Cambridge, UK. rusts. Molecular Ecology 2, 113-118. Stanley, T.D. & Ross, E.M. (1989). Flora of South­ Hibbert, D.S. & Binder, M. (2001). Evolution of eastern Queensland. Volume 3. Queensland Marine Mushrooms. Biological Bulletin 201, 319— Department of Primary Industries, Brisbane, 322. Australia. Huynh, T.T., McLean, C.B., Coates, F. & Lawrie, Taylor, D.L. & Bruns, T.D. (1997). Independent, A.C. (2004). Effect of developmental stage and specialized invasions of ectomycorrhizal mutualism peloton morphology on success in isolation of by two nonphotosynthetic orchids. Proceedings of mycorrhizal fungi in Caladenia formosa the National Academy of Sciences 94,4510^1515. (Orchidaceae). Australian Journal of Botany 52, Taylor, D.L. & Bruns, T.D. (1999). Population, 231-241. habitat and genetic correlates of mycorrhizal Leake, J.R. (1994). The biology of myco- specialization in the 'cheating' orchids heterotrophic ('saprophytic') plants. New Corallorhiza maculata and C. mertensiana. PhytologistUl, 171-216. Molecular Ecology 8, 1719-1732. McCormick, M.K., Whigham, D.F. & O'Neill, J. Taylor, D.L., Bruns, T.D., Szaro, T.M. & Hodges, (2004). Mycorrhizal diversity in photosynthetic S.A. (2003). Divergence in mycorrhizal speciation terrestrial orchids. New Phytologist 163,425-438. within Hexalectris spicata (Orchidaceae), a non- McKendrick, S.L., Leake, J.R., Taylor, D.L. & Read, photosythetic desert orchid. American Journal of D.J. (2000a). Symbiotic germination and Botany 90,1168-1179. development of myco-heterotrophic plants in Warcup, J.H. (1971). Specificity of mycorrhizal nature: ontogeny of Corallorhiza trifida and association in some Australian terrestrial orchids. characterization of its mycorrhizal fungi. New New Phytologist 70,41^16. Phytologist 145, 523-537. Warcup, J.H. (1973). Symbiotic germination of some McKendrick, S.L., Leake J.R. & Read, D.J. (2000b). Australian orchids. New Phytologist 72, 387-392. Symbiotic germination and development of myco- Warcup, J.H. (1981). The mycorrhizal relationships heterotrophic plants in nature: transfer of carbon of Australian orchids. New Phytologist 87, 371— from ectomycorrhizal Salix repens and Betula 381. pendula to the orchid Corallorhiza trifida through Warcup, J.H. (1985). Rhizanthella gardneri shared hyphal connections. New Phytologist 145, (orchidaceae), its Rhizoctonia endophyte and close 523-537. association with Melaleuca uncinata (Myrtaceae) Milligan, M.J. & Williams, P.G. (1988). The in Western Australia. New Phytologist 99, 273- mycorrhizal relationship of multinucleate 280. rhizoctonias from non-orchids with Microtis Warcup, J.H. (1988). Mycorrhizal associations of (Orchidaceae). New Phytologist 108,205-209. isolates of Sebacina vermifera. New Phytologist Perkins, A.J. & McGee, P.A. (1995). Distribution of 110,227-231. the orchid mycorrhizal fungus, Rhizoctonia solani, Warcup, J.H. (1990). Mycorrhizas, in R.J. Bates & in relation to its host, Pterostylis acuminata, in the J.Z. Weber (eds), Orchids of South Australia, pp. field. Australian Journal of Botany 43, 565-575. 21-26. Flora and Fauna of South Australia Perkins, A.J., Masuhara, G. & McGee, P.A. (1995). Handbook Committee, Adelaide, Australia. Specificity of the associations between Microtis Warcup, J.H. (1991). The Rhizoctonia endophytes of parviflora (Orchidaceae) and its mycorrhizal fungi. Rhizanthella (Orchidaceae). Mycological Research Australian Journal of Botany 43, 85—91. 95, 656-659. Pope, E.J. & Carter, D.A. (2001). Phylogenetic White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990). placement and host specificity of mycorrhizal Amplification and direct sequencing of fungal isolates belonging to AG-6 and AG-12 in the ribosomal RNA genes for phylogenetics, in Rhizoctonia solani species complex. Mycologia 93, M.A. Innis, D.H. Gelfand, J.J. Sninsky & T.J. 712-719. White (eds), PCR Protocols: a Guide to Methods Rasmussen, H.N. (2002). Recent developments in the and Applications, pp. 315-322, Academic Press, study of orchid mycorrhiza. Plant and Soil 244, San Diego, USA. 149-163. Zelmer, CD., Cuthbertson, L. & Currah, R.S. (1996). Selosse, M-A., WeiB, M., Jany, J-L. & Tillier, A. Fungi associated with terrestrial orchid (2002). Communities and populations mycorrhizas, seeds and protocorms. Mycoscience of sebacinoid basidiomycetes associated with the 37,439-448. GJK,<.73£JJS;:JTL' ::... EROSPORUMREDISCOVERED?

Heino Lepp

PO Box 38, Belconnen, ACT 2616.

Abstract

A possible specimen of Ceratobasidium sphaerosporum has been collected in South Australia.

H. Lepp (2005). Ceratobasidium sphaerosporum rediscovered? Australasian Mycologist 24 (1): 20-22.

Introduction

Warcup & Talbot (1971) described Ceratobasidium Specimen studied: SOUTH AUSTRALIA: sphaerosporum, obtained as a perfect state from Pooginook Conservation Park, 45 km (by road) E of Rhizoctonia isolates from north Queensland orchid Morgan, on Morgan Road; on fallen, long-dead roots. Roberts (1999) noted that the species was still eucalypt branch in mallee scrub with Triodia; 23 known only from those north Queensland orchid May 2003; HLepp 4058 (CANB 649904). roots and that the holotype was lost. Field work in a mallee area in South Australia has produced a possible specimen of C. sphaerosporum. The Discussion specimen has been deposited in the Australian National Herbarium (CANB) at the Centre for Plant Figure lc shows the only cystidia I found, despite Biodiversity Research in Canberra. In this paper I further searching in several areas of the sporocarp. describe that collection and compare its features with The genus is described as lacking cystidia and, given those of similar taxa. Measurements and drawings their general absence, I would take the three as are from material rehydrated in approximately 5% simply some aberrant growths in a specimen that KOH (with a dash of phloxine). otherwise matches Ceratobasidium. The basidia, very much broader than the hyphae, and the lack of much Specimen description development beyond the basal hyphae would rule out Thanatephorus. Oliveonia is similar to Ceratobasidium cf. sphaerosporum Warcup & Ceratobasidium and has cystidiate species, but with P.H.B. Talbot New Phytol. 70: 38 (1971). Figure agglutinated and narrower hyphae. Note that I could 1. not trace the three cystidia back to their sources, so The sporocarp covers about two square centimetres cannot rule out the possibility that they are a and appears as a pale grey bloom to the naked eye, contaminant. farinose under a hand lens. Hyphal system: monomitic; hyphae hyaline, smooth, undamped, Warcup & Talbot (1980) described another spherical- branching at right-angles and growing as an open spored species (C. globisporum, also based on weave, thin-walled (except for the basal hyphae, isolates from North Queensland orchids), with which are up to 0.8 um thick), 4.0-6.4 um diameter. considerably larger dimensions of all its structures'. Spores: globose or subglobose (the length/breadth Ceratobasidium globisporum and C. sphaerosporum ratio in the range 1.0-1.1), smooth, thin-walled, may simply be the extremes of a single taxon inamyloid; 6.4—8.0 urn (excluding the apiculus, up to (Roberts 1999). Boidin & Gilles (2000) reported a 0.8 um long), germinating by repetition. Basidia: single collection of Ceratobasidium cf. globisporum barrel-shaped to sphaeropedunculate (with the stem 'sur bois tres degrade' (i.e. on well-rotted wood) lateral in some cases), with 4 sterigmata, 14—17 x from Reunion, in the south-west Indian Ocean. 8.8-12 urn. Cystidia: three narrow, almost hyphidial- like growths were present, two fairly short (Fig. lc). Scale bar = 10 um

Figure 1. Ceratobasidium cf. sphaerosporum (HL4058). A, Basidia (hashed area is debris). B, Spores. C, Basal hyphae, basidioles and cystidia.

Table 1. Comparison of the micro-features of taxa in the C. sphaerosporum/C. globisporum complex. HL4058 C. sphaerosporum C. globisporum C. cf. globisporum Warcup & Talbot Warcup & Talbot Boidin & Gilles (1971) (1980) (2000) Hyphae 4.0-6.4 pm; hyaline, basal Up to 6 pm wide, the 7-9 pm wide; the Basal hyphae 13- hyphae with walls to basal hyphae lightly walls of the basal 14 pm wide; walls 0.8 pm thick coloured and with walls hyphae lightly about 1 pm thick about 0.8 pm thick coloured and 1— 1.5 pm thick Spores (Sub)globose, 6.4-8.0 pm Spherical 6.5-9 pm Spherical, 10— Spherical, 8.8-13 or (+ apiculus, up to 0.8 pm diameter; with a 12 pm diameter, or 9-13 x 8.5-12 pm long) prominent hilum sphaeroidal 10-12 (-14) x 10-12 pm Basidia Barrel-shaped to Sphaero- or Obovate to Subcylindrical, 19— sphaeropedunculate, 14— pyropedunculate, 11- subspherical 15-22 26 x 10.5-12.5 pm; 17 x 8.8-12 pm; with 14.5 x 8-10 pm; with x 13-16 pm, with 4 with 2^1 sterigmata 4 sterigmata that are up to 4 sterigmata that are up sterigmata that are that are 12-18(-24) 13 pm long to 6 pm long 6-11 (-20) pm long pm long

Ceratobasidium globisporum is known only from the the collections of which have since been moved to north Queensland material and its holotype also either the State Herbarium of South Australia (AD) could not be found (Roberts 1999). The holotypes of or the Plant Pathology Herbarium of the Orange both C. globisporum and C. sphaerosporum had been Agricultural Institute (DAR). Graham Bell (AD, pers. deposited at the University of Adelaide herbarium, comm.) and Michael Priest (DAR, pers. comm.) have confirmed that the holotypes have still not been Acknowledgements found. The 'Filamentous fungi database' I thank the South Australian Department of http://www.cbs.knaw.nl/databases/search_filam.htm Environment and Heritage for allowing collecting in of the Centraalbureau voor Schimmelcultures (CBS) Pooginook Conservation Park, and Graham Bell and notes that CBS holds cultures obtained from the Michael Priest for checking for the holotypes noted original isolates by Warcup & Talbot. A web and above. literature search yielded just one other Ceratobasidium species with globose spores, C. setariae (Sawada) Oniki et al. However, its spores References range from globose to ellipsoid (9-14 [-20] x 7-13 Boidin, J. & Gilles, G. (2000). Basidiomycetes [-18], with a length/breadth ratio from 1.0 to 1.3), its Aphyllophorales de I'ile de la Reunion. XXI-suite. basidia are bi-sterigmate and it is known only as a Mycotaxon 75,357-387. cereal pathogen (Roberts 1999). Roberts, P. (1999). Rhizoctonia-forming fungi: A taxonomic guide. Royal Botanic Gardens, Kew. Table 1 compares the micro-features of taxa in the Warcup, J.H. & Talbot, P.H.B. (1971). Perfect states C. sphaerosporum/C. globisporum complex, with the of Rhizoctonias associated with orchids. II. New descriptions taken from the indicated sources. In all Phytologist 70, 35^40. cases the spores are hyaline, smooth-walled and Warcup, J.H. & Talbot, P.H.B. (1980). Perfect states inamyloid, and the hyphae are smooth and of Rhizoctonias associated with orchids. III. New undamped. Phytologist 86, 267-272. ANNUAL GENERAL MEETING AND ELECTION OF COUNCIL MEMBERS OF TH= .4MS — CALL FOR NOMINATIONS

As the Society is not holding a conference this year, the Council has decided to hold this year's AGM as an email meeting to allow as many members as possible to participate. This meeting will take place between 14— 18 November 2005.

If you wish to participate, please register your intent by sending an email to the Secretary, Jerry Cooper ([email protected]), no later than 1 September 2005. Also, please email Jerry with any agenda items you have for the AGM by the same date. The agenda will be published on the AMS website and in the next issue of the journal along with detailed instructions as to the procedure for participating in the meeting.

The current council membership, and period of eligibility, is as follows:

President - Wieland Meyer (Nov. 2002 - Nov. 2005) Vice-President - Geoff Ridley (Nov. 2002 - Nov. 2005) Treasurer - David Ratowsky (Sep. 2001 - Sep. 2007) Secretary - Jerry Cooper (Nov. 2002 - Nov. 2008) Councillor - Teresa Lebel (Nov. 2004 - Nov. 2010) Councillor - Dee Carter (Nov. 2002 - Nov. 2008)

The Australasian Mycological Society is now calling for nominations for membership of council, particularly with respect to the position of President being vacated by Wieland Meyer, Vice-President being vacated by Geoff Ridley, and Secretary being vacated by Jerry Cooper.

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Dr Jerry Cooper Secretary Australasian Mycological Society Landcare Research Lincoln 8152 New Zealand

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12.3 The positions of President and Vice-President are held on an annual basis and restricted to 3 consecutive terms. The remaining positions are restricted to 6 consecutive terms. 13.1b Each nomination shall be made in writing, signed by 2 members of the society and accompanied by written consent of the candidate. 13.1c Each nomination shall be in the hands of the Secretary at least 8 weeks before the AGM. CALL FGL'. ?i%PEHS

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EFEREES FOR 2004

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r:r - mm MEMSE^ OF THE i-u^-i-usiM: n r ZOIOGICJ-.L I^CIETT

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8th International Mycological Congress, Cairns, Queensland 2006 http://www.sapmea.asn.au/conventions/imc8