Bushland fungi of the Busselton area

This report presents data from the Perth Urban Bushland Fungi (PUBF) Project and the Centre of Excellence for Climate Change and Woodland and Forest Health weekend workshop held on 27 and 28 June 2009 at Busselton, in the South west of Western Australia. The event was organized to survey fungi in the local tall Tuart forest but due to stormy weather conditions that weekend, safety issues dictated that it was too dangerous to enter the Tuart forest. Instead, the groups searched for fungi in two reserves near Busselton dominated by Peppermint woodland: on Saturday at Locke Nature Reserve, a DEC managed woodland reserve, and on Sunday at Captain Baudin Reserve, a coastal reserve managed by the Shire of Busselton. The weekend was organised with the assistance of the Busselton Naturalists’ Club and the Friends of Island Point. Thirty nine people took part in the Saturday foray and workshop, and 34 people endured very rainy and wintery conditions on Sunday to survey fungi at Captain Baudin Reserve. At each foray site, the participants were divided into five foray groups which were led by volunteer Leaders from the PUBF Project. This was the first ever survey of fungi for both of the Reserves

Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations

Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR - RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR - RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR - RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR - RFLP tool for identifying basidiomycetes associated with plants from field samples

Interspecific and intraspecific variation of ectomycorrhizal fungi associated with Eucalyptus ecosystems as revealed by ribosomal DNA PCR-RFLP

Gondwanan vegetation, and the Australian region in particular, is species rich for ectomycorrhizal fungi in epigeous and hypogeous forms with over 100 species recorded in small (1 ha) patches of forests. Distinguishing co-occurring ectomycorrhizal fungi as root associations in native (natural or wildlands) vegetation or plantations and discriminating them from other larger basidiomycetes, e.g. wood and leaf litter decomposer fungi, places large demands on molecular identification, especially if interspecific similarities and intraspecific variation occur in target sequences. One hundred and nine species of larger basidiomycetes from a single forest location were characterised by PCR-RFLP profiles of two genomic regions (nuclear rDNA ITS and mtLSU). Over one-third of the species for which multiple isolates were tested showed intraspecific variation in either one or both genomic regions. This remarkably high variation questions previous assumptions about intraspecific ITS sequence variation and highlights the value of integrated molecular and morphological databases including voucher specimens. It also emphasises the value of molecular investigations that use more than one genomic region. Interspecific similarities were common among the Cortinariaceae, especially in the ITS region. Discrimination of most Cortinariaceae species was achieved using variation in the mtLSU region in conjunction with the ITS. This new information raises the possibilities that the ITS sequence is more conserved and the mtLSU more variable than among species of the other 23 families. In the other families, interspecific ITS variation was greater and the mtLSU profiles grouped species within families. The high variation in the two genomic regions indicated possible differences in the fungal population structure between two adjacent, differently managed blocks of Eucalyptus marginata forest. The significance of this variation to ecology, biodiversity assessment and ecosystem management are discussed

Ectomycorrhiza formation in Eucalyptus. III. Superificial ectomycorrhizas initiated by Hysterangium and Cortinarius species

Cortinarius and Hysterangium species are a dominant component of the macrofungi in eucalypt forests of Western Australia, and their hyphae occupy 10 % of the soil surface area. Anatomical studies of jarrah (Eucalyptus marginata Donn ex Sm.) and karri (E. diversicolor F. Muell) roots collected in association with hyphae of Cortinarius globuliformis Boug., C. ochraceus Clel. and Hysterangium inflatum Rodway showed mycorrhizal structures with mantles one to five cells thick and Hartig nets penetrating to the hypodermis. These superficial ectomycorrhizas were similar in size to those of non‐colonized feeder roots and lacked the expanded cortex and broad mantle of pyramidal ectomycorrhizas formed by Laccaria laccata (Scop, ex Fr.) Berk. & Br. Colonized host roots responded with polyphenol accumulation in the epidermis/hypodermis and lignification of the outer cortex for both the superficial and pyramidal types

Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests?

A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiß and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata

Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina, Casuarina and Eucalyptus

The effect of inoculating seedlings of Eucalyptus grandis, Allocasuarina littoralis and Casuarina equisetifolia with two isolates of Pisolithus and two isolates of Scleroderma from under eucalypts was examined in a glasshouse trial. Ectomycorrhizas formed extensively on Eucalyptus (23–46% fine roots ectomycorrhizal) and Allocasuarina (18–51% fine roots ectomycorrhizal). On Casuarina, the fungi were either unable to colonize the rhizosphere (one isolate of Pisolithus), or sheathed roots, resembling ectomycorrhizas, formed on 1–2% of the fine roots. Colonization of roots by one isolate of Scleroderma resulted in the death of Casuarina seedlings. Inoculation with fungi increased shoot dry weight by up to a factor of 32 (Eucalyptus), 4 (Allocasuarina) and 3 (Casuarina). Ectomycorrhizas formed in associations with Eucalyptus and Allocasuarina had fully differentiated mantles and Hartig nets in which the host and fungal cells were linked by an extensive fibrillar matrix. Sheathed roots in Casuarina lacked a Hartig net, and the epidermis showed a hypersensitive reaction resulting in wall thickening and cell death. The sheaths are described as mantles since the density and arrangement of the hyphae in the sheaths was similar to that in mantles of the eucalypt ectomycorrhizas. The intercellular carbohydrate matrix was not produced in the Casuarina mantle in association with Pisolithus, hence the mantle was not cemented to the root. These structures differ from poorly compatible associations described previously for Pisolithus and Eucalyptus. The anatomical data indicate that ectomycorrhizal assessment based on surface morphological features may be misleading in ecological studies because compatible and incompatible associations may not be distinguishable