I.C. Tommerup

False-negative isolations or absence of lesions may cause mis-diagnosis of diseased plants infected with Phytophthora cinnamomi

In a series of growth cabinet, glasshouse and field experiments, tissue samples from living clonal Eucalyptus marginata (jarřah) were incubated immediately after sampling on agar (NARPH) selective for Phytophthora. Phytophthora cinnamomi was recovered 3–6 months after inoculation from 50% of samples with lesions and 30% of symptomless samples. However, up to 11% of samples with and without lesions and from which P. cinnamomi was not initially isolated contained viable pathogen. This was shown by removing tissue which had not produced any growth of P. cinnamomi on NARPH plates, cutting it into smaller sections, washing in sterile deionised water repeatedly for 9 days, and replating. Plating stem or bark tissue directly onto NARPH produced false-negative results for nine P. cinnarnomi isolates and six jarrah clones. The behaviour of the pathogen indicates that it could be present as dormant structures, such as chlamydospores, that need to be induced to germinate. Alternatively, fungistatic compounds in the tissue needed to be removed to allow the pathogen to grow. These results have important implications for disease diagnosis and management, disease-free certification and quarantine clearance.

Three clonal lineages of Phytophthora cinnamomi in Australia revealed by microsatellites

ABSTRACT The genetic structure of populations of Phytophthora cinnamomi, a pathogen of an enormous variety of woody plants, was investigated using microsatellites. Three intensively sampled disease sites in southwest Australia were analyzed along with a large culture collection of Austra-lian isolates and some isolates from elsewhere in the world. The mutation in the four microsatellite loci analyzed revealed spatial patterns at the disease sites that correlated with the age of the infestation. Only three clonal lineages were identified in Australian populations and these same clonal lineages were present in worldwide populations, where it is suggested that a limited number of clonal lineages have spread in most regions. No evidence for sexual reproduction between these clonal lineages in Australia has been found even though the pathogen has the opportunity. Instead, mitotic recombination is frequent within the clonal lineages. The implications of this are discussed.

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

Abstract. 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.

Reliability of RAPD fingerprinting of three basidiomycete fungi, Laccaria, Hydnangium and Rhizoctonia

Randomly amplified polymorphic DNA (RAPD) profiles are currently being developed for Laccaria and Hydnangium species and Rhizoctonia solani. The technique is increasingly being used to differentiate fungal isolates. As for the polymerase chain reaction (PCR) from which it was derived, the conditions necessary for reproducible RAPD products have received attention. However, in contrast to the PCR reaction, the technique relies on non-specific primers and as a consequence the reaction conditions are not necessarily as specific as they are in PCR. Compared with PCR products, RAPD fingerprints were therefore more sensitive to reaction and thermocycle conditions. RAPD products produced using 10 and 17–23 mer primers were visualized on ethidium bromide stained polyacrylamide electrophoresis gels. Factorial experiments showed RAPD patterns were altered by changes in various reaction mixture components including the concentration of non-DNA impurities, the number and concentration of primers and the DNA polymerase enzyme type, source and concentration. These factors, particularly the enzyme source, reacted differently with the magnesium chloride concentration. Fluorescent dye labelled primers were used with internal lane standards in a DNA sequencing system to assess accurately the molecular weights and relative amounts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on patterns visualized on ethidium bromide stained non-denaturing polyacrylamide gels. The temperature at which DNA was denatured in the first cycles altered the fingerprints. Reaction mixture temperatures of 94° or higher, compared with 91–93°, caused loss of visual yield of some products, particularly those greater than 500 base pairs, and increased the yield of others. Reproducibility of RAPD patterns, when the reaction mixture and temperature profile factors were varied, was facilitated by cross reference to fluorescently labelled bands separated with internal lane standards in a DNA sequencing system. Reproducibility of fingerprint patterns in a standard reaction mixture was achieved, with different thermocycle programmes and in different thermocyclers when the temperature profile was reproduced, suggesting that particular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used.

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.

Phenotypic variation in a clonal lineage of two Phytophthora cinnamomi populations from Western Australia

Seventy-three isolates of Phytophthora cinnamomi were collected from diseased Eucalyptus marginata (jarrah) and Corymbia calophylla (marri) trees in two forest communities in the southwest of Western Australia. Both populations of P. cinnamomi were examined for phenotypic and genotypic variation. Microsatellite DNA analysis showed that all isolates were of the same clonal lineage. We show, for the first time for P. cinnamomi, that morphological and pathogenic variation between populations of the clonal lineage are very broad and continuous. The phenotypes examined included growth rates and colony morphology on potato dextrose agar at different temperatures, sporangial and gametangial morphology, ability to form lesions in detached jarrah and marri stems, and ability to cause deaths of clonal jarrah plants in a glasshouse trial. Phenotype variation was derived asexually. All phenotypes investigated varied independently from one another. Cluster analysis of 24 morphological and pathogenicity phenotypes identified two main clusters of isolates corresponding to each population. The ability to cause deaths in both populations ranged from killing all plants within 59 d to plants being symptomless 182 d after inoculation.

Hydrogel bead inocula for the production of ectomycorrhizal eucalypts for plantations

Inocula of eleven eucalypt ectomycorrhizal fungi, produced by the culture of mycelia within hydrogel beads, were found to be of high efficacy as propagules. The beads were discrete units approximately 2·5 mm in diameter. SEM examination revealed a dense profusion of intact mycelia within the beads. All beads produced mycelial outgrowth within 1–3 d when plated on agar medium and a single bead was sufficient for the initiation of mycorrhiza in the roots of aseptic seedlings and micropropagated plantlets. Depending on fungal species, the beads can be stored for at least 7 months without losing their capacity as propagules.

Sequence variation in the rDNA ITS of Australian Armillaria species and intra-specific variation in A. luteobubalina

In contrast to northern hemisphere species, Australia’s Armillaria species have high variation in the rDNA Internal Transcribed Spacer (ITS) region. Isolates of the five different Australian species, five species from south-eastern Australia and one, A. luteobubalina, from Western Australia had distinct ITS sequences and PCR-RFLP profiles. Phylogenetic analyses grouped A. hinnulea away from the other Australian Armillaria species and with the seven northern hemisphere species. The 28 ITS sequences from A. luteobubalina isolates, 27 from Western Australia, gave rise to four distinct polymorphic groups. This ITS variation was not related to geographical locality, host species or mating phenotype.

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.

Laccaria fraterna, a common ectomycorrhizal fungus with mono- and bi-sporic basidia and multinucleate spores: comparison with the quadristerigmate, binucleate spored L. laccata and the hypogeous relative Hydnangium carneum

Examination of Laccaria collections from Western Australia revealed that the most common species L. fraterna had basidia which were monosporic, bisporic or very occasionally trisporic, in contrast to L. laccata which was quadrisporic. For L. fraterna , all post-meiotic nuclei migrated into the spores leaving basidia anucleate. Spores of monosterigmate basidia had four nuclei, and bisterigmate basidia usually had two. Post-meiotic mitosis occurred after spores were delimited and prior to morphological maturation. Mature spores had up to eight nuclei. That most spores had compatible mating-type nuclei was indicated by most single germinated spores having clamped mycelium with dikaryotic cells. In Hydnangium carneum , a hypogeous relative of Laccaria , post-meiotic mitosis occurred in the basidium and two nuclei migrated into each of the four spores. Migration patterns indicated that some spores had compatible mating-type nuclei, as in L. fraterna . Allocation of compatible mating types to spores partly explains why L. fraterna ‘weed-like’ behaviour and is a pioneer in plantations of eucalypts on former farmland or rehabilitated mine sites. Mycorrhizas of these fungi have been synthesized aseptically with several species of Eucalyptus .