M. Glen

Puccinia psidii: a threat to the Australian environment and economy a review

Puccinia psidii causes a rust disease on a broad range of hosts in the Myrtaceae and Heteropyxidaceae. It is native to South America where it can cause severe disease in eucalypt plantations and other introduced Myrtaceae. The pathogen has recently expanded its geographical range to Hawaii, increasing concerns about the potential for an incursion in Australia. This paper reviews the taxonomy, biology, impact and options for control of P. psidii. It also discusses the probable impact if an incursion were to occur in Australia and the preparations that must be made to mitigate adverse consequences.

Uredo rangelii, a taxon in the guava rust complex, newly recorded on Myrtaceae in Australia.

Uredo rangelii (myrtle rust) is reported for the first time in Australia — detected on 22 April 2010 — from Agonis flexuosa, Callistemon viminalis and Syncarpia glomulifera. This taxon is morphologically distinct from Puccinia psidii, the cause of guava rust, but DNA sequence data place it in the P. psidii complex. Surveys up to the end of May 2010 have detected U. rangelii on cultivated shrubs and trees at four properties (two cut flower farms and two wholesale nurseries) on the Central Coast of New South Wales, with no records in native forest thus far. Containment operations are currently underway on infected properties.

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.

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.

Development of nested polymerase chain reaction detection of Mycosphaerella spp. and its application to the study of leaf disease in Eucalyptus plantations

ABSTRACT Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm(2) or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

A latitudinal cline in disease resistance of a host tree

The possible drivers and implications of an observed latitudinal cline in disease resistance of a host tree were examined. Mycosphaerella leaf disease (MLD) damage, caused by Teratosphaeria species, was assessed in five Eucalyptus globulus (Tasmanian blue gum) common garden trials containing open-pollinated progeny from 13 native-forest populations. Significant population and family within population variation in MLD resistance was detected, which was relatively stable across different combinations of trial sites, ages, seasons and epidemics. A distinct genetic-based latitudinal cline in MLD damage among host populations was evident. Two lines of evidence argue that the observed genetic-based latitudinal trend was the result of direct pathogen-imposed selection for MLD resistance. First, MLD damage was positively associated with temperature and negatively associated with a prediction of disease risk in the native environment of these populations; and, second, the quantitative inbreeding coefficient (QST) significantly exceeded neutral marker FST at the trial that exhibited the greatest MLD damage, suggesting that diversifying selection contributed to differentiation in MLD resistance among populations. This study highlights the potential for spatial variation in pathogen risk to drive adaptive differentiation across the geographic range of a foundation host tree species.

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.

Ganoderma and Amauroderma species associated with root-rot disease of Acacia mangium plantation trees in Indonesia and Malaysia

Fungal sporocarps and cultures associated with signs and symptoms of root-rot disease were collected from Acacia mangium and other tropical hardwood species. The collections were identified by either morphological characters and/or by phylogenetic analysis based on DNA sequences as Ganoderma philippii, G. mastoporum, G. aff. steyaertanum, G. australe and Amauroderma rugosum. Phylogenetic analysis unequivocally placed in the G. philippii clade four sequences amplified from A. mangium root and butt tissue showing clear signs of red root-rot disease (roots are covered by a red rhizomorphic skin). Whereas G. philippii was the most frequently encountered fungal species in A. mangium with red root-rot disease, this study indicates that other fungal species related to G. mastoporum may cause root-rot disease with very similar symptoms. An isolate (FRIM 138) that had caused red root-rot disease in artificial inoculations carried out before this study and was presumed to be G. philippii, is here determined to be closely related to G. mastoporum, G. cupreum and G. sinense. A Ganoderma species associated with a yellow-brown root-rot disease killing trees in an A. mangium plantation in Central Java, previously identified as G. lucidum, is shown by phylogenetic analysis to be closely related to G. steyaertanum, though some morphological characters vary from the original description of that species.

Evidence for different QTL underlying the immune and hypersensitive responses of Eucalyptus globulus to the rust pathogen Puccinia psidii

The rust Puccinia psidii infects many species in the family Myrtaceae. Native to South America, the pathogen has recently entered Australia which has a rich Myrtaceous flora, including trees of the ecologically and economically important genus Eucalyptus. We studied the genetic basis of variation in rust resistance in Eucalyptus globulus, the main plantation eucalypt in Australia. Quantitative trait loci (QTL) analysis was undertaken using 218 genotypes of an outcross F2 mapping family, phenotyped by controlled inoculation of their open pollinated progeny with the strain of P. psidii found in Australia. QTL analyses were conducted using a binary classification of individuals with no symptoms (immune) versus those with disease symptoms, and in a separate analysis dividing plants with disease symptoms into those exhibiting the hypersensitive response versus those with more severe symptoms. Four QTL were identified, two influencing whether a plant exhibited symptoms (Ppr2 and Ppr3), and two influencing the presence or absence of a hypersensitive reaction (Ppr4 and Ppr5). These QTL mapped to four different linkage groups, none of which overlap with Ppr1, the major QTL previously identified for rust resistance in Eucalyptus grandis. Candidate genes within the QTL regions are presented and possible mechanisms discussed. Together with past findings, our results suggest that P. psidii resistance in eucalypts is quantitative in nature and influenced by the complex interaction of multiple loci of variable effect.