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Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Calonectria leaf blight (CLB) is one of the best-known diseases of Eucalyptus spp., particularly in Asia and South America. Recently, typical symptoms of leaf and shoot blight caused by Calonectria spp. Were observed in a Eucalyptus plantation in the YunNan Province of southwestern China. Isolations were made from diseased leaves and top soil collected below the diseased trees to determine the causal agent of the disease and to consider the distribution characteristics of the Calonectria species. This resulted in 417 isolates, of which 228 were from leaves and 189 were from soil. Based on comparisons of DNA sequences for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (β-tubulin) gene regions, as well as morphological characteristics, 11 Calonectria species were identified. These included Calonectria aciculata (0.7 %), Ca. colhounii (1.2 %), Ca. eucalypti (10.6 %) and Ca. honghensis (43.2 %) in the Ca. colhounii species complex, and Ca. aconidialis (15.3 %), Ca. asiatica (9.8 %), Ca. hongkongensis (1.0 %), Ca. ilicicola (6.0 %), Ca. kyotensis (0.5 %), and Ca. yunnanensis (11.3 %) in the Ca. kyotensis species complex. In addition, a novel species, accounting for 0.5 % of the isolates, was discovered and is described here as Ca. dianii sp. nov. in the Ca colhounii species complex. Most (99.1 %) of the isolates collected from the leaves resided in the Ca. colhounii species complex and a majority (95.8 %) of those from the soils were in Ca. kyotensis species complex. These results suggest that Calonectria spp. in the Ca. colhounii species complex infecting leaves might be adapted to that niche and that those in the Ca. kyotensis species complex are better adapted to a soil habitat., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Eucalyptus scab and shoot malformation caused by Elsinoë necatrix is an emerging disease and a serious threat to the global commercial forestry industry. The disease was first discovered in North Sumatra, Indonesia, and now requires a simple and effective method for early pathogen detection. In this study, a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for E. necatrix . A unique region in a secondary metabolite gene cluster was used as a target for the assay. To test robustness of the assay, LAMP was verified in 15 strains of E. necatrix . A specificity test against 23 closely related Elsinoë species and three fungal species commonly isolated on Eucalyptus showed that the LAMP assay exclusively identified E. necatrix isolates. The assay had a high level of sensitivity, able to detect 0.01 ng (approximately 400 target copies) of pure E. necatrix DNA. Furthermore, using a simple DNA extraction method, it was possible to use this assay to detect E. necatrix in infected Eucalyptus leaves., Competing Interests: The author(s) declare no conflict of interest.

Shoot and leaf blight caused by Teratosphaeria destructans is one of the most devastating foliar diseases on Eucalyptus . Therefore, breeding for resistance to this disease is considered urgent. Differences in susceptibility to T. destructans have been observed in the field but a robust inoculation protocol has, until recently, been unavailable and a disease scoring method for precise phenotyping has not been established. A first objective of this study was to determine the optimal conidial concentration for T. destructans inoculations on a susceptible Eucalyptus host. This concentration was then used to determine differences in susceptibility of six genotypes of Eucalyptus grandis × E. urophylla to the pathogen by assessing the percentage of infected stomata using electron microscopy and the percentage of leaf area covered by lesions (PLACL) using image processing. In addition, we developed a disease susceptibility index (SI) of six categories ranging from highly resistant (SI = 0) to highly susceptible (SI = 1.5 to 2). The more resistant genotypes were moderately resistant, with an SI value of 0.49 to 0.54 and a PLACL of 6.5 to 9%. In contrast, the more susceptible genotype scored an SI of 1.52 and PLACL of 48%. Host susceptibility was also assessed relative to the sporulation of the pathogen. This showed that the percentage of sporulation was not significantly correlated with host resistance. Overall, the results provide the basis for rigorous screening and selection of resistant genotypes to the disease caused by T. destructans using artificial inoculation., Competing Interests: The author(s) declare no conflicts of interest.

Calonectria leaf blight caused by Calonectria spp. is among the most serious diseases affecting the health and sustainability of Eucalyptus plantations in southern China. Recent outbreaks of this disease in GuangDong Province prompted a need to identify the species involved. Typical symptoms of Calonectria leaf blight were observed on 2-year-old Eucalyptus urophylla × E. grandis trees in a plantation in the ZhaoQing region. In total, 38 Calonectria isolates were collected from 32 diseased trees. All isolates were identified using DNA sequence analyses of the translation elongation factor 1-α ( tef1 ), β-tubulin ( tub2 ), calmodulin ( cmdA ), and histone H3 ( his3 ) gene regions. Phylogenetic analyses revealed that Calonectria queenslandica was the dominant species, accounting for 81.6% of the isolates collected. Other species isolated included C. pseudoreteaudii (10.5%), C. reteaudii (5.3%), and C. aconidialis (2.6%). This is the first report of C. queenslandica in China and all isolates had identical sequences in all four gene regions. PCR amplification using primers targeting the MAT1-1-1 and MAT1-2-1 genes in all C. queenslandica isolates revealed that only the MAT1-2 idiomorph was present. The results suggest that C. queenslandica was introduced into the sampled area with very limited genetic diversity. Pathogenicity tests were conducted on two Eucalyptus genotypes widely planted in the GuangDong Province using isolates representing all species collected. The results showed that these species could all cause disease but the predominance of C. queenslandica on infected trees suggests that it is the major driver of the disease problem studied. Different Eucalyptus genotypes used in the pathogenicity tests differed in susceptibility to infection by the Calonectria spp. tested, providing opportunities to avoid leaf blight by deploying disease-tolerant planting stock.

The genus Calonectria includes many aggressive plant-pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive to Eucalyptus in China but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

Leaf blight caused by Teratosphaeria destructans is one of the most important diseases of Eucalyptus planted in the subtropics and tropics. In contrast, the better-known T. epicoccoides , though also a primary pathogen of Eucalyptus , causes less damage to trees in these areas. Although T. destructans is an aggressive pathogen, nothing is known about its infection biology. In this study, the conditions for infection and disease development caused by T. destructans and T. epicoccoides were evaluated and compared on a Eucalyptus grandis × E. urophylla hybrid clone. The optimal temperature for germination ranged from 25 to 30°C for T. destructans and 15 to 20°C for T. epicoccoides . The germination of these pathogens was favored under conditions of light and high levels of RH. Penetration by T. destructans and T. epicoccoides occurred via stomata, and the hyphae colonized the intercellular spaces of infected leaves. Symptoms were clearly visible 3 weeks after inoculation by both pathogens, and reproductive structures started to develop in substomatal cavities at 4 weeks after inoculation. The results of this study will facilitate the establishment of rapid screening trials based on artificial inoculations aimed at reducing the impact of disease caused by T. destructans.

Teratosphaeria destructans is one of the most aggressive foliar pathogens of Eucalyptus. The biological factors underpinning T. destructans infections, which include shoot and leaf blight on young trees, have never been interrogated. Thus, the means by which the pathogen modifies its host environment to overcome host defences remain unknown. By applying transcriptome sequencing, the aim of this study was to compare gene expression in a South African isolate of T. destructans grown on nitrogen-deficient and complete media. This made it possible to identify upregulated genes in a nitrogen-starved environment, often linked to the pathogenicity of the fungus. The results support the hypothesis that nitrogen starvation in T. destructans likely mirrors an in planta genetic response. This is because 45% of genes that were highly upregulated under nitrogen starvation have previously been reported to be associated with infection in other pathogen systems. These included several CAZymes, fungal effector proteins, peptidases, kinases, toxins, lipases and proteins associated with detoxification of toxic compounds. Twenty-five secondary metabolites were identified and expressed in both nitrogen-deficient and complete conditions. Additionally, the most highly expressed genes in both growth conditions had pathogenicity-related functions. This study highlights the large number of expressed genes associated with pathogenicity and overcoming plant defences. As such, the generated baseline knowledge regarding pathogenicity and aggressiveness in T. destructans is a valuable reference for future in planta work., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Teratosphaeria gauchensis and T. zuluensis are closely related fungi that cause Teratosphaeria (previously Coniothyrium) stem canker disease on Eucalyptus species propagated in plantations for commercial purposes. This disease is present in many countries in which Eucalyptus trees are planted, and continues to spread with the international trade of infected plant germplasm., Taxonomy: Fungi, Ascomycota, Pezizomycotina, Dothideomycetes, Dothideomycetidae, Capnodiales, Teratosphaeriaceae, Teratosphaeria., Identification: The causal agents form dark masses of pycnidia that are visible on the surface of distinct stem cankers that typically form on young green stem tissues. Accurate diagnosis of the causal agents requires DNA sequence data., Host Range: Nine species of Eucalyptus are known to be affected. Of these, E. grandis and its hybrids, which include some of the most important planting stock globally, appear to be particularly vulnerable., Disease Symptoms: Small necrotic lesions develop on young green stem tissue. These lesions coalesce to form large cankers that exude gum. Epicormic shoots develop below the girdling canker and, in severe cases, trees die., Useful Websites: Mycobank, https://www.mycobank.org; Publications of the Forestry and Agricultural Biotechnology Institute (FABI), https://www.fabinet.up.ac.za/index.php/journals., (© 2018 The Authors. Molecular Plant Pathology Published by BSPP and John Wiley & Sons Ltd.)

The Eucalyptus stem canker pathogen Teratosphaeria zuluensis was discovered in South Africa in 1988 and it has subsequently been found in several other African countries as well as globally. In this study, the population structure, genetic diversity and evolutionary history of T. z uluensis were analysed using microsatellite markers to gain an enhanced understanding of its movement in Africa. Isolates were collected from several sites in Malawi, Mozambique, Uganda and Zambia. Data obtained were compared with those previously published for a South African population. The data obtained from 334 isolates, amplified across eight microsatellite loci, were used for assignment, differentiation and genetic diversity tests. STRUCTURE analyses, θ st and genetic distances revealed the existence of two clusters, one dominated by isolates from South Africa and the other by isolates from the Zambezi basin including Malawi, Mozambique and Zambia. High levels of admixture were found within and among populations, dominated by the Mulanje population in Malawi. Moderate to low genetic diversity of the populations supports the previously held view that the pathogen was introduced into Africa. The clonal nature of the Ugandan population suggests a very recent introduction, most likely from southern Africa.

During routine surveys for possible fungal pathogens in the rapidly expanding plantations of Eucalyptus and Cunninghamia lanceolata in China, numerous isolates of unknown species in the genus Ceratocystis (Microascales) were obtained from tree wounds. In this study we identified the Ceratocystis isolates from Eucalyptus and Cunninghamia in the GuangDong, GuangXi, FuJian and HaiNan Provinces of South China based on morphology and through comparisons of DNA sequence data for the ITS, partial β-tubulin and TEF-1α gene regions. Morphological and DNA sequence comparisons revealed two previously unknown species residing in the Indo-Pacific Clade. These are described here as Ceratocystis cercfabiensis sp. nov. and Ceratocystis collisensis sp. nov. Isolates of Ceratocystis cercfabiensis showed intragenomic variation in their ITS sequences and four strains were selected for cloning of the ITS gene region. Twelve ITS haplotypes were obtained from 17 clones selected for sequencing, differing in up to seven base positions and representing two separate phylogenetic groups. This is the first evidence of multiple ITS types in isolates of Ceratocystis residing in the Indo-Pacific Clade. Caution should thus be exercised when using the ITS gene region as a barcoding marker for Ceratocystis species in this clade. This study also represents the first record of a species of Ceratocystis from Cunninghamia.

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.

The genus Celoporthe was first described when C. dispersa was discovered in South Africa associated with dieback and cankers on trees in the Myrtales. Four additional species were recently described from Eucalyptus and Syzygium cumini in China as well as S. aromaticum and Eucalyptus in Indonesia. Inoculation trials have shown that all Celoporthe species, including those that have not been found on Eucalyptus species in nature, are pathogenic to Eucalyptus and they are thus potentially threatening to commercial Eucalyptus forestry. New isolates, morphologically similar to Celoporthe, have been collected from S. legatti in South Africa and S. guineense in Zambia. Multigene phylogenetic analyses based on DNA sequences of the ITS region, TEF1α gene and two areas of the β-tubulin gene revealed additional cryptic species in Celoporthe. Phylogenetic data were supported by morphological differences. These resulted in the description of two previously unknown species of Celoporthe, namely C. fontana and C. woodiana, for two of these cryptic groups, while the third group represented C. dispersa. These species all can readily infect Eucalyptus as well as several species of Syzygium, the latter of which are native to Africa.

Human-associated introduction of pathogens and consequent invasions is very evident in areas where no related organisms existed before. In areas where related but distinct populations or closely related cryptic species already exist, the invasion process is much harder to unravel. In this study, the population structure of the Eucalyptus leaf pathogen Teratosphaeria nubilosa was studied within its native range in Australia, including both commercial plantations and native forests. A collection of 521 isolates from across its distribution was characterized using eight microsatellite loci, resulting in 112 multilocus haplotypes (MLHs). Multivariate and Bayesian analyses of the population conducted in structure revealed three genetically isolated groups (A, B and C), with no evidence for recombination or hybridization among groups, even when they co-occur in the same plantation. DNA sequence data of the ITS (n = 32), β-tubulin (n = 32) and 27 anonymous loci (n = 16) were consistent with microsatellite data in suggesting that T. nubilosa should be considered as a species complex. Patterns of genetic diversity provided evidence of biological invasions by the pathogen within Australia in the states of Western Australia and New South Wales and helped unravel the pattern of invasion beyond Australia into New Zealand, Brazil and Uruguay. No significant genetic differences in pathogen populations collected in native forests and commercial plantations were observed. This emphasizes the importance of sanitation in the acquisition of nursery stock for the establishment of commercial plantations., (© 2012 Blackwell Publishing Ltd.)

The genus Ceratocystis includes important fungal pathogens of trees, including Eucalyptus spp. Ironically, very little is known regarding the diversity or biology of Ceratocystis species on Eucalyptus species in Australia, where most of these trees are native. The aim of this study was to survey for Ceratocystis spp., and their possible insect associates, on eucalypts in Australia and thus to establish a foundation of knowledge regarding these fungi on the continent. Collections were made in three states of Australia from wounds on trees, as well as from nitidulid beetles associated with these wounds. Ceratocystis spp. were identified based on morphology and multigene sequence comparisons. Of the 54 isolates obtained, two previously unknown species of Ceratocystis were found and these are described here as Ceratocystis corymbiicola sp. nov. and Ceratocystis tyalla sp. nov. Furthermore, the distribution of Ceratocystis pirilliformis is expanded to include Eucalyptus spp. in Tasmania.

DNA extraction from minute hymenopterans and their larvae is difficult and challenging because of their small size indicating a low amount of starting material. Hence, 11 DNA extraction methods were compared to determine their efficacy in isolating DNA. Success of each method was scored on a 2% agarose gel after PCR of the cox 1 mitochondrial locus. A silica-membrane-based approach was the most successful, followed by a method using a combination of incubation buffers and a method using magnetic beads. The method using buffers was the most cost- and time effective. Using this method, larvae from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline) in the gall-inhabiting complex., (© 2011 Blackwell Publishing Ltd.)

Many species in the Cryphonectriaceae cause diseases of trees, including those in the genera Eucalyptus and Syzygium. During disease surveys on these trees in southern China, fruiting structures typical of fungi in the Cryphonectriaceae and associated with dying branches and stems were observed. Morphological comparisons suggested that these fungi were distinct from the well known Chrysoporthe deuterocubensis, also found on these trees in China. The aim of this study was to identify these fungi and evaluate their pathogenicity to Eucalyptus clones/species as well as Syzygium cumini. Three morphologically similar fungal isolates collected previously from Indonesia also were included in the study. Isolates were characterized based on comparisons of morphology and DNA sequence data for the partial LSU and ITS nuclear ribosomal DNA, β-tubulin and TEF-1α gene regions. After glasshouse trials to select virulent isolates field inoculations were undertaken to screen different commercial Eucalyptus clones/species and S. cumini trees for susceptibility to infection. Phylogenetic analyses showed that the Chinese isolates and those from Indonesia reside in a clade close to previously identified South African Celoporthe isolates. Based on morphology and DNA sequence comparisons, four new Celoporthe spp. were identified and they are described as C. syzygii, C. eucalypti, C. guangdongensis and C. indonesiensis. Field inoculations indicated that the three Chinese Celoporthe spp., C. syzygii, C. eucalypti and C. guangdongensis, are pathogenic to all tested Eucalyptus and S. cumini trees. Significant differences in the susceptibility of the inoculated Eucalyptus clones/species suggest that it will be possible to select disease-tolerant planting stock for forestry operations in the future.

The Cryphonectriaceae accommodates some of the world's most important tree pathogens, including four genera known from native and introduced Myrtales in Africa. Surveys in the past 3 y in southern Africa have led to the discovery of cankers with fruiting structures resembling those of the Cryphonectriaceae on trees in the Myrtales in Namibia, South Africa, Swaziland and Zambia. These fungi were identified with morphological characteristics and DNA sequence data. For the first time we report Chrysoporthe austroafricana from Namibia and on Syzygium guineense and Holocryphia eucalypti in Swaziland on a Eucalyptus grandis clone. The host and geographic ranges of Celoporthe dispersa are expanded to include S. legatti in South Africa and S. guineense in Zambia. In addition a monotypic genus, Latruncellus aurorae gen. sp. nov., is described from Galpinia transvaalica (Lythraceae, Myrtales) in Swaziland. The present and other recent studies clearly emphasize the limited understanding of the diversity and distribution of fungi in the Cryphonectriaceae in Africa.

Chrysoporthe cubensis is one of the most important pathogens of Eucalyptus. Based on phylogenetic evidence and geographic origin, isolates of this fungus are known to reside in distinct 'South America' and 'Southeast Asia' clades. In this study, reproductive isolation amongst these isolates of C. cubensis was tested using gene flow statistics for 12 polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. Gene flow statistics between populations, and relative to the closely related Chrysoporthe austroafricana, were low and not significantly different (P<0.05). Additionally, phylogenetic analyses of DNA sequence data for four gene regions convincingly distinguished the two subclades of C. cubensis. Isolates in the Southeast Asian subclade are described in the new species, Chrysoporthe deuterocubensis. Chrysoporthe cubensis and C. deuterocubensis represent closely related fungi that are thought to be native to South America and Southeast Asia, respectively. A technique is presented that allows for rapid differentiation between these species and that will aid in quarantine procedures to limit their spread to new environments., (Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Aims: This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay., Methods and Results: Several molecular techniques including 16S rRNA and rpoB gene sequencing and DNA-DNA hybridization were used to characterize the gram-negative, facultatively anaerobic, slime-producing bacterial strains isolated along with Pantoea species from Eucalyptus. Hypersensitivity reactions (HR) and pathogenicity tests were performed on tobacco and Eucalyptus seedlings, respectively. The isolates clustered closely with the type strain of Enterobacter cowanii in both phylogenetic trees constructed. The DNA-DNA similarity between the isolates and the type strain of Ent. cowanii ranged from 88% to 92%. A positive HR was observed on the tobacco seedlings, but no disease symptoms were visible on the inoculated Eucalyptus seedlings., Conclusions: Enterobacter cowanii was isolated from trees with symptoms of bacterial blight although strains of this bacterial species do not appear to be the causal agent of the disease., Significance and Impact of the Study: This study provides the first report of Ent. cowanii isolated from Eucalyptus. Its presence in Eucalyptus tissue suggests that it is an endophyte in trees showing symptoms of blight.

Background: Teratosphaeria nubilosa is a serious leaf pathogen of several Eucalyptus spp. This review considers the taxonomic history, epidemiology, host associations and molecular biology of T. nubilosa., Taxonomy: Kingdom Fungi; Phylum Ascomycota; Class Dothideomycetes; Order Capnodiales; Family Teratosphaeriaceae; genus Teratosphaeria; species nubilosa., Identification: Pseudothecia hypophyllous, less so amphigenous, ascomata black, globose becoming erumpent, asci aparaphysate, fasciculate, bitunicate, obovoid to ellipsoid, straight or incurved, eight-spored, ascospores hyaline, non-guttulate, thin walled, straight to slightly curved, obovoid with obtuse ends, medially one-septate, slightly constricted at the median septum, tapering to both ends, ascospore germination type F, germinating from both ends, germ tubes growing parallel to the long axis of the spore with distortion of the primary ascospore cell., Host Range: Teratosphaeria nubilosa is a primary pathogen of several Eucalyptus spp., including E. botryoides, E. bicostata, E. bridgesiana, E. cypellocarpa, E. dunnii, E. globulus ssp. bicostata, E. globulus ssp. globulus, E. globulus ssp. maidenii, E. globulus ssp. pseudoglobulus, E. grandis, E. gunnii, E. nitens, E. pilularis, E. quadrangulata, E. viminalis, E. grandis x E. resinifera and E. urophylla x E. globulus., Disease Symptoms: Leaf spots predominantly occur on juvenile Eucalyptus foliage; however, T. nubilosa has also recently been found on mature Eucalyptus foliage. Leaf spots are amphigenous, varying in size from small spots that are round to irregular. Lesions enlarge and coalesce to form larger blotches over the leaf surface. Initial lesions appear as pale-green spots surrounded by purple margins and, once mature, are generally yellow to pale brown with dark-brown raised borders., Useful Websites: Mycobank, http://www.mycobank.org; Mycosphaerella identification website, http://www.cbs.knaw.nl/mycosphaerella/BioloMICS.aspx.

A recent study to determine the cause of collar and root rot disease outbreaks of cold tolerant Eucalyptus species in South Africa resulted in the isolation of two putative new Phytophthora species. Based on phylogenetic comparisons using the ITS and beta-tubulin gene regions, these species were shown to be distinct from known species. These differences were also supported by robust morphological characteristics. The names, Phytophthora frigida sp. nov. and Phytophthora alticola sp. nov. are thus provided for these taxa, which are phylogenetically closely related to species within the ITS clade 2 (P. citricola, P. tropicali and P.multivesiculata) and 4 (P. arecae and P. megakarya), respectively. Phytophthora frigida is heterothallic, and produces stellate to rosaceous growth patterns on growth medium, corraloid hyphae, sporangia with a variety of distorted shapes and has the ability to grow at low temperatures. Phytophthora alticola is homothallic and has a slower growth rate in culture. Both P. frigida and P. alticola are pathogenic to Eucalyptus dunnii. In pathogenicity tests, they were, however, less pathogenic than P. cinnamomi, which is a well-known pathogen of Eucalyptus in South Africa.

Species of Phaeophleospora are anamorphs of Mycosphaerella and they include some of the most serious foliar pathogens of Eucalyptus spp. grown in plantations worldwide. Pathogens assigned to this genus and occurring on Eucalyptus spp. were previously treated in Kirramyces and they are also phylogenetically closely related to other anamorphs of Mycosphaerella residing in the genus Colletogloeopsis. The primary aim of this study was to consider the appropriate taxonomic placement of these species. To achieve this goal, morphological characteristics and DNA sequence data from the ITS and translation EF1-alpha gene regions were used to compare the type species P. eugeniae, Phaeophleospora spp. and Colletogloeopsis spp. occurring on eucalypts, using ex-type cultures and herbarium specimens. Phylogenetic data and morphological comparisons supported the separation of P. eugeniae from Phaeophleospora species occurring on eucalypts. The name Phaeophleospora is retained for P. eugeniae and the name Kirramyces is resurrected for the species occurring on eucalypts (genera Eucalyptus, Corymbia, and Angophora). Sequence data from the type specimens of two previously described species of Kirramyces, K. lilianiae and K. delegatensis, show they reside in a clade with other Kirramyces spp. Morphological and DNA sequence comparisons also showed that there is considerable overlap between species of Phaeophleospora and Colletogloeopsis from eucalypts. Based on these findings, Colletogloeopsis is reduced to synonymy with the older Kirramyces and the description of Kirramyces is emended to include species with aseptate, as well as multiseptate, conidia produced in acervuli or pycnidia. Two new species of Kirramyces, K. angophorae and K. corymbiae, are also described.

The majority of Eucalyptus species are native to Australia, but worldwide there are over 3 million ha of exotic plantations, especially in the tropics and subtropics. Of the numerous known leaf diseases, three species of Phaeophleospora can cause severe defoliation of young Eucalyptus; Phaeophleospora destructans, Phaeophleospora eucalypti and Phaeophleospora epicoccoides. Phaeophleospora destructans has a major impact on seedling survival in Asia and has not, as yet, been found in Australia where it is considered a serious threat to the biosecurity of native eucalypts. It can be difficult to distinguish Phaeophleospora species based on symptoms and micromorphology and an unequivocal diagnostic tool for quarantine purposes would be useful. In this study, a multiple gene genealogy of these Phaeophleospora species and designed specific primers has been constructed to detect their presence from leaf samples. The phylogenetic position of these Phaeophleospora species within Mycosphaerella was established. They are closely related to each other and to other important Eucalyptus pathogens, Mycosphaerella nubilosa, Mycosphaerella cryptica and Colletogloeopsis zuluensis. The specific primers developed can now be used for diagnostic and screening purposes within Australia.

Botryosphaeria spp. are common endophytes of woody plants, and they also include some serious pathogens of Eucalyptus and Acacia species. Numerous anamorphs have been associated with Botryosphaeria, of which the species Fusicoccum are amongst the most common. Here, we characterize two new Fusicoccum species, isolated from Eucalyptus and Acacia trees in Venezuela, based on morphological features in culture and comparisons of DNA sequence data. The two taxa named Fusicoccum andinum and F. stromaticum spp. nov, reside in two well-supported clades (BS values = 100%) based on a combined data set of the ITS of the rDNA operon and translation elongation factor 1-alpha (EF1- alpha) gene sequences. The conidia of F. andinum are unusually large amongst Botryosphaeria anamorphs, and peripherally resemble those of B. mamane and B. melanops. F. stromaticum is characterized by large conidiomata in cultures, growth at 35 degrees C and slightly thickened conidial walls, which is different to most other Fusicoccum spp. No teleomorphs were observed for these fungi, but DNA sequence data show that they are anamorphs of Botryosphaeria.

Coniothyrium zuluense causes a serious canker disease of Eucalyptus in various parts of the world. Very little is known regarding the taxonomy of this asexual fungus, which was provided with a name based solely on morphological characteristics. In this study we consider the phylogenetic position of C. zuluense using DNA-based techniques. Distance analysis using 18S and ITS regions revealed extensive sequence divergence relative to the type species of Coniothyrium, C. palmarum and species of Paraconiothyrium. Coniothyrium zuluense was shown to be an anamorph species of Mycosphaerella, a genus that includes a wide range of Eucalyptus leaf and stem pathogens. Within Mycosphaerella it clustered with taxa having pigmented, verruculose, aseptate conidia that proliferate percurrently and sympodially from pigmented conidiogenous cells arranged in conidiomata that vary from being pycnidial to acervular. The genus Colletogloeopsis is emended to include species with pycnidial conidiomata, and the new combination Colletogloeopsis zuluense is proposed. This is also the first report of the pathogen from China where it is associated with stem cankers on Eucalyptus urophylla.

Species within the genus Botryosphaeria include some of the most widespread and important pathogens of woody plants, and have been the focus of numerous taxonomic studies in recent years. It is currently accepted that anamorphs of Botryosphaeria belong to two distinct genera, Fusicoccum and Diplodia. Species within the genus Fusicoccum commonly produce aseptate, hyaline conidia. In the present study, fungi were isolated from foliage and wood of Eucalyptus in native forests and plantations in Australia. Although these fungi produced Dichomera anamorphs in culture, they clustered within the Fusicoccum clade of Botryosphaeria based on their ITS sequence data. Four species, Botryosphaeria dothidea, B. parva, B. ribis and B. australis produced Dichomera conidia in culture. The Dichomera synanamorphs are described for these four species of Botryosphaeria. In addition, falling within the Fusicoccum clade of Botryosphaeria, two species were found to be distinct from previously described Botryosphaeria spp. based on their ITS sequences, but synonymous with D. versiformis and D. eucalypti. These observations are currently unique to isolates from host trees within the genus Eucalyptus in Australia, and the pleoanamorphic nature of these species is discussed.

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.

The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-alpha gene (EF 1-alpha) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length:width > 1.5), in contrast to the dark brown conidia of Coniella (avg. length:width < or = 1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study.

Coniothyrium canker caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus species in sub-tropical regions of South Africa. This disease is typified by necrotic bark lesions that coalesce to form large kino-impregnated cankers along the stems of trees. The strategy currently used to manage Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Thus, forest managers are interested in the genetic diversity of the pathogen and its potential to overcome disease resistance in planting stock. In this study, 344 isolates of C. zuluense from different plantation regions in South Africa were compared on the basis of colony colour, conidial morphology, growth characteristics on agar and pathogenicity to a susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were similar in size and shape. The fungus is slow growing in culture, which is indicative of its apparent biotrophic habit, with optimum growth observed at 30 degrees C. Isolates of C. zuluense displayed considerable variation in colony colour and pathogenicity in inoculation trials. Variation in morphology and pathogenicity amongst isolates suggests that C. zuluense has been present in South Africa for an extended period of time, or that it is changing rapidly due to strong directional selection pressures.

Wilt and death of an E. grandis × E. urophylla variety was recently observed in the Zululand region of KwaZulu-Natal, South Africa. Symptoms on the dying trees included a streaking pattern of discolouration in the sapwood and cambium of stems and roots. A fungus resembling a Ceratocystis sp. was consistently found sporulating on diseased material and isolated from the symptomatic root and stem tissue. DNA sequence analyses of the Ceratocystis isolates, including multiple gene regions, identified the isolates as C. eucalypticola. Multiple ITS types were identified among the isolates sequenced, similar to those found in other, closely related, Ceratocystis species. Artificial inoculations under field conditions confirmed the pathogenicity of the isolates. This is the first report of a Ceratocystis sp. causing a wilt disease of Eucalyptus in South Africa. [ABSTRACT FROM AUTHOR]