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Butternut (Juglans cinerea L. [Juglandaceae]) is a native tree in the US and Canada that is in rapid decline due to an introduced fungal pathogen (Ophiognomonia clavigignenti juglandacearum [Oc-j]). Resistance to the disease has not been identified, and conservation efforts for this nut-producing species have concentrated on the ex situ cryogenic storage of germplasm. The conventional method for growing butternut uses the nut. In general, Juglans species exhibit seed dormancy, which can be broken by cold, moist stratification at 1-4 °C (32-39.2 °F) for 90-120 d. Stratified nuts usually germinate within 4 wk, with variation among seeds. Average germination by the conventional method is approximately 60% for J. cinerea. A new in vitro germination protocol has been developed for the production of butternut seedlings that results in high germination and acclimatization success (conversion of germinants growing on medium to seedlings growing in soil) for both cryogenically and non-cryogenically stored embryonic axes (root-shoot axis). This protocol eliminates the need for conventional nut stratification; produces decontaminated tissue via surface sterilization, such that seedlings are visibly canker-free; increases germination; and enables long-term cryogenic storage. [ABSTRACT FROM AUTHOR]

Summary: The use of a molecular assay for quantifying conidia of Ophiognomonia clavigignenti‐juglandacearum, the fungal pathogen responsible of butternut canker, was investigated. Purified DNA from conidia collected on glass fibre filters of a passive rain collectors was quantified using a TaqMan real‐time quantitative polymerase chain reaction (qPCR) assay. The qPCR assay could specifically discriminate the target species from all other North American known species of Ophiognomonia, and it was sensitive enough to repeatedly detect one conidium. A linear relationship between numbers of conidia and qPCR Ct values was determined, and used to assess the sporulation of the pathogen under trees that were released to promote their vigour. In total, 977 samples of field‐captured conidia from 49 trees, at two locations, and from two successive growing seasons were analysed. No significant difference of sporulation was observed under control and release treatments. However, our results demonstrated that qPCR assay was reliable for detecting and quantifying O. clavigignenti‐juglandacearum from environmental samples, which will be useful to assess further control methods for this disease. [ABSTRACT FROM AUTHOR]

Butternut trees dying from a canker disease were first reported in southwestern Wisconsin in 1967. Since then, the disease has caused extensive mortality of butternut throughout its North American range. The objectives of this study were to quantify changes in butternut populations and density across its range and identify habitat characteristics of sites where butternut is surviving in order to locate regions for potential butternut restoration. The natural range of butternut ( Juglans cinerea L.) extends over a large region of eastern N. America encompassing New Brunswick south to North Carolina, north to Minnesota, and southwest to Missouri. Despite the species' large range, it is typically not a common tree, comprising a relatively minor component of several different forest types. We evaluated change in butternut abundance and volume from current and historic data from 21 states in the eastern United States. We related abundance and volume at two time periods to a suite of ecological and site factors in order to characterize site conditions where butternut survived. We also assessed the current level of butternut mortality across its range. Since the 1980s, the number of butternut trees and butternut volume have decreased by 58% and 44%, respectively, across its US range. Substantial relative decreases in tree numbers and volume occurred in most ecoregion sections. Five environmental variables were found to be significant predictors of butternut presence. The potential impacts of butternut canker are particularly acute as the canker pathogen invasion pushes a rare tree species toward extinction, at least at a local scale. Based on the results presented here, large-diameter maple/beech/birch stands in dry, upland sites in eastern Minnesota, western Wisconsin, and upstate New York appear to offer the most favorable conditions for butternut growth and survival and thus may be the best stands for planting resistant butternut trees. [ABSTRACT FROM AUTHOR]

Diaporthe ampelina, causal agent of Pho-mopsis cane and leaf spot of grapevine (Vitis vinifera L.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthe species, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthe community in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaporthe isolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthe species from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricana and D. novem in North America. Our findings also expand the host ranges of D. ambigua to apricot and willow, D. australafricana to almond and willow, D. chamaeropis to grapevine and willow, D. foeniculina to willow and D. novem to almond. The generalists D. ambigua and D. eres were the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelina was the most pathogenic species to grapevine wood. [ABSTRACT FROM AUTHOR]

Butternut (Juglans cinerea L.) trees are being extirpated from their natural range by an epidemic caused by a fungal pathogen. Widespread mortality is reminiscent of past epidemics on American chestnut (Castanea dentata (Marsh.) Borkh.) and American elm (Ulmus americana L.). Butternut has remained relatively understudied, resulting in unsampled areas and gaps in our understanding of this forest epidemic and the future outlook of this species in North America. The previously unsampled area consisting of the northeastern United States was surveyed for the presence of J. cinerea, and several population health metrics were recorded, including recruitment, disease pressure, and hybridization. A total of 252 butternut trees were sampled. Analysis indicates that there is insufficient J. cinerea recruitment to maintain population sizes. Further compounding low recruitment, butternut saplings demonstrate elevated levels of disease impact from the fungal pathogen Ophiognomonia clavigignentijuglandacearum Broders & Boland. Natural hybridization of butternut with introduced congenics such as Juglans ailantifolia Carrière is strongly associated with lower disease impact. Hybrid trees displayed an average of 2.4 cankers per tree compared with 4.5 cankers for nonhybrid butternut. Further niche and resistance studies are required to assess whether butternut hybrids can replace butternut in a natural setting. It still remains uncertain whether tree size or habitat affect disease impact; however, smaller trees, often residing in riparian habitats, were found to have a greater number of cankers. The data presented here, combined with past studies, provide critical information for use in butternut management strategy plans. [ABSTRACT FROM AUTHOR]

The abundance of butternut ( Juglans cinerea L.) trees has severely declined rangewide over the past 50 years. An important factor in the decline is butternut canker, a disease caused by the fungus Ophiognomonia clavigigenti- juglandacearum, which has left the remaining butternuts isolated and sparsely distributed. To manage the remaining populations effectively, information regarding how butternut's population genetic structure is affected by environmental and historical factors is needed. In this study, we assessed genetic structure and diversity of 161 butternut trees from 19 adjacent watersheds in the southern portion of butternut's range using 12 microsatellite markers. We assessed the genetic diversity and genetic differentiation among trees grouped at various spatial scales. Our goal was to use historical abundance and land use data for these watersheds, which are now all a part of the Great Smoky Mountains National Park (GSMNP), to understand the ecological and evolutionary forces that challenge the conservation and management of butternut. In general, butternuts within the 19 neighboring watersheds were all part of one continuous population, with gene flow throughout. Significant genetic differentiation was detected between some groups of trees, but the differentiation was quite small and may not represent an ecologically significant distinction. The mean heterozygosity in all watersheds remained high, despite extensive mortality. Overall, genetic diversity and rare alleles were evenly distributed across all watersheds, with some variability in subpopulations containing butternut-Japanese walnut hybrids ( Juglans x bixbyi or buarts). These results indicate that management of this species should focus on protection from future hybridization with Japanese walnut, promotion of regeneration, and persistence of all remaining butternut trees, which still retain high levels of genetic diversity. [ABSTRACT FROM AUTHOR]

The article reflects on a study conducted within Great Smoky Mountains National Park (GSMNP) to examine the population of Juglans cinerea L. (butternut) to protect it and for its restoration. It mentions that J. cinerea is infected by Ophiognomonia clavigignenti-juglandacearum, a non native pathogen, which causes butternut canker disease. The study found that recruitment of J. cinerea trees has declined severely over the past 30 years because of less-intensive contemporary disturbance regime.

The genetic consequences of natural in situ recovery for rare or threatened species are not as well understood as the impact of population bottlenecks, fragmentation and admixture, particularly the mechanisms by which genetic diversity is lost or preserved as populations recover. Here we examine how mating patterns, dispersal and ecologically constrained regeneration influences genetic diversity and kinship in a naturally regenerating population of a threatened temperate forest tree, Juglans cinerea L. (butternut). Butternut regeneration is now rare throughout the native range due to the butternut canker, a lethal fungal disease from Asia, and land use changes. In this study of one of the only known regenerating patches large enough for kinship and parentage analysis, we used 12 microsatellite markers, direct and inferred parentage analyses and Bayesian clustering of 152 trees to show that natural regeneration at this site resulted in loss of allele richness due to a small number of parents, most of which are spatially proximal to the regenerants. Of the 116 potential parents tested, one contributed 20.8 % and the top four contributed 71.1 % of the gametes in 36 regenerants. Parent-parent and parent-offspring distances revealed limited pollen and seed dispersal (<100 m). Regenerants were highly related and spatially clustered in sibling groups. Proximity to the regenerating patch was the most significant factor in parental success. Our results suggest that in situ regeneration of forest trees with limited propagule dispersal and specific site requirements may be insufficient to preserve native genetic diversity in protected areas with few suitable sites. [ABSTRACT FROM AUTHOR]

Species of Ophiognomonia are leaf-inhabiting endophytes, pathogens, and saprobes that infect plants in the families Betulaceae, Fagaceae, Juglandaceae, Lauraceae, Malvaceae, Platanaceae, Rosaceae, Salicaceae, and Sapindaceae. Based on extensive collecting, this species-rich genus is now known to have a world wide distribution in primarily temperate areas, although some species are known from the subtropics. Analyses of DNA sequences from three markers including guanine nucleotide-binding protein subunit beta-like protein (MS204), translation elongation factor 1α ( tef-1α), and the ITS region including ITS1, 5.8 S rDNA and ITS2 regions (ITS) were used to define phylogenetic species in Ophiognomonia. Host plant association correlated with these species. Twenty-five new species of Ophiognomonia and two new combinations are proposed with descriptions and illustrations. In addition, descriptions and illustrations are provided for 12 other species of Ophiognomonia. A key is provided to the 45 currently accepted species of Ophiognomonia. The disposition of additional names in Ophiognomonia is also discussed. [ABSTRACT FROM AUTHOR]

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217 000 reads, 80% of which collapsed into 16 125 754 bases in 30 339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. E software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability. [ABSTRACT FROM AUTHOR]

Butternut ( Juglans cinerea L.) is a temperate deciduous hardwood native to the eastern USA and southern Canada valued for its nuts and wood. Butternut's survival is threatened by butternut canker, a disease caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Field observations indicate that trees commonly called buartnut (a hybrid of butternut and its close congener Japanese walnut ( Juglans ailantifolia × J. cinerea)) may be more resistant to butternut canker than is either parental species. Hybrids are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. We used pair-wise combinations of 40 random primers to screen bulked DNA pools of butternut, Japanese walnut, and buartnuts to identify genomic regions unique to Japanese walnut. We ultimately identified one ITS region marker, one chloroplast marker, one mitochondrial marker, and six nuclear markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. We confirmed that hybrids have a complex genetic history and present features of the parental species in all possible combinations. These results will assist in the identification and testing of (non-hybrid) butternut for breeding and reintroduction of the species to its former habitats. [ABSTRACT FROM AUTHOR]

Abstract: Sirococcus clavigignenti-juglandacearum (Sc-j), which causes a canker disease on butternut, is largely responsible for the decline of this tree in the United States and Canada. The original description of the species was based on anamorphic characters because the teleomorph is unknown. Recent phylogenetic investigations have found that Sc-j is not a member of the genus Sirococcus, and accurate taxonomic classification is required. The objective of this study is to use sequence data to determine the phylogenetic placement of Sc-j within the Gnomoniaceae, Diaporthales. Isolates were recovered from infected Juglans ailantifolia var. cordiformis (heartnut), Juglans cinerea (butternut), and Juglans nigra (black walnut) in Ontario and the eastern United States. The genes coding for β-tubulin, actin, calmodulin, internal transcribed spacers 1 and 2, and the translation elongation factor 1-alpha from 28 isolates of Sc-j and representatives of the major lineages within the Gnomoniaceae were evaluated. There was no difference in the sequences of the five genes among the isolates of Sc-j studied, indicating a recent introduction followed by asexual reproduction and spread via conidia. The phylogenetic analyses demonstrate this fungus does not belong to the genus Sirococcus, and provides strong support (99% MP and 100% NJ bootstrap values, and 100% Bayesian posterior probabilities) for its inclusion in the genus Ophiognomonia, thereby supporting a reclassification of the butternut canker fungus to Ophiognomonia clavigignenti-juglandacearum. [ABSTRACT FROM AUTHOR]

Butternut canker, caused by the fungal pathogen Sirococcus clavigignenti-juglandacearum, is present throughout the range of butternut (Juglans cinerea) and is the primary cause for its decline. A quick and reliable method for identification of S. clavigignenti-juglandacearum would provide a valuable tool for the detection of the pathogen on propagative material to avoid spread, as well as assist studies targeted at the epidemiology of this pathogen, in particular the dissemination of the pathogen by seeds of the butternut. The objective of this study was to develop a diagnostic assay to detect S. clavigignenti-juglandacearum in butternut plant tissue. The primers were developed using an alignment of internal transcribed spacer (ITS) sequences from isolates of S. clavigignenti-juglandacearum and several closely related species. These primers were tested on J. cinerea, 48 isolates of S. clavigignenti-juglandacearum recovered from diseased trees, and 26 species of other fungi recovered from butternut tissue. The primers amplified a product from the DNA of all isolates of S. clavigignenti-juglandacearum, detected its DNA at a concentration as low as 1 pg/pl, and detected the pathogen at a concentration of 1 x 1O spore/ml. The primers developed in this study will be a valuable tool for the detection of S. clavigignenti-juglandacearum present on butternut seeds, and as a rapid diagnostic tool for early detection of the pathogen on butternut trees. [ABSTRACT FROM AUTHOR]

Butternut ( Juglans cinerea L.) is an eastern North American forest tree severely threatened by an exotic fungal pathogen, Sirococcus clavigignenti-juglandacearum. We report here 13 nuclear microsatellites for genetic evaluation of the remaining natural populations. Summary statistics are reported for individuals from a population of butternuts in central Kentucky ( N = 63). All markers were polymorphic, with an average of 13.7 alleles per locus observed. Four loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium ( P < 0.05). [ABSTRACT FROM AUTHOR]

Abstract: A severe canker disease has been causing decline and death of Corymbia calophylla in the southwest of Western Australia (WA) for some years, but the causal agent has never been investigated. However, there have been historical reports dating back to the 1920s of a canker disease of amenity planted C. ficifolia caused by ‘Sporotrichum destructor’, though the description and Latin diagnosis were never published. It has been suggested that there may be links between this species and the genus Quambalaria, a group containing leaf and shoot pathogens of species of Eucalyptus and Corymbia. The aim of this study was to investigate the identity of the pathogen historically attributed to canker disease of C. ficifolia, determine whether this pathogen is responsible for the current epidemic of C. calophylla canker, and whether it is synonymous with Quambalaria. Surveys examined the range of Quambalaria spp. on Corymbia spp. endemic to southwest WA. Their phylogenetic relationship to Q. cyanescens, Q. eucalypti, and Q. pitereka was examined using rLSU and ITS sequence data. Morphological characters were also compared. Sequences confirmed that Q. cyanescens and Q. pitereka are present in southwest WA, with the latter associated with leaf and shoot disease. A third group isolated from cankers represent a new species of Quambalaria. Comparisons of disease symptoms and conidiogenesis indicate this species is synonymous with ‘S. destructor’. The species is formally described here as Q. coyrecup sp. nov. [Copyright &y& Elsevier]

The fungus Sirococcus clavigignenti-juglandacea rum causes butternut canker in North America and is not known to kill or seriously affect tree species other than butternut (Juglans cinerea). However, this putative exotic pathogen has been found on branches of black walnut (J. nigra) and heartnut (J. ailantifolia var. cordiformis), raising questions regarding the potential host range of the pathogen and its spread to new areas on tree hosts other than butternut. Results of artificial inoculations of several hardwood species indicated that the pathogen, although not causing lethal cankers, has the ability to colonize and survive in the wood of several genera, including Quercus, Carya, Corylus, Prunus, and Castanea. Several commercially important Persian walnut (J. regia) cultivars tested were moderately or highly susceptible, underscoring the importance of preventing the movement of the pathogen into commercial Juglans plantations in California, where its ability to cause disease and damage trees is unknown. Evidence for the presence of variation in aggressiveness among isolates of S. clavigignenti-juglandacearum was detected based on differences in canker lengths resulting from inoculations with two isolates of the pathogen. In a field test of several previously named butternut cultivars, all were found to be highly susceptible to the disease, calling into question the value of these selections for nut pro- duction or future restoration uses. [ABSTRACT FROM AUTHOR]

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An attempt is made to determine the cause of a new canker and dieback disease affecting poplar hybrids (Populus spp. ) growing in Ontario. Since the disease symptoms appear to be similar to those of a bacterial infection of poplars in Eruope caused by Xanthomonas populi Ridé, bacterial species were investigated as possible causal agents of this new disease, Strains of Erwinia herbicola and Erwinia carotovora were associated with branch cnakers on affected trees. The hypersensitive reaction on tobacco (Nicotiana tabacum var, Havana) resulting from artificial inoculations of strains of E, carotovora suggests the phytopathogenicity of these strains. Early results form inoculations into rooted poplar cuttings indicated that both strains may be capable of causing discolouration and decay of xylem tissues but these inoculations did not result in canker formation. Both of these opportunistic organisms may be potential pathogens of poplar under certain conditions. [ABSTRACT FROM AUTHOR]

Vegetative compatibility of strains of Diaporthe ambigua has not previously been examined. Single ascospore and single ascus strains, originating from individual apple, pear and plum rootstocks, were paired on freshly prepared oatmeal agar to determine if vegetative incompatibility could be detected in D. ambigua. Barrage reactions were evident as black lines along the zone of mycelial contact between expanding colonies (vegetative incompatibility reaction). Strains from cankers within an area were of numerous vegetative compatibility groups (VCGs). Strains from adjacent rootstocks usually differed in VCG. D. ambigua has the ability to outcross, and does so, despite its apparent homothallic nature. DsRNA-containing strains of D. ambigua developed a broad, clear zone when paired with a dsRNA-containing strain from a different VCG. [ABSTRACT FROM AUTHOR]

Butternut trees (Juglans cinerea) clonally propagated via grafting from source trees with putative resistance to butternut canker were inoculated monthly with two isolates of Sirococcus clavigignenti-juglandacearum in a field planting in Minnesota. Significant differences in resulting canker length were found among (i) month of inoculation, (ii) accession, and (iii) isolate. Results support the suggestion that canker-free butternut occasionally found growing among butternut severely affected by butternut canker potentially have disease resistance and may have value for genetic conservation and restoration of the species. Developing an effective resistance screening technique will require additional information on the natural infection process and testing under various climatic conditions across the range of butternut. [ABSTRACT FROM AUTHOR]

Butternut ( Juglans cinerea L.), an early successional riparian hardwood species native to Canada and the United States, is under serious threat from a nonnative fungal pathogen, Ophiognomonia clavigignenti-juglandacearum . Since it was first reported in Canada in 1990, this fungal pathogen has spread rapidly and established in New Brunswick in 1997. Apart from the first report in 1997 and another in 2004, no surveys have been conducted to assess the spread of the pathogen in the province. The purpose of this research was to survey butternut throughout its range in New Brunswick, evaluate disease occurrence as well as tree health, and investigate the impact of different topographic and tree health factors on canker incidence. Results showed that the disease has spread throughout the range of butternut in New Brunswick. The disease likely only recently (2007) spread to the northeastern-most populations, given that lower rates of canker occurrence and higher health ratings are found further away from the point of initial occurrence of the disease. Although canker incidence is high throughout the province, tree dieback is minimal, and trees still producing nuts could support opportunities for ex situ conservation. Because of the rate of pathogen spread in the province, implementing a tree improvement strategy might be the only means for maintaining the butternut genome on the landscape.