SummaryBronze leaf disease (BLD) of species and hybrids of aspens, grey and white poplars (Genus Populus, section Populus) is a systemic disease incited by the fungus Apioplagiostoma populi (E.K. Cash & A.M. Waterman) M.E. Barr that results in a characteristic bronze to dark brown pigmentation of infected leaves in late summer and early fall. Branches on affected trees die over a period of several years, disfiguring trees and eventually leading to mortality of susceptible trees planted in landscapes. In this study, we describe the histopathology of the disease and fungus development relative to external symptoms on affected trees. A. populi hyphae were observed in leaf vessels, lateral veins and xylem vessels of stems and roots of affected trees. Hyphae were also observed in veins and vessels of asymptomatic leaves. Xylem vessels of growth rings on dead branches were completely occluded with A. populi near the vascular cambium and together with the ray parenchyma were stained a bronze colour. Evidence of putative toxin damage to mesophyll cells, not colonized by hyphae, was observed and may contribute to the characteristic tissue staining observed. The development of the fungus was consistent with previous reports, but in this study, we report additional details including perithecial development and ascospore release from predominantly abaxial leaf surfaces. We also report for the first time the isolation and growth of the pathogen in pure culture and systemic infection of roots.

Abstract 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.

Butternut canker, caused by the fungus Ophiognomonia clavigignenti-juglandacearum, primarily kills butternut (Juglans cinerea). Rain splash and local air currents are the primary means of conidia dispersal but that does not explain its long-distance spread and infection of isolated trees. Dispersal by insect or animal vectors or plant material likely necessitates the ability for conidia to tolerate drying for a period of time over variable temperature and humidity conditions. The objective of this study was to determine the influence of temperature and humidity on conidial germination and survival of air-dried conidia. Conidia collected from 1-month-old cultures germinated on water agar over a wide range of temperatures (4 to 32°C) and were viable after brief periods at 36°C when returned to a lower temperature. Viability of air-dried conidia held on nylon membranes at various temperatures and humidities varied from less than a day at 28°C and 90% relative humidity (RH) to a mean of 15 days at 20°C and 80% RH. RH had the least effect on viability at 12°C, with conidia remaining viable for 7 days at most humidity levels tested. Conidia held at 100% RH began germinating on the membranes after 21 days. Conidia in a water suspension remained viable for 168 days at all temperatures tested. These results suggest that O. clavigignenti-juglandacearum conidia may remain viable on the surface of a vector or plant material and seed for over 2 weeks, given the proper conditions, or for much longer if in water or in an environment of saturated humidity. This potential may, in part, explain the frequent presence of the disease on isolated trees.

For most wild species affected by exotic pests or pathogens, the relative importance of heritable genetic differences in determining apparent variation in disease resistance is unknown. This is true in particular for butternut, a North American hardwood affected by butternut canker disease and undergoing demographic contraction. Little is known about site effects on butternut decline, in part because long-term monitoring data are lacking. We collected detailed disease phenotypes and multilocus microsatellite genotypes for all surviving individuals in a large natural population of butternut in 2003 (n=302) and 2012 (n=113). Two analytical methods, correlations between pairwise phenotypic similarity and pairwise relatedness, and estimation of among-family variance, both indicated weak heritability of disease-related traits and no heritability for overall tree health in the population. Additionally, an analysis of spatial data collected in 2001 (n=341) and 2012 (n=113) demonstrated that drier, upland sites contribute to increased likelihood of survival. We conclude that genetic differences among wild butternut individuals contributed little to observed variance in survival over 10 years but fine-scale site differences were useful predictors of butternut mortality.

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.

A rapid and reliable technique is needed for identifying butternut trees (Juglans cinerea) with resistance to butternut canker. We investigated the potential of a bark extract bioassay to detect levels of resistance to Ophiognomonia clavigignenti-juglandacearum (Oc-j), the causal agent of butternut canker. Both reagent grade naphthoquinones and crude bark extracts of Juglans species inhibited germination of Oc-j conidia. A disc diffusion bioassay was used to study the level of inhibition by these bark extracts and results indicated extensive variation within and between butternut and other species of Juglans tested. In many months over a 3 year period, bark from butternut trees selected for apparent disease resistance could be distinguished from that of unselected trees. Inhibition of conidia germination roughly correlated to the level of resistance observed in field inoculations of the trees. Quantification of the naphthoquinone compounds juglone and plumbagin in butternut bark was performed using ultra-high performance liquid chromatography mass spectrometry. While the concentrations of these two compounds varied by month and by individual tree, juglone levels correlated well with the bark extract bioassay in some months. These results suggest that juglone concentration may account in part for the observed range of inhibition observed in the bioassay and variation in canker resistance among selections of butternut field inoculated with Oc-j. The bark extract bioassay described in the following report may have potential use for selecting resistant butternut for conservation and restoration purposes.

Entoleuca mammata (Wahlenb.) J.D. Rogers & Y.M. Ju (syn. Hypoxylon mammaturn (Wahlenb.) P. Karst.) causes the most damaging canker disease of quaking aspen (Populus tremuloides Michx.) Hypoxylon canker in many areas of North America. A study by Anderson (1964) in the Lake States (Michigan, Minnesota and Wisconsin) estimated that Hypoxylon canker killed 1-2% of the aspen volume each year, which is equivalent to 31% of the net annual growth. The estimated yearly volume loss in Ontario (Canada) was 2 million m3 (Pitt et al., 2001). Bigtooth aspen (P. grandidentata Michx.) is occasionally infected but the disease is not nearly as damaging to this species as it is to quaking aspen. Quaking and bigtooh aspens are the most abundant commercially and ecologically important species in the region. Aspens are critical to numerous wildlife species for food and habitat, and contribute substantially to many local and regional economies. It was estimated in 1971 that tree mortality attributed to Hypoxylon canker in the Lake States was equal to a loss of 4.4 million US$ a year at harvest (Ma*, 1972). Since that time, the value of aspen for pulp and paper and solid wood products has significantly increased, with a corresponding increase in its utilization by the forest industry. Hypoxylon canker is widely distributed in the northeastern and Lake States regions of the USA but less common in the western USA (Hinds, 1985) and, for reasons unknown, it is absent from Alaska (Hinds and Laurent, 1978). The disease is widely distributed in Canada (Bier, 1940). In Europe, it has been reported aEfecting European aspen (P. tremula L.) from Finland, Germany and Sweden (Miller, 1961), and from France (Pinon, 1979). Other hosts in Europe indude white poplar (P. alba L.), black cottonwood (l? balsamifea L. subsp. trichocarpa (Tom &A. Gray ex Hook) Brayshaw) and hybrid aspen (l? tremula x l? tremuloides) (Kasanen et al., 2004). The fungus has also been collected in Italy and Switzerland (Kasanen et al., 2004). Various hybrid Populus spp. are occasionally damaged by E. mammafa. Hypoxylon cankers were found on plantation-grown trees of P. nigra L. var. betulifolia x P. nigra 'Volga', P. nigra var. betulifolia x P. balsamifera subsp. trichocarpa, P. maximowiczii A. Henry x (P. xberolinensis C. Koch), P. balsamifera subsp. balsamifera x (P. xberolinensis), and P. deltoides Bartram ex Marsh. x P. nigra 'Incrassata' (Ostry and McNabb, 1986). Experimental evidence

Damage from Sirococcus and Dip Iodic shoot blights of red pine is widespread and periodically severe in the Lake States. An outbreak of shoot blight occurred in red pine sapling plantations across northern Wisconsin, northern Minnesota, and the Upper Peninsula of Michigan in 1993. We established monitoring plots in red pine sapling plantations severely affected by shoot blight in Michigan and Wisconsin to assess the influence of residual overstory red pine and the presence of Sirococcus and Diplodia shoot blights on disease severity and their long-term impact on tree survival and growth. Fourteen years after the initial outbreak, many red pine saplings had recovered from serious damage (> 1/3 of new shoots affected) that had occurred in a single outbreak year followed by lower levels of disease in subsequent years. However, the most severely affected trees sustained higher mortality and reduced growth. Forking or development of crooks of the main stem was common if terminal leaders were killed by shoot blight. Results from this study suggest that the impact of a shoot blight outbreak on red pine saplings largely depends on the presence or absence of an inoculum source in residual overstory trees, the species of pathogen present in the stand, and the initial disease severity.

To better understand the potential long-term effects of biomass harvesting on biodiversity, the polyporoid fungi community was characterized from 120 plots at four aspen-dominated forests in Minnesota. Four deadwood variables (substrate species, substrate type, decay class, and diameter class) were recorded for each polyporoid species occurrence. A total of 2358 polyporoid occurrences, representing 86 species, were recorded on 16 tree species. Eight species (Trichaptum biforme, Bjerkandera adusta, Trametes hirsuta, Phellinus tremulae, Fomes fomentarius, Irpex lacteus, Fomitopsis ochracea and Antrodia serialis) made up 67% of occurrences. Four polyporoid species (Funalia trogii, Pycnoporellus fulgens, Rigidoporus crocatus and Skeletocutis chrysella) are potentially rare and/or threatened in the Lake States. Similarity indices and non-metric multidimensional scaling demonstrated that diameter class was the most important deadwood variable influencing polyporoid species occurrence. Aspen-dominated systems show great potential for biomass production, but these forests also support a species-rich community of polyporoid fungi, including potentially rare species.

Bronze leaf disease (BLD) affects several Populus species in North America but is particularly damaging to hybrids in section Populus (4). BLD, caused by the fungus Apioplagiostoma populi (syn. Plagiostoma populi) described by Cash and Waterman (1), takes its name from the characteristic dark purple to brown pigmentation of infected leaves. A. populi has not been cultured on artificial media either from diseased tissues or cast ascospores. An anamorph has not been conclusively identified but spores from blister-like acervuli on symptomatic leaves have been suggested to function as spermatia (3). In an attempt to describe the imperfect stage of A. populi, collections of diseased leaves of P. alba × sieboldii and P. alba × grandidentata'Crandon' growing in a plantation near Rosemount, MN, were made on September 14, 2011 (leaves attached to shoots, rolled inward), October 12, 2011 (leaves attached or on the ground, tightly rolled inward), and November 1, 2011 (most leaves on the ground), and examined in the laboratory for fungal development. Leaf laminae from the September 14 collection were uniformly covered with erumpent, subcuticular blister-like acervuli on the adaxial surface only, containing unicellular, colorless spores ranging in size from 2.2 to 10.0 × 2.0 to 5.0 μm (mean 3.6 × 6.2 μm) (n = 100). Attempts to germinate and obtain cultures from these spores on common artificial media were unsuccessful. On leaves collected October 12, the blister-like acervuli were predominantly empty or releasing spores and immature perithecia of A. populi were present. Leaves collected on November 1 contained immature perithecia but were heavily colonized by surface saprophytic fungi and no intact blister-like acervuli were present. DNA was extracted from bulked samples of spores removed from the blister-like acervuli in leaves from multiple trees collected on September 14 using the DNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD). The internal transcribed spacer (ITS2) region of rDNA was PCR amplified and sequenced with primers ITS3 and ITS4. BLASTn searches revealed that sequences from three independent bulked samples were homologous (99% identity, 353 of 355 nucleotides) to A. populi from isolated perithecia (GenBank Accession No. GU205341) (2). The inability of the spores to germinate, the timing of their development and release, and the tight, inward roll of infected leaves facilitating their spread across the upper leaf surface suggest that these spores function as spermatia in the life cycle of A. populi and the blister-like acervuli in which they develop are spermogonia. References: (1) E. K. Cash and A. M. Waterman. Mycologia 49:756, 1957. (2) L. M. Kawchuk et al. Plant Disease 94:377, 2010. (3) W. A. Sinclair and H. H. Lyon, Comstock Publishing Associates, Cornell University Press, Ithaca, NY, 2005. (4) J. A. Smith et al. Plant Disease 86:462, 2002.

Increasing the diversity of species and structure of red pine (Pious resinosa) is often a management goal in stands simplified by practices such as fire suppression and plantation management in many areas of the Great Lakes Region. One approach to diversification is to convert predominantly even-aged, pure red pine stands to multi-cohort, mixed-species forests through variable overstory retention at harvest. Based on limited empirical evidence, pathologists have advised against this multi-cohort approach in stands where pathogens causing damaging shoot blight diseases are established. We examined disease incidence among planted red, jack (Pious banksiana), and white pine (Pious strobus) in a variable retention harvest and understory woody vegetation removal (brushing) experiment in northern Minnesota. The experiment included four overstory treatments (dispersed and two aggregated overstory retention treatments and a control, N = 4) that were split by an understory brushing treatment (yes or no). Prior to harvest in 2003, the fungal pine pathogens Diplodia pinea, Sirococcus conigenus and Armillaria solidipes (syn. Armillaria ostoyae) were common on the study site. Within 6 years after harvest, these pathogens reduced the survival of planted red, white and jack pine, potentially interfering with long-term management objectives. Across all treatments, shoot blight incidence was generally higher in dead red and jack pine than white pine seedlings and was predominantly caused by D. pinea. The disease killing white pine seedlings was predominately Armillaria root rot. Overstory treatment affected the percentage of jack and white pine seedling mortality attributable to shoot blight, but not the more susceptible red pine, with greater overstory retention resulting in greater disease incidence. Understory brushing had no effect on the incidence of shoot blight on seedlings. We expect disease to continue to influence stand structure and composition across all treatments. Our study results highlight the need for forest managers to assess long-term risk of potentially damaging pathogens in red pine stands prior to harvest and use that information to guide decisions regarding silvicultural practices to increase age and species diversity. Published by Elsevier B.V.

Crown dieback and mortality of black ash (Fraxinus nigra) has been noted across the range of the species in North America for several decades. Causes of dieback and mortality are not definitive, but may be related to spring drought or excessive moisture. Where black ash is the dominant tree species in the forest, continued dieback and mortality may result in open, non-forest systems in the future. There is only limited research that has examined tree regeneration in black ash-dominated forests and none in stands experiencing dieback and mortality. Such studies are needed to better understand likely successional dynamics of impacted stands. Our objective was to quantify tree regeneration of black ash and other species in stands with and without crown dieback and mortality, to elucidate potential successional trajectories with decline and loss of black ash. We assessed 54 stands in Minnesota exhibiting a range of black ash crown dieback and mortality. Dieback and mortality in the black ash sapling layer were positively correlated with the same condition in the overstory, suggesting that saplings may not replace dead overstory trees. The next most abundant sapling-sized species was speckled alder, a non-canopy species, indicating that replacement tree species for black ash are not currently abundant. Black ash seedling densities were generally low, suggesting that a pool of advance regeneration is not available to replace black ash. Other canopy potential species were limited in the seedling layer, while abundance of shrubs was generally high. Our results suggest a high potential for loss of a tree layer in these stands and conversion to open, shrub dominated systems. Published by Elsevier B.V.

Macrofungi are distinguished from other fungi by their spore-bearing fruit bodies (mushrooms, conks, brackets). These fungi are critical in forests, causing disease, and wood and litter decay, recycling nutrients, and forming symbiotic relationships with trees. This guide is intended to assist in identifying macrofungi and provide a description of the ecological functions of some of the most frequently encountered macrofungi in aspen-birch, northern hardwood, lowland conifer, and upland conifer forests in the Midwest and Northeast.

Many, but not all, forest pathologists use "decline" to describe forest tree diseases of complex etiology. We contend that this distinction from abiotic or biotic diseases is completely arbitrary, has caused undue confusion, and provides no practical insights for forest managers. All diseases are complex and can be characterized within the conceptual framework of the disease triangle. Why do we use a simple label ("decline") to describe disease situations of complex abiotic and biotic origin when we need to know which damaging agents are present, whether the environment is conducive for disease progression, and host susceptibility over time to understand the origins and management of disease? We propose that forest pathologists discontinue the use of "decline" as a distinct category of disease. Furthermore, we suggest that new diseases should be named based on the affected host, characteristic symptom, and, once known, major determinant. We believe that clearer communication in describing complex diseases is a prerequisite to finding effective management options.

Butternut (Juglans cinerea) is being killed throughout its native range by the fungus Sirococcus clavigignenti-juglandacearum (Scj). In recent years, many disease-free trees have been determined to be complex hybrids with an admixture of Japanese walnut (J. ailantifolia). We challenged 5-year-old trees from two progeny tests with Scj in 2008. Th e fi rst test (northern Indiana), planted in 2003, had 37 diverse families (n=319). Th irty-two of these seedling families were derived from a grafted orchard. Five additional families were collected from hybrid trees. Th e second test (southern Indiana), planted in 2004, had 12 pure butternut half-sib families collected from a woodlot with: 4 resistant, 4 moderately resistant, 4 susceptible, and 1 resistant hybrid families (n=213). Resistance ratings were based on the disease status of the mother trees when the seed was harvested in the fall of 2002. Eleven black walnut (J. nigra) trees were also included. In early fall of 2008, trees were inoculated with two strains of Scj obtained from branch cankers on trees in two locations in Indiana. Th e trees were scored 8 months after inoculation for canker incidence and severity. Some trees in the fi rst test were naturally infected by Scj and resulting canker incidence and severity were recorded. Butternut hybrid families were more resistant to natural infection than the pure butternut families. Eight months after inoculation, canker incidence and severity varied signifi cantly among butternut hybrid families and Scj strain but not among pure butternut families.