Keith Woeste

Population structure of Geosmithia morbida , the causal agent of thousand cankers disease of walnut trees in the United States

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.

Susceptibility of Walnut and Hickory Species to Geosmithia morbida

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Long-term human impacts on genetic structure of Italian walnut inferred by SSR markers

Life history traits, historic factors, and human activities can all shape the genetic diversity of a species. In Italy, walnut (Juglans regia L.) has a long history of cultivation both for wood and edible nuts. To better understand the genetic variability of current Italian walnut resources, we analyzed the relationships among the genetic structure of local walnut populations (inferred by SSR markers) and human migrations along ancient routes, using the territory of Royal Tratturo Candela-Pescasseroli (RT) as a case study. Sixteen J. regia provenances were collected along RT and compared with 13 Italian provenances and the landrace Sorrento. Although the level of SSR polymorphism we observed was moderately high, AMOVA revealed that most of the diversity was located within individuals (92.58%), and geographical differentiation was low (Dest = 0.076). Evidence for human-mediated domestication bottleneck events was detected in about 95% of walnut provenances. A Bayesian approach divided 456 walnut samples into three clusters: (1) Sorrento genotypes, (2) trees from the island of Sicily, and (3) the remaining germplasm. The UPGMA tree based on Neis distances distinguished northeastern provenances and weakly grouped 12 of 16 provenances of RT. The observed genetic differences derived mainly from gradations in allele frequencies. Separation of the Sicilian provenance from the mainland may be explained in terms of founder effects and prolonged geographic isolation. Two contrasting forces, selection, and frequent inter-regional transfer of propagules, appear to drive the patterns of genetic variability for J. regia.

DNA markers identify hybrids between butternut (Juglans cinerea L.) and Japanese walnut (Juglans ailantifolia Carr.)

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.

Genetic diversity of butternut (Juglans cinerea) and implications for conservation

The management of threatened and endangered species can be improved by understanding their patterns of genetic diversity and structure. This paper presents the results of the first analysis of the population genetics of butternut (Juglans cinerea L.) using nuclear microsatellites. Butternut once was an economically and ecologically valuable hardwood, but now its numbers are rapidly decreasing throughout its native range in eastern North America. By genotyping butternut trees (n = 157) from five populations at seven highly polymorphic loci, we determined that the remaining genetic diversity for the species is considerably higher than previously estimated (mean HE = 0.75). Populations were nearly genetically homogenous (FST = 0.025), with no evidence of isolation by distance. These results indicate that butternut retains a large amount of genetic diversity, and that it is not too late to implement strategies to conserve local butternut populations.

Ancient Humans Influenced the Current Spatial Genetic Structure of Common Walnut Populations in Asia

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. It is generally accepted that J. regia survived and grew spontaneously in almost completely isolated stands in its Asian native range after the Last Glacial Maximum. Despite its natural geographic isolation, J. regia evolved over many centuries under the influence of human management and exploitation. We evaluated the hypothesis that the current distribution of natural genetic resources of common walnut in Asia is, at least in part, the product of ancient anthropogenic dispersal, human cultural interactions, and afforestation. Genetic analysis combined with ethno-linguistic and historical data indicated that ancient trade routes such as the Persian Royal Road and Silk Road enabled long-distance dispersal of J. regia from Iran and Trans-Caucasus to Central Asia, and from Western to Eastern China. Ancient commerce also disrupted the local spatial genetic structure of autochthonous walnut populations between Tashkent and Samarkand (Central-Eastern Uzbekistan), where the northern and central routes of the Northern Silk Road converged. A significant association between ancient language phyla and the genetic structure of walnut populations is reported even after adjustment for geographic distances that could have affected both walnut gene flow and human commerce over the centuries. Beyond the economic importance of common walnut, our study delineates an alternative approach for understanding how the genetic resources of long-lived perennial tree species may be affected by the interaction of geography and human history.

Rethinking the history of common walnut (Juglans regia L.) in Europe: Its origins and human interactions

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its high-quality wood and nuts. It is generally accepted that after the last glaciation J. regia survived and grew in almost completely isolated stands in Asia, and that ancient humans dispersed walnuts across Asia and into new habitats via trade and cultural expansion. The history of walnut in Europe is a matter of debate, however. In this study, we estimated the genetic diversity and structure of 91 Eurasian walnut populations using 14 neutral microsatellites. By integrating fossil pollen, cultural, and historical data with population genetics, and approximate Bayesian analysis, we reconstructed the demographic history of walnut and its routes of dispersal across Europe. The genetic data confirmed the presence of walnut in glacial refugia in the Balkans and western Europe. We conclude that human-mediated admixture between Anatolian and Balkan walnut germplasm started in the Early Bronze Age, and between western Europe and the Balkans in eastern Europe during the Roman Empire. A population size expansion and subsequent decline in northeastern and western Europe was detected in the last five centuries. The actual distribution of walnut in Europe resulted from the combined effects of expansion/contraction from multiple refugia after the Last Glacial Maximum and its human exploitation over the last 5,000 years.

Evaluation of four herbicides and tillage for weed control on third year growth of tree seedlings

Abstract Azafenidin (AZ), pendimethalin, simazine, and sulfometuron (SF) were applied alone and in combination to black cherry, black walnut, eastern white pine, flowering dogwood, northern bayberry, northern red oak, Siberian crabapple, white ash, white oak, and yellow poplar seedlings grown for 2 yr in the field. There were significant differences in diameter, height, and stem volume among the treatments for every species after the third growing season. Tillage significantly increased tree growth over controls for most species. No single herbicide treatment ranked best for all tree species but comparison of the mean ranks of the treatments for all species indicated that AZ resulted in the best growth. Post–bud-break applications of SF and AZ were no better than untreated controls for most species. Herbicide treatments did not affect tree survival over years but exhibited potential for better growth in establishment years. Nomenclature: Azafenidin; pendimethalin; simazine; sulfometuron; black cherry, Prunus serotina Ehrh.; black walnut, Juglans nigra L.; eastern white pine, Pinus strobus L.; flowering dogwood, Cornus florida L.; northern bayberry, Myrica pensylvancia L.; northern red oak, Quercus rubra L.; Siberian crabapple, Malus baccata (L.) Borkh; white ash, Fraxinus americana L.; white oak, Quercus alba L.; yellow poplar, Liriodendon tulipifera L.

Conservation Status of a Threatened Tree Species: Establishing a Baseline for Restoration of Juglans cinerea L. in the Southern Appalachian Mountains, USA

ABSTRACT: To mitigate the loss of native tree species threatened by non-native pathogens, managers need to better understand the conservation status of remaining populations and the conditions that favor successful regeneration. Populations of Juglans cinerea L. (butternut), a wide-ranging riparian species, have been devastated by butternut canker, a disease caused by a non-native fungal pathogen. We assessed J. cinerea within Great Smoky Mountains National Park (GSMNP) to determine post-disease survivorship and health, recruitment history, environmental conditions associated with survival, and the extent of hybridization with a non-native congener. Monitoring records were used to locate and collect data for 207 J. cinerea trees in 19 watersheds. Tree cores were collected from a subset of individuals to assess recruitment history. We sampled vegetation plots within areas that contained J. cinerea to assess site conditions and overstory species composition of characteristic habitat. We collected leaf samples for genetic analysis to determine the frequency of hybridization. Our reassessment of monitoring records suggests that J. cinerea abundance in GSMNP has declined due to butternut canker and thirty years of poor regeneration. Populations displayed continuous recruitment following Park establishment (1934) until around 1980, after which regeneration declined drastically. Ordination analysis revealed that J. cinerea in the contemporary forest was associated with greater distance from homesites and reduced basal area of competing species. Hybrids comprised a small portion of sampled trees. The presence of healthy trees and low rate of hybridization suggest that these trees may contribute to the development of disease-resistant genotypes for future restoration efforts.

Vegetative and Adaptive Traits Predict Different Outcomes for Restoration Using Hybrids

Hybridization has been implicated as a driver of speciation, extinction, and invasiveness, but can also provide resistant breeding stock following epidemics. However, evaluating the appropriateness of hybrids for use in restoration programs is difficult. Past the F1 generation, the proportion of a progenitor’s genome can vary widely, as can the combinations of parental genomes. Detailed genetic analysis can reveal this information, but cannot expose phenotypic alterations due to heterosis, transgressive traits, or changes in metabolism or development. In addition, because evolution is often driven by extreme individuals, decisions based on phenotypic averages of hybrid classes may have unintended results. We demonstrate a strategy to evaluate hybrids for use in restoration by visualizing hybrid phenotypes across selected groups of traits relative to both progenitor species. Specifically, we used discriminant analysis to differentiate among butternut (Juglans cinerea L.), black walnut (J. nigra L.), and Japanese walnut (J. ailantifolia Carr. var. cordiformis) using vegetative characters and then with functional adaptive traits associated with seedling performance. When projected onto the progenitor trait space, naturally occurring hybrids (J. × bixbyi Rehd.) between butternut and Japanese walnut showed introgression toward Japanese walnut at vegetative characters but exhibited a hybrid swarm at functional traits. Both results indicate that hybrids have morphological and ecological phenotypes that distinguish them from butternut, demonstrating a lack of ecological equivalency that should not be carried into restoration breeding efforts. Despite these discrepancies, some hybrids were projected into the space occupied by butternut seedlings’ 95% confidence ellipse, signifying that some hybrids were similar at the measured traits. Determining how to consistently identify these individuals is imperative for future breeding and species restoration efforts involving hybrids. Discriminant analysis provides a useful technique to visualize past selection mechanisms and current variation in hybrid populations, especially when key ecological traits that distinguish progenitors are unknown. Furthermore, discriminant analysis affords a tool to assess ecological equivalency of hybrid populations and breeding program efforts to select for certain traits and monitor the amount of variability of those traits, relative to progenitors.