Genus Castanea Research Articles

Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control.

Cryphonectria parasitica (Murr.) Barr. is a Sordariomycete (ascomycete) fungus in the family Cryphonectriaceae (Order Diaporthales). Closely related species that can also be found on chestnut include Cryphonectria radicalis, Cryphonectria naterciae and Cryphonectria japonica. Major hosts are species in the genus Castanea (Family Fagaceae), particularly the American chestnut (C. dentata), the European chestnut (C. sativa), the Chinese chestnut (C. mollissima) and the Japanese chestnut (C. crenata). Minor incidental hosts include oaks (Quercus spp.), maples (Acer spp.), European hornbeam (Carpinus betulus) and American chinkapin (Castanea pumila). Cryphonectria parasitica causes perennial necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees, eventually leading to wilting of the plant part distal to the infection. Chestnut blight cankers are characterized by the presence of mycelial fans and fruiting bodies of the pathogen. Below the canker the tree may react by producing epicormic shoots. Non-lethal, superficial or callusing cankers on susceptible host trees are usually associated with mycovirus-induced hypovirulence. After the introduction of C. parasitica into a new area, eradication efforts by cutting and burning the infected plants/trees have mostly failed. In Europe, the mycovirus Cryphonectria hypovirus 1 (CHV-1) acts as a successful biological control agent of chestnut blight by causing so-called hypovirulence. CHV-1 infects C. parasitica and reduces its parasitic growth and sporulation capacity. Individual cankers can be therapeutically treated with hypovirus-infected C. parasitica strains. The hypovirus may subsequently spread to untreated cankers and become established in the C. parasitica population. Hypovirulence is present in many chestnut-growing regions of Europe, either resulting naturally or after biological control treatments. In North America, disease management of chestnut blight is mainly focused on breeding with the goal to backcross the Chinese chestnut's blight resistance into the American chestnut genome.

A New Species of Dryocosmus Giraud (Hymenoptera: Cynipidae: Cynipini) in Japan and Korea—First Record of Eastern Palearctic Dryocosmus Species Showing Alternation of Generations on Section Cerris Oaks

Dryocosmus kunugiphagus Ide and Abe, n. sp. associated with a section Cerris oak, Quercus acutissima Carruth., is described in Japan and Korea. Although section Cerris oaks are the main host plants of Western Palearctic species of Dryocosmus Giraud, hitherto known Eastern Palearctic ones exhibit host specificity with plants of the genus Castanea Mill. and the subgenus Cyclobalanopsis (Oerst.) of the genus Quercus L. Therefore, the present study served as the first record of a Dryocosmus species being associated with section Cerris oaks in the Eastern Palearctic region. The species was previously identified as Spathegaster sp., Andricus inflator Hartig, and Neuroterus boni-henrici Dettmer in Japan; however, examination of adults that emerged from galls resulted in the description of a new Dryocosmus species. Collection and emergence dates indicated the occurrence of a prolonged diapause in the asexual generation of D. kunugiphagus.

Fingerprinting of Fagaceae individuals using intermicrosatellite markers.

The genera Castanea (chestnuts), Fagus (beechs) and Quercus (oaks) are the only genera of the family Fagaceae distributed throughout the temperate forests of the northern hemisphere (Kremer et al. 2007). Comprising more than 900 species and nine genera (Manos et al. 2001), this family is considered highly complex at the phylogenetic level. However, its taxonomic classification is presently being widely revised due to the availability of new molecular data. Inter-simple sequence repeats (ISSRs) have been used in different Fagaceae species for genetic diversity analyses, DNA fingerprinting and estimation of interspecific and intraspecific relationships (Goulao et al. 2001; Carvalho et al. 2009; Coutinho et al. 2014). ISSRs could be used alone or in combination with other markers such as the simple sequence repeats (SSRs) (Lopez-Aljorna et al. 2007). Recent technological progresses made in single-locus markers reduced the importance of multi-locus markers. SSRs are the marker system of choice for genetic diversity evaluation in species with reduced polymorphism, otherwise, both SSRs and ISSRs could be used for the same (Lopez-Aljorna et al. 2007; Gomes et al. 2008; Kumar and Sharma 2011; Jia et al. 2011; Noormohammadi et al. 2013). Many expressed sequence tags (EST) were developed for Fagaceae species such as Quercus petraea, Q. robur, C. sativa and F. sylvatica (NCBI dbEST, http://www.ncbi.nlm.nih.gov/dbest/index. html). The ESTs are useful for comparative mapping studies but their transferability is dependent on the occurrence of point mutations in the primer annealing sites, and could lead to some uncertainty in phylogenetic inferences. On the other hand, ISSRs are ubiquitous, simple, affordable and produce a large number of polymorphic fragments (increasing their resolving power and polymorphic information content), thus being suitable for genetic diversity assessment of distant and closely related species and for the estimation of phylogenies.

Taxonomic status and genetic differentiation of Hyrcanian Castanea based on noncoding chloroplast DNA sequences data

The taxonomy and phylogenetic relationships among Castanea species were investigated using sequence data from the chloroplast trnL-F and trnH-psbA intergenic spacer regions. Samples included Castanea specimens of uncertain taxonomic affinity that were collected in the Hyrcanian forest of northern Iran. The trnL-F data were more informative than trnH-psbA, having seven parsimony-informative sites. A low level of haplotype diversity was detected within Hyrcanian samples and the whole species of the genus Castanea. In the trnL-F dataset, Castanea sativa and Castanea mollissima have unique character states that differentiate them from other species of Castanea. The genus Castanea was recovered as a monophyletic with high to moderate support when inferred from combined trnH-psbA and trnL-F spacer data. Two main lineages received minimal support in the trnL-F analysis, whereas trnH-psbA could not distinguish different species of Castanea from each other. Finally, low levels of haplotype diversity was found within small remnant stands of Castanea in the Hyrcanian forest, indicating that genetic erosion may increase the extinction risk for these valuable trees.

Microsatellite markers reveal a strong geographical structure in European populations of Castanea sativa (Fagaceae): Evidence for multiple glacial refugia

Large-scale studies on the genetic diversity of forest trees are relevant for the inventory, conservation, and management of genetic resources and provide an insight into the geographical origins of the species. This approach is appropriate to use with Castanea sativa, a tree of great economic importance and the only species from the genus Castanea in Europe. The history of C. sativa was deduced from fossil pollen data, but the large-scale genetic structure of this species needs to be elucidated. We evaluated the genetic diversity of C. sativa to define previously unclarified genetic relationships among the populations from Turkey and those from Greece and western Europe. The influence of natural events such as glaciations and human impact in terms of species distribution are discussed. • Wild chestnut trees (779) were sampled in 31 European sites. Six polymorphic microsatellites were used for the analysis. A set of measures of intra- and interpopulation genetic statistics were calculated. The population structure was inferred by using a Bayesian approach. • The population structure showed a genetic divergence between the eastern (Greek and Turkish) and western (Italian and Spanish) populations. Two gene pools and a zone of gene introgression in Turkey were revealed. • The inferred population structure shows a strong geographical correspondence with the hypothesized glacial refugia and rules out the migration of the chestnut from Turkey and Greece to Italy. The homogeneous gene pool observed in Italy and Spain could have been originated from common refugia along with human-mediated colonization.

Description of Two New Species of Dryocosmus (Hymenoptera: Cynipidae: Cynipini) Inducing Galls on the Strictly Asian Subgenus Cyclobalanopsis of the Genus Quercus, with a Key to Species of Dryocosmus in East Asia

ABSTBACT Two new species of Dryocosmus (Hymenoptera: Cynipidae: Cynipini), D. sefuriensis Ide, Wachi et Abe, sp. nov. and D. sakureiensis Ide, Wachi et Abe, sp. nov., are described from Japan. A key to species of Dryocosmus in East Asia is given. Although the members of Dryocosmus have been known to induce galls on the subgenus Quercus of the genus Quercus and the genera Castanea, Castanopsis, Chrysolepis, and Lithocarpus of Fagaceae, this is the first record of this gall wasp genus from the strictly Asian subgenus Cyclobalanopsis of the genus Quercus. The discovery of two new species of Dryocosmus associated with Q. (C.) acuta suggests the exceptionally diverse host range of Dryocosmus and the species richness of Cynipini on this plant subgenus in Asia.

Chestnut Breeding in the United States for Disease and Insect Resistance.

The genus Castanea (family Fagaceae) is found in north temperate climates around the world, and is highly prized in many different cultures for its nutritious nuts and valuable timber. Selection for larger, better-tasting nuts has been ongoing in Asia and Europe for centuries. Early trade routes moved European chestnut trees (C. sativa) west of their native range (in the Caucasus mountains), and the Romans then moved them across their empire to provide support posts for grapevines, as well as for the nuts. Cultivar selection in Turkey, Italy, Spain, and Portugal has been extensive, and regional favorites developed. The many uses of the wood of American chestnut made this "all purpose" tree extremely valuable in its native range in North America. Nut production was important as a food source for rural families and many species of birds and animals. The other American species in the genus Castanea are classed as chinquapins, and may be divided into several or lumped as a single species. The small nuts from these trees and bushes serve primarily as mast for wildlife. Two serious diseases of chestnut trees changed the direction of chestnut research in the United States. Ink disease, caused by the root pathogen Phytophthora cinnamomi, was discovered to be the cause of widespread death of chestnuts and chinquapins in the southern United States, which had been observed since about 1850. This imported pathogen probably came into the southern United States before 1824. The second chestnut disaster was the introduction of chestnut blight disease, which was first found in the United States in 1904. The pathogen causing the lethal cankers is an Ascomycete now known as Cryphonectria parasitica. The longest continuing chestnut breeding program in the United States is in Connecticut.

Genomics of Fagaceae

An overview of recent achievements and development of genomic resources in the Fagaceae is provided, with major emphasis on the genera Castanea and Quercus. The Fagaceae is a large plant family comprising more than 900 species belonging to 8–10 genera. Using a wide range of molecular markers, population genetics and gene diversity surveys were the focus of many studies during the past 20 years. This work set the stage for investigations in genomics beginning in the early 1990s and facilitated the application of genetic and quantitative trait loci mapping approaches. Transferability of markers across species and comparative mapping have indicated tight macrosynteny between Quercus and Castanea. Omic technologies were more recently developed and the corresponding resources are accessible via electronic and physical repositories (expressed sequence tag sequences, single-nucleotide polymorphisms, candidate genes, cDNA clones, bacterial artificial chromosome (BAC) libraries) that have been installed in North America and Europe. BAC libraries and physical maps were also constructed in Castanea and Quercus and provide the necessary resources for full nuclear genome sequencing projects that are currently under way in Castanea mollissima (Chinese chestnut) and Quercus robur (pedunculate oak).

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with approximately 1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 A, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)(8) barrel fold and each pair swapping the 8th alpha-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH.ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.

PHYLOGENY OF GENUS CASTANEA MILLER AND IDENTIFICATION OF C. LATIFOLIA (SORD.) IN 45,000-YEAR-OLD SAMPLES FROM VENICE LAGOON

The phylogeny of the genus Castanea Miller and its biogeographic history are presented. The sequence of the trnL-trnF chloroplast DNA region was determined for 4 species of the genus Castanea and for fossil samples including C. latifolia Sord., a species considered extinct based on palynological evidence. The fossil material was collected from a site of North-East Italy dating back to a period between the final phases of the last Interglacial and the beginning of the last Wurmian glaciation (45,000-year-old). The DNA sequences amplified from the fossil sample indicate that they belong to the genus Castanea but not to the extant Castanea species. The optimal solution of phylogeny and dispersal-vicariance analyses indicated that the ancestral area of speciation of Castanea was the Middle East. The lineage first diverged for dispersion into two sublineages, one with an ancestral area in the Middle East and the second with an ancestral area in Eastearn Asia, which later became fragmented by a vicariant event separating the existent species present in the Middle-East, Europe and Eastern Asia. At least a dispersal event from the sublineage Eastearn was directed towards North America.

Development of Simple Sequence Repeat Markers in Chinese Chestnut and Their Characterization in Diverse Chestnut Cultivars

To develop and characterize valid simple sequence repeat (SSR) markers in chestnut (Castanea spp.), we used a selective hybridization method to perform SSR sequence enrichment in the genomic library of chinese chestnut (Castanea mollissima). From 47 sequences of the enriched library, we designed 24 primer pairs for SSR polymerase chain reaction (PCR). SSR PCR was performed for 23 chinese chestnut cultivars. Among the 24 primers, 22 primers amplified their respective SSR loci; 21 primer pairs yielded polymorphic fragments across the cultivars. Among these 21 primer pairs, 17 primer pairs amplified single SSR loci with polymorphisms. Multilocus amplification patterns were observed in the other four primers. For the corresponding 17 SSR loci, the number of alleles per locus was two to 13, and the average number of alleles was 7.19. The observed heterozygosity (number of genotypes of the heterozygote/total number of genotypes scored at that locus) ranged from 0.00 to 0.87 (average = 0.68), and the expected heterozygosity ranged from 0.00 to 0.87 (average = 0.68). One of the SSR loci—ICMA014—was a unique locus, because the primer pair for this locus did not amplify any fragment in any of the cultivars, except in Qianchi, which was used to construct the SSR-enriched genomic library. Cross-species amplifications using the 17 primer pairs were also successful in the other species of genus Castanea. A dendrogram depicting the relationships among the genotypes on the basis of genetic distances was generated using the unweighted pair group method with arithmetic mean cluster analysis. The genotypes were clearly separated into three large groups, which reflected the three cultivated species, namely, C. mollissima, C. crenata, and C. sativa.

AN EVALUATION OF SSR MARKERS ISOLATED FROM CORYLUS AVELLANA FOR GENETIC ANALYSIS WITHIN FOUR ASIAN SPECIES OF THE GENUS CASTANEA

The potential of SSR markers isolated from European hazelnut, Corylus avellana, for the genetic study of chestnut was evaluated. High conservation of primer binding sites was confirmed in the genus Castanea of the Fagaceae. Seventy percent of 33 SSR primer pairs successfully amplified good PCR products from 30 genotypes of four species of the genus Castanea naturally distributed in Asia. Sequencing results for PCR products showed that fragment length variation was mainly caused by variable repeat number in microsatellites. Allele segregation at SSR loci was observed with a mapping population of chestnut. Forty percent of loci tested in one of two parents were heterozygous. Our study indicated that hazelnut SSR markers were a useful tool for genetic analysis of population and construction of genetic map in the genus Castanea.

COMPARATIVE PHYLOGEOGRAPHY OF CASTANEA SPECIES

The genus Castanea consists of seven species that are disjunctly distributed in Asia, Europe and North America. Phylogenetic analysis of Castanea using DNA sequence data from six variable regions of the chloroplast genome indicated migration of extant Castanea species from Asia westward to Europe and North America. Cycles of Pleistocene glaciation and environmental events like the spread of chestnut blight were major determinants of historical migration and current genetic diversity in North American Castanea species. Chloroplast and nuclear sequence information revealed two lineages within American chestnut (C. dentata), one which is closely related to Allegheny chinkapin (C. pumila var. pumila) haplotypes from the southern Appalachian region, indicative of hybridization in refuge zones. The other American chestnut lineage expanded in northerly direction following the Last Glacial Maximum, and was accompanied by a gradual loss of diversity. Ozark chinkapins (C. pumila var. ozarkensis) show high sequence homology with the most frequently occurring Allegheny chinkapin haplotype from Virginia and North Carolina. Ancestral Allegheny chinkapin haplotypes can be detected in populations along the Gulf Coast, while haplotypes in Virginia share regions of the chloroplast genome with Ozark and ancestral haplotypes, suggesting multiple colonization routes and suture zones of chloroplast lineages in the Appalachian Mountains. This knowledge will be useful for development of conservation management strategies and successful re-introduction of blight resistant American chestnut into the Appalachian mountain ecosystem.

Molecular evidence for an Asian origin and a unique westward migration of species in the genus Castanea via Europe to North America

The genus Castanea (Fagaceae) is widely distributed in the deciduous forests of the Northern Hemisphere. The striking similarity between the floras of eastern Asia and those of eastern North America and the difference in chestnut blight resistance among species has been of interest to botanists for a century. To infer the biogeographical history of the genus, the phylogeny of Castanea was estimated using DNA sequence data from different regions of the chloroplast genome. Sequencing results support the genus Castanea as a monophyletic group with Castanea crenata as basal. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene followed by intercontinental dispersion and divergence between the Chinese and the European/North American species during the middle Eocene and a split between the European and the North American species in the late Eocene. The differentiation within North America and China might have occurred in early or late Miocene. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and Castanea dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.

Molecular Evidence for an Asian Origin and a Unique Westward Migration of Species in the Genus Castanea via Europe to North America

The genus Castanea (Fagaceae), which contains three sections and seven species, is widely distributed in the deciduous forests of the Northern Hemisphere. The phylogeny of Castanea was estimated using DNA sequence data from five different regions of the chloroplast genome. Sequencing results support the genus Castanea as a paraphyletic group with C. crenata, the Japanese chestnut, representing an early divergence in the genus. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene, followed by intercontinental dispersion and divergence between the Chinese and European/North American species during the Oligocene and a split between the European and North American species in the early Miocene. The differentiation within North America and China might have occurred in late Miocene or early Pliocence. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and C. dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.

The Genetic Resources of European Sweet Chestnut (Castanea sativa Miller) in Andalusia, Spain

Sweet chestnut (Castanea sativa Miller) is the only native species of the genus Castanea in Europe. A collecting mission has been carried out in Andalusia (South of Spain) with the purpose to evaluate of the genetic variability of this species in this region. During the field trips, two different types of chestnut trees have been detected: one of clonal nature (varieties obtained by graft) and other of sexual nature (trees derived from nut). For the former, 156 trees were located in plantations in the Genal river Valley (Malaga) and the Natural Park of `Sierra de Aracena y Picos de Aroche' (Huelva), where 43 varietal denominations were recorded. Thirty out of them had never been mentioned. For the latter, 29 stands were located in Almeria, Cordoba, Granada, Jaen, and Malaga. This work reveal that this species present a high degree of genetic variability. Nevertheless, their preservation is seriously threatened in the most zones due to the varietal substitution, the low profit or the abandonment, which determinate the urgent necessity to safeguard these genetic resources.

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.

065 Genetic Diversity of the Allegheny Chinkapin (Castanea pumila var. pumila)

American species in the genus Castanea are susceptible to chestnut blight, caused by the Asian fungus Cryphonectria parasitica. This disease spread throughout the natural range of the American chestnut and reduced the species from a timber and nut producing tree to an understory shrub. The lesser known member of the genus, the chinkapin, has also been affected by this disease and a conservation plan is needed. Genetic diversity within and between geographic populations of the Allegheny chinkapin was evaluated to provide baseline genetic information pertinent to conservation of the species. Nuts of Allegheny chinkapin trees from populations in Mississippi, Florida, Alabama, Virginia, and Ohio were collected and evaluated for isozyme and RAPD marker polymorphism. The genetic diversity of these populations will be compared with that of Ozark chinkapin and American chestnut populations. Conservation strategies will be discussed.

Genetic Diversity of Castanea pulia var. ozarkensis

The genus Castanea includes several species, some of which, like the American chestnut (C. dentata) and chinkapin (C. pumila), are susceptible to chestnut blight, caused by the Asian fungus Cryphonectria parasitica. Blight spread throughout the natural range of the American chestnut, destroying several billion trees within the past 50 years. Although the plight of the American chestnut is well-known, the chinkapin has been neglected. Taxonomic studies indicated two varieties, the Ozark chinkapin, var. ozarkensis, limited to the Ozark Highlands of Arkansas, Missouri, and Oklahoma, and the Allegheny chinkapin, var. pumila, found from New Jersey to Florida and Texas. The genetic diversity within and between 11 geographic populations of the Ozark chinkapin was evaluated to provide baseline genetic information pertinent to the conservation and restoration of this species. Nuts or dormant buds of chinkapin trees were evaluated for isozyme and RAPD polymorphism. The genetic variability of the Ozark chinkapin populations was relatively high when compared to the American chestnut, and most of the diversity resides within the populations. Conservation considerations for restoration of the Ozark chinkapin will be discussed.