Pityophthorus Juglandis Research Articles

Colonization of Artificially Stressed Black Walnut Trees by Ambrosia Beetle, Bark Beetle, and Other Weevil Species (Coleoptera: Curculionidae) in Indiana and Missouri.

Thousand cankers disease (TCD) is a new disease of black walnut (Juglans nigra L.) in the eastern United States. The disease is caused by the interaction of the aggressive bark beetle Pityophthorus juglandis Blackman and the canker-forming fungus, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley & Tisserat, carried by the beetle. Other insects also colonize TCD-symptomatic trees and may also carry pathogens. A trap tree survey was conducted in Indiana and Missouri to characterize the assemblage of ambrosia beetles, bark beetles, and other weevils attracted to the main stems and crowns of stressed black walnut. More than 100 trees were girdled and treated with glyphosate (Riverdale Razor Pro, Burr Ridge, Illinois) at 27 locations. Nearly 17,000 insects were collected from logs harvested from girdled walnut trees. These insects represented 15 ambrosia beetle, four bark beetle, and seven other weevil species. The most abundant species included Xyleborinus saxeseni Ratzburg, Xylosandrus crassiusculus Motschulsky, Xylosandrus germanus Blandford, Xyleborus affinis Eichhoff, and Stenomimus pallidus Boheman. These species differed in their association with the stems or crowns of stressed trees. Multiple species of insects were collected from individual trees and likely colonized tissues near each other. At least three of the abundant species found (S. pallidus, X. crassiusculus, and X. germanus) are known to carry propagules of canker-causing fungi of black walnut. In summary, a large number of ambrosia beetles, bark beetles, and other weevils are attracted to stressed walnut trees in Indiana and Missouri. Several of these species have the potential to introduce walnut canker pathogens during colonization.

Wingnut (Juglandaceae) as a new generic host forPityophthorus juglandis(Coleoptera: Curculionidae) and the thousand cankers disease pathogen,Geosmithia morbida(Ascomycota: Hypocreales)

AbstractThe walnut twig beetle (WTB),Pityophthorus juglandisBlackman (Coleoptera: Curculionidae), vectors a fungus,Geosmithia morbidaKolařík, Freeland, Utley, and Tisserat (Ascomycota: Hypocreales), which colonises and kills the phloem of walnut and butternut trees,JuglansLinnaeus (Juglandaceae). Over the past two decades, this condition, known as thousand cankers disease (TCD), has led to the widespread mortality ofJuglansspecies in the United States of America. Recently the beetle and pathogen were discovered on severalJuglansspecies in northern Italy. Little is known about the extra-generic extent of host acceptability and suitability for the WTB. We report the occurrence of both the WTB andG. morbidain three species of wingnut,Pterocarya fraxinifoliaSpach,Pterocarya rhoifoliaSiebold and Zuccarini, andPterocarya stenopterade Candolle (Juglandaceae) growing in the United States Department of Agriculture-Agricultural Research Service, National Clonal Germplasm Repository collection in northern California (NCGR) and in the Los Angeles County Arboretum and Botanic Garden in southern California, United States of America. In two instances (once inP. stenopteraand once inP. fraxinifolia) teneral (i.e., brood) adult WTB emerged and were collected more than four months after infested branch sections had been collected in the field. Koch’s postulates were satisfied with an isolate ofG. morbidafromP. stenoptera, confirming this fungus as the causal agent of TCD in this host. A survey of the 37PterocaryaKunth accessions at the NCGR revealed that 46% of the trees had WTB attacks and/or symptoms ofG. morbidainfection. The occurrence of other subcortical Coleoptera associated withPterocaryaand the first occurrence of the polyphagous shot hole borer, a species nearEuwallacea fornicatusEichhoff (Coleoptera: Curculionidae), inJuglansare also documented.

A New Species of Theocolax Westwood (Hymenoptera: Pteromalidae: Cerocephalinae) Reared from Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae)

The new species Theocolax americanus McEwen, n. sp. (Hymenoptera, Pteromalidae, Cerocephalinae), is described from the United States of America. A description is provided with a diagnosis and comparison to similar species. A key to differentiate the Theocolax species recorded in the USA is included and notes given on new morphological features. Theocolax americanus is thought to attack the walnut twig beetle (Pityophthorus juglandis Blackman), which transmits thousand cankers disease. Sequencing of CO1 for the new species, other Theocolax species found in the U.S.A., Neocalosoter pityphthori Ashmead (Hymenoptera, Pteromalidae, Cerocephalinae), and P. juglandis is reported.

Phylogeography of the walnut twig beetle, Pityophthorus juglandis, the vector of thousand cankers disease in North American walnut trees.

Thousand cankers disease (TCD) of walnut trees (Juglans spp.) results from aggressive feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, accompanied by inoculation of its galleries with a pathogenic fungus, Geosmithia morbida. In 1960, WTB was only known from four U.S. counties (in Arizona, California, and New Mexico), but the species has now (2014) invaded over 115 counties, representing much of the western USA, and at least six states in the eastern USA. The eastern expansion places TCD in direct proximity to highly valuable (> $500 billion) native timber stands of eastern black walnut, Juglans nigra. Using mitochondrial DNA sequences, from nearly 1100 individuals, we examined variation among 77 samples of WTB populations across its extended range in the USA, revealing high levels of polymorphism and evidence of two divergent lineages. The highest level of genetic diversity for the different lineages was found in the neighboring Madrean Sky Island and Western New Mexico regions, respectively. Despite their proximity, there was little evidence of mixing between these regions, with only a single migrant detected among 179 beetles tested. Indeed, geographic overlap of the two lineages was only common in parts of Colorado and Utah. Just two haplotypes, from the same lineage, predominated over the vast majority of the recently expanded range. Tests for Wolbachia proved negative suggesting it plays no role in "driving" the spread of particular haplotypes, or in maintaining deep levels of intraspecific divergence in WTB. Genotyping of ribosomal RNA corroborated the mitochondrial lineages, but also revealed evidence of hybridization between them. Hybridization was particularly prevalent in the sympatric areas, also apparent in all invaded areas, but absent from the most haplotype-rich area of each mitochondrial lineage. Hypotheses about the specific status of WTB, its recent expansion, and potential evolutionary origins of TCD are discussed.

Geosmithia morbida Found on Weevil Species Stenomimus pallidus in Indiana

The canker pathogen Geosmithia morbida is known to be transmitted to Juglans species by the bark beetle Pityophthorus juglandis, and to lead to development of thousand cankers disease. In an Indiana-wide trap-tree survey of ambrosia and bark beetles and weevils colonizing stressed Juglans nigra, G. morbida was detected on three Stenomimus pallidus weevils emerged from two trees on one site. This is the first report of the pathogen in Indiana and the first report of the fungus from an insect species other than P. juglandis. Accepted for publication 22 October 2014. Published 12 January 2015.

Development of microsatellite loci in Pityophthorus juglandis, a vector of thousand cankers disease in Juglans spp.

Using next-generation sequencing, 18 microsatellite loci were developed and characterized for walnut twig beetle, Pityophthorus juglandis, a vector of thousand cankers disease (TCD) affecting Juglans spp. Although all Juglans species are susceptible to TCD infection, native populations of J. nigra and J. cinerea, which is endangered in Canada, are most susceptible and threatened by habitat loss. Novel primers amplified di-, tri-, and tetra nucleotide repeats and detected 4–14 alleles per locus. Averaged observed and expected heterozygosity was 0.22 and 0.67, respectively. Our results indicate that P. juglandis microsatellite loci can be used to investigate genetic diversity and population structure of this vector across a widespread geography. These markers will be useful tools for evaluating genetic structure of P. juglandis population outbreaks and developing appropriate conservation strategies. Microsatellite loci obtained in this study can also be utilized to determine relationships of P. juglandis to other closely related Pityophthorus spp.

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.

Status of thousand cankers disease on eastern black walnut in the easternUnitedStates at two locations over 3 years

SummaryThousand cankers disease (TCD), a lethal fungal dieback of eastern black walnut (Juglans nigra), caused byGeosmithia morbida, and spread by the walnut twig beetle,Pityophthorus juglandis, was documented in 2009 to be very destructive in the western United States and was identified in the native range ofJ. nigraat Knoxville, Tennessee, in 2010, and in 2011 at Richmond, Virginia. Beginning late 2010, we studied branch dieback levels (per cent live crown) and newTCDsymptom development at these two quarantined locations monthly for 3 years. Of the 106 trees studied (53 at each location), 31 trees had low live crown ratings of 70 to 0% with little change over the 3 years of the study. One per cent of the trees developed new symptoms on a per‐year basis. Thus, a moderate level ofTCD(mean = 76% live crown) was present in these two locations, and most trees were in a quiescent or dormantTCDcondition for 3 years, an important finding not previously reported. We found newTCDsymptoms developed in Richmond in 2011 and 2012 when precipitation from January 1 to the end of August was low (60–64 cm), and not when the precipitation in Richmond was higher (99 cm). In late 2012, in Richmond, soil water potential assays indicated that some black walnut trees were under severe physiological stress (−15 bars). In contrast, in 2013, high precipitation levels (99 and 130 cm) and high soil water potentials (−0.1 to −3 bars) at both locations were associated with extensive new foliage and stem growth and recovery fromTCD. Further research is needed on water relationships in regard toTCDand black walnut health.

First Report of Geosmithia morbida on English Walnut and its Paradox Rootstock in California.

Geosmithia morbida, the causal agent of thousand cankers disease (TCD), is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, causing decline in eastern black walnut, Juglans nigra (4), and canker development on many Juglans species (5). In the summer of 2012, a survey for TCD incidence in English walnut, J. regia, in orchards in California identified many trees with WTB activity and characteristic TCD symptoms. Both the J. regia scion and its Paradox hybrid rootstock (J. hindsii× J. regia) were affected. In some cases, trees exhibited bleeding on the bark surface from WTB entrance holes. Removal of the outer bark revealed cankers in the phloem around the WTB galleries. Two samples were taken from scions and three samples were collected from rootstocks of trees in orchards in northern California. Pieces (~3 to 4 mm2) of symptomatic tissue were placed in acidified potato dextrose agar (APDA), and the plates were incubated for 4 to 5 days at 30°C. Samples exhibiting fungal growth similar in morphology to G. morbida were transferred to PDA plates to obtain pure cultures and then processed to obtain single-spore cultures. Culture morphology for five single-spore isolates (Gm103, Gm104, Gm105, Gm107, and Gm108) was similar to that described by Kolařík et al. (4) for G. morbida. Conidiophores were penicillate and verrucose. Conidia were narrowly cylindrical, 5.2 ± 0.06 × 2.2 ± 0.04 μm (n = 50). Single-spore isolates were then grown in 1% yeast extract glucose liquid culture for 7 to 10 days. DNA was extracted and the ITS region was amplified, including the 5.8S region by using primers ITS1F/ITS4. Sequences were assembled and deposited in GenBank under accessions KJ664793 to KJ664797. Sequences were compared to those in GenBank; all sequences matched (99 to 100% identity) the ITS sequences of G. morbida strain CBS 124663. Pathogenicity tests were performed on 28-cm-long detached branches of J. regia. Four branches per isolate were inoculated with a 5-mm-diameter mycelial plug from a 2-week-old culture. Branches were incubated at room temperature (23 ± 2°C) in a humidified container for 3 weeks, and then canker lengths were measured. Pieces of the cankered area were placed in APDA and incubated as described above with G. morbida re-isolated from the cankers for all of the isolates, completing Koch's postulates. Average canker lengths ranged from 48.6 ± 4.3 to 72.1 ± 7.1 mm. Re-isolated G. morbida exhibited the same growth and reproductive structure morphology in culture on PDA as the original cultures. TCD in association with WTB has been observed in California English walnut orchards since 2008 (1,2,3). However, this is the first report for completion of Koch's postulates and morphological and molecular confirmation of G. morbida in J. regia and the Paradox rootstock, the predominant rootstock used in commercial orchards. TCD is a concern to the walnut industry in California with over 245,000 bearing acres reported in 2012.

First Record of Thousand Cankers Disease Fungal Pathogen Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans regia in Europe.

Thousand cankers disease (TCD) is a disease complex caused by the fungus Geosmithia morbida Kolařik (Ascomycota, Hypocreales) and its vector Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae; walnut twig beetle, WTB). Since the mid-1990s, the disease was responsible for widespread mortality of many walnut species in the United States (4). After the first detection of TCD on black walnut (Juglans nigra L.) in Italy (3), an extensive survey was activated in cooperation with the Regional Phytosanitary Service. In May 2014, early TCD symptoms (4) were observed on English walnuts (J. regia L.). Canopies showed yellowing, wilting, and dieback of the youngest twigs, and a number of small brown cankers. Longitudinal and radial sections sampled through the cankers revealed gray to brown discoloration of both phloem and bark, and the presence of bark beetle galleries. Xylem discoloration was never observed. From one ~20-year-old European walnut growing in a garden neighboring an infected black walnut plantation (Santorso, Vicenza, 45°72' N, 11°40' E), a number of 1- to 2.5-cm-diameter twigs showing cankers up to 2 cm long surrounding bark beetle holes were collected. Whitish mycelium producing verticillate conidiophores was detected inside the insect galleries. From the necrotic margin of eight cankers previously surface-sterilized with 3% sodium hypochlorite, two 4-mm-wide chips per canker were placed on potato dextrose agar and incubated at 28 ± 1°C in the dark. Slow growing lobate, plane, yellowish-ocher colonies with hyaline mycelium appeared in 5 days. After subculturing to the same medium, growth features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolarik's description of G. morbida (2) were observed. The ITS region of rDNA from the fungus strain LM14GM001-JR was amplified by using ITS1F and ITS4 primers and sequenced obtaining a 387-bp gene fragment. BLAST analysis showed 99% identity to the G. morbida strain U19 (GenBank Accession No. KF808301.1) for 384 bp, and 99% identity to the G. morbida strain LM13GM001-JN previously isolated from J. nigra in Italy (3). From the same samples, two emerging beetles were collected and identified as P. juglandis both morphologically (5) and genetically by DNA extraction following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxidase I was then amplified by using universal primers (1) and sequenced to obtain a 614-bp fragment of the gene. BLAST analysis showed 100% identity to P. juglandis based on comparison with KJ451422. A few other English walnuts with both the fungus and WTB were also found close to other infected black walnut plantations. To our knowledge, this is the first record of G. morbida and P. juglandis on J. regia in Europe, where the tree is cultivated for both fruit and timber production, as well as a traditional landscape tree. Voucher specimens are stored in the TeSAF herbarium and in the DAFNAE insect collection.

Crepuscular flight activity of an invasive insect governed by interacting abiotic factors.

Seasonal and diurnal flight patterns of the invasive walnut twig beetle, Pityophthorus juglandis, were assessed between 2011 and 2014 in northern California, USA in the context of the effects of ambient temperature, light intensity, wind speed, and barometric pressure. Pityophthorus juglandis generally initiated flight in late January and continued until late November. This seasonal flight could be divided approximately into three phases (emergence: January–March; primary flight: May–July; and secondary flight: September–October). The seasonal flight response to the male-produced aggregation pheromone was consistently female-biased (mean of 58.9% females). Diurnal flight followed a bimodal pattern with a minor peak in mid-morning and a major peak at dusk (76.4% caught between 1800 and 2200 h). The primarily crepuscular flight activity had a Gaussian relationship with ambient temperature and barometric pressure but a negative exponential relationship with increasing light intensity and wind speed. A model selection procedure indicated that the four abiotic factors collectively and interactively governed P. juglandis diurnal flight. For both sexes, flight peaked under the following second-order interactions among the factors when: 1) temperature between was 25 and 30°C and light intensity was less than 2000 lux; 2) temperature was between 25 and 35°C and barometric pressure was between 752 and 762 mba (and declined otherwise); 3) barometric pressure was between 755 and 761 mba and light intensity was less than 2000 lux (and declined otherwise); and 4) temperature was ca. 30°C and wind speed was ca. 2 km/h. Thus, crepuscular flight activity of this insect can be best explained by the coincidence of moderately high temperature, low light intensity, moderate wind speed, and low to moderate barometric pressure. The new knowledge provides physical and temporal guidelines for the application of semiochemical-based control techniques as part of an IPM program for this invasive pest.

First Report of Geosmithia morbida in North Carolina: The Pathogen Involved in Thousand Cankers Disease of Black Walnut.

In the past decade, black walnut (Juglans nigra) trees throughout western North America have suffered from widespread branch dieback and canopy loss, causing substantial tree mortality (2,3). The fungus, Geosmithia morbida, vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, has been associated with this devastating disease known as Thousand Cankers Disease (TCD) (2,3). In August of 2012, branch samples from TCD symptomatic black walnut trees (5 to 10 cm in diameter and 15 to 30 cm long) were collected on the North Carolina side of the Great Smoky Mountain National Park (GRSM) in Cataloochee Cove (35°37.023' N, 83°07.351' W) and near the Big Creek Campground (35°45.290' N, 83°06.473' W), in Haywood County. Five symptomatic trees near the Big Creek Campground and three from Cataloochee Cove displayed typical TCD signs including progressive crown thinning, branch flagging, and branch dieback; however, insect holes were not observed. Samples were double bagged in Ziploc plastic bags, sealed in a 19-liter plastic bucket, and transported to the University of Tennessee. Outer bark was removed from the samples and small, elliptical, necrotic cankers were observed. Wood chips (3 to 4 mm2) from cankers were excised and placed on 1/10 strength potato dextrose agar amended with 30 mg/liter streptomycin sulfate and 30 mg/liter chlortetracycline HCL and incubated on a 12-h dark/light cycle at 22°C for 5 to 7 days. Fungal isolates were tentatively identified as G. morbida by using culture morphology, and characteristics of conidiophores and conidia (2). The isolated fungus from the Cataloochee Cove location was grown in 1/10 strength potato dextrose broth at room temperature for 2 weeks. Isolates from Big Creek Campground were contaminated and were not analyzed further. Fungal colonies were tan to light yellow. Conidia were tan, subcylindrical, and catenulate. Conidiophores were multibranched, verticillate, and verrucose. To verify the morphological data, DNA was extracted from fungal mycelia using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's published protocol. Isolates from Cataloochee Cove were characterized using ITS1 and ITS4 universal primers (4). The putative G. morbida isolate (GenBank Accession No. KC461929) had ITS sequences that were 100% identical to the G. morbida type isolate CBS124663 (FN434082.1) (2). Additionally, fungal DNA from Cataloochee Cove was amplified using G. morbida-specific microsatellite loci (GS04, GS27, and GS36) (1). PCR products were analyzed with the QIAxcel Capillary Electrophoresis System (Qiagen) and were similar to those previously published (2). To date, all confirmed cases of TCD in the native range of black walnut have been in urban areas, along rural roadsides and/or fence rows. The report in North Carolina is the first finding of G. morbida, the causal agent of TCD, in a forest setting.

Discovery of Walnut Twig Beetle, Pityophthorus juglandis, Associated with Forested Black Walnut, Juglans nigra, in the Eastern U.S.

Thousand cankers disease (TCD) is an insect-mediated disease of walnut trees (Juglans spp.) involving walnut twig beetle (Pityophthorus juglandis) and a fungal pathogen (Geosmithia morbida). Although first documented on walnut species in the western U.S., TCD is now found on black walnut (J. nigra) in five states in the eastern U.S. Most collections of P. juglandis or G. morbida are from trees in agriculturally- or residentially-developed landscapes. In 2013, 16 pheromone-baited funnel traps were deployed in or near black walnuts in forested conditions to assess the risk of infestation of forested trees by P. juglandis. Four of the 16 funnel traps collected adult P. juglandis from three forested areas (one in North Carolina and two in Tennessee). These collections, while in forested settings, may still be strongly influenced by human activities. The greatest number of P. juglandis (n = 338) was collected from a forested location in an urbanized area near a known TCD-positive tree. The other two forested locations where P. juglandis (n = 3) was collected were in areas where camping is common, and infested firewood may have introduced P. juglandis unintentionally into the area. Future studies to assess P. juglandis on more isolated forested walnuts are planned.

First Record of Thousand Cankers Disease Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans nigra in Europe.

Thousand cankers disease (TCD) of walnut is responsible for widespread mortality of black walnut (Juglans nigra L.) in the United States since the mid-1990s (2). The disease is caused by the fungus Geosmithia morbida Kolařik (Ascomycota, Hypocreales), vectored by the walnut twig beetle Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae). In September 2013, TDC was observed in northeastern Italy (Bressanvido, Vicenza, 45°39' N, 11°38' E) in black walnuts of different ages: ~80-year-old plants growing in a garden and 17-year-old trees belonging to a nearby walnut plantation for timber production. Main symptoms were yellowing, wilting, twig and branch dieback, and a high number of small bark cankers (3). Longitudinal and radial sections collected through the cankers revealed gray to brown discoloration of both phloem and outer bark, and the presence of bark beetle galleries radiating from the mating chamber and developing horizontally (across the wood grain), and vertical (along the grain) larval galleries. Occasionally, discoloration involved the outward xylematic tissues. Fungal fruiting bodies were not found on or near the cankers. Whitish mycelium, sometimes producing verticillate conidiophores, was frequently detected inside galleries. A number of 1- to 3-cm diameter twigs showing cankers up to 2 cm long surrounding bark beetle penetration holes were randomly collected. From samples, emerging beetles were identified as P. juglandis both morphologically (4) and genetically. DNA extraction was carried following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxydase I was then amplified using universal primers (1) and sequenced, obtaining 627 bp. BLAST analysis showed 100% identity to P. juglandis. Sequences were finally deposited in the BoldSystem database (GenBank Accession No. KF725084). From the necrotic margin of six cankers previously surface-sterilized with 3% sodium hypochlorite, two 3-mm-wide chips per canker were placed on potato dextrose agar and incubated at 23 ± 1°C in the dark. Among a variety of microorganisms, slow growing lobate, plane, yellowish-ochre colonies with hyaline mycelium appeared in 6 days. After subculturing to the same medium, growing features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolařik's description of G. morbida (2) were observed. Same result was obtained culturing the mycelium growing inside the galleries. The ITS region of rDNA was amplified using ITS1F and ITS4 primers and sequenced, obtaining 597 bp. BLAST analysis showed 100% identity to G. morbida strain U173 (HF546283.1) for 558 bp. To our knowledge, this is the first record of TCD and P. juglandis to Europe, where walnut species (mainly J. regia, J. nigra, and their hybrids) are intensively cultivated for timber production. This finding is therefore of particular importance. An intensive survey of the disease is suggested, both to assess fungus/beetle presence and to verify possible pathways of introduction, likely associated to importation of infested/infected timber from native Nearctic regions. Voucher specimens are stored in the TeSAF herbarium (fungus) and in the DAFNAE insect collection.

First record of Pityophthorus solus (Blackman, ) in Europe

First records of Pityophthorus solus (Blackman, ) (Coleoptera: Curculionidae: Scolytinae) for Europe are reported. Twenty one specimens were obtained unintentionally during surveys for Ips sexdentatus (Boemer, 1767) and for Pityophthorus juglandis Blackman, using multiple funnel traps baited with specific attractants for both species in the Basque Country (Northern Spain).

Efficacy of Heat Treatment for the Thousand Cankers Disease Vector and Pathogen in Small Black Walnut Logs

Thousand cankers disease, caused by the walnut twig beetle (Pityophthorus juglandis Blackman) and an associated fungal pathogen (Geosmithia morbida M. Kolarík, E. Freeland, C. Utley, and N. Tisserat), threatens the health and commercial use of eastern black walnut (Juglans nigra L.), one of the most economically valuable tree species in the United States. Effective phytosanitary measures are needed to reduce the possibility of spreading this insect and pathogen through wood movement. This study evaluated the efficacy of heat treatments and debarking to eliminate P. juglandis and C. morbida in J. nigra logs 4-18 cm in diameter and 30 cm in length. Infested logs were steam heated until various outer sapwood temperatures (60, 65, and 70 degrees C in 2011; 36, 42, 48, 52, and 56 degrees C in 2012) were maintained or exceeded for 30-40 min. In 2011, all heat treatments eliminated G. morbida from the bark, but logs were insufficiently colonized by P. juglandis to draw conclusions about treatment effects on the beetle. Debarking did not ensure elimination of the pathogen from the sapwood surface. In 2012, there was a negative effect of increasing temperature on P. juglandis emergence and G. morbida recovery. G. morbida did not survive in logs exposed to treatments in which minimum temperatures were 48 degrees C or higher, and mean P. juglandis emergence decreased steadily to zero as treatment minimum temperature increased from 36 to 52 degrees C. A minimum outer sapwood temperature of 56 degrees C maintained for 40 min is effective for eliminating the thousand cankers disease vector and pathogen from walnut logs, and the current heat treatment schedule for the emerald ash borer (60 degrees C core temperature for 60 min) is more than adequate for treating P. juglandis and G. morbida in walnut firewood.

Attraction of Walnut Twig Beetle Pityophthorus juglandis (Coleoptera: Curculionidae) to the Fungus Geosmithia morbida

Geosmithia morbida causes thousand cankers disease of Juglans nigra and it is transmitted by the walnut twig beetle (WTB, Pityophthorus juglandis). Along with WTB, an ambrosia beetle, Xyleborinus saxeseni, is commonly associated with thousand cankers disease in the later stages of J. nigra decline, although X. saxeseni is not a known vector of G. morbida. We initiated studies to determine whether WTBs or X. saxeseni were attracted to volatiles produced by G. morbida and other bark fungi in a laboratory choice-test setting. There was no difference between the numbers of emerged WTB adults that were collected in tubes containing potato dextrose agar colonized by G. morbida and those in tubes containing agar only. More adult X. saxeseni were collected in tubes containing agar only compared with G. morbida. Walnut twig beetle larvae migrated more frequently toward an agar plug colonized by G. morbida and Fusarium solani compared with an un-colonized agar plug. No larval preference was observed when agar plugs colonized by F. solani and G. morbida, or G. morbida and Penicillium solitum, were placed in the same petri dish. These results suggest that WTB larvae are attracted to bark fungi in general, but not specifically to G. morbida. Accepted 27 June 2014. Published 13 August 2014.

Genetic differentiation and spatial structure of Geosmithia morbida, the causal agent of thousand cankers disease in black walnut (Juglans nigra)

The main objectives of this study were to evaluate genetic composition of Geosmithia morbida populations in the native range of black walnut and provide a better understanding regarding demography of the pathogen. The fungus G. morbida, and the walnut twig beetle, Pityophthorus juglandis, have been associated with a disease complex of black walnut (Juglans nigra) known as thousand cankers disease (TCD). The disease is manifested as branch dieback and canopy loss, eventually resulting in tree death. In 2010, the disease was detected in black walnut in Tennessee, and subsequently in Virginia and Pennsylvania in 2011 and North Carolina in 2012. These were the first incidences of TCD east of Colorado, where the disease has been established for more than a decade on indigenous walnut species. A genetic diversity and population structure study of 62 G. morbida isolates from Tennessee, Pennsylvania, North Carolina and Oregon was completed using 15 polymorphic microsatellite loci. The results revealed high haploid genetic diversity among seven G. morbida populations with evidence of gene flow, and significant differentiation among two identified genetic clusters. There was a significant correlation between geographic and genetic distance. Understanding the genetic composition and demography of G. morbida can provide valuable insight into recognizing factors affecting the persistence and spread of an invasive pathogen, disease progression, and future infestation predictions. Overall, these data support the hypotheses of two separate, highly diverse pathogen introductions into the native range of black walnut.

The Effect of Temperature on Survival ofPityophthorus juglandis(Coleoptera: Curculionidae)

The walnut twig beetle (Pityophthorus juglandis Blackman) vectors Geosmithia morbida, the causal agent of thousand cankers disease in Juglans, and is particularly damaging to Juglans nigra L. (black walnut). Native hosts of P. juglandis are distributed in the southwestern United States where winter temperatures tend to be higher than those found within the native range of black walnut. To better understand temperature effects on survival of P. juglandis, we initiated studies to determine: 1) seasonal variations in cold tolerance, as measured by the supercooling point (SCP), and 2) upper and lower lethal temperatures (LT). In the lower LT study, Xyleborinus saxeseni (Ratzeberg) was tested for comparison. Insects were either exposed to increasing or decreasing temperatures and then checked for survival. Upper and lower LTs were estimated using a logistic model. For the SCP study, data were grouped into seasons. Seasonal mean SCPs were highest in summer (-15.4°C) and lowest in fall (-18.1°C). The upper lethal limit estimations required to kill 99% of the population (LT99) for adults and larvae were 52.7 and 48.1°C, respectively, and lower limit LT99 estimations for adults and larvae were -18.1 and -18.7°C, respectively. The lower median LT (LT50) of X. saxeseni was -24.7°C. These studies, as well as beetle survival in infested Colorado trees where temperatures reached -29°C in February 2011, suggest P. juglandis could survive the winter in much of the native range of black walnut, but may be limited in trees where temperatures regularly exceed the lower LT.

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.