Influence of varieties of hemp, Cannabis sativa (Rosales: Cannabaceae), and fertilization rates on damage caused by corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae).

Industrial hemp, Cannabis sativa L., production has been negatively impacted by larvae of corn earworm, Helicoverpa zea (Boddie), which feed on developing inflorescences. Adult H. zea oviposit on hemp once flowers develop, and late-instar larvae can cause serious loss to both quality and yield. A 2-year study to assess the influence of hemp variety and fertilization practices on damage caused by H. zea was conducted. Differences in damage ratings among varieties were observed in both years; however, the rate of nitrogen applied did not influence biomass yield or damage rating. These results indicate that increasing nitrogen fertility may not be an effective means of cultural control for mitigating damage from H. zea. Floral maturity was very influential on damage caused by H. zea as late-maturing varieties had much less floral injury than those which matured early in outdoor field trials. Some cannabinoids were also correlated to damage rating, but this relationship was due to late-maturing plants with immature flowers low in cannabinoid concentrations receiving less floral injury. Based on these results, the selection of high-yielding varieties that flower when ovipositional activity of H. zea is expected to decline should be the first step in an integrated pest management program for hemp production. This research expanded our knowledge of the role of fertility rate, varietal characteristics, cannabinoid profile, and floral maturity on damage caused by H. zea to hemp. Findings from this research will allow growers to make more informed agronomic decisions before planting to improve hemp production.

First report of Diplodia corticola causing dieback of white oak (Quercus alba) in Tennessee.

Diplodia corticola is a fungal pathogen causing oak dieback in Quercus (oak) spp. in parts of North America, northern Africa, and Europe (Ferreira et al., 2021; Smahi et al., 2017; Tsopelas et al., 2018). In August 2021, a single mature white oak (Q. alba) exhibiting wilt symptoms, vascular discoloration, and interveinal chlorosis was observed in Cove Lake State Park in Campbell County, Tennessee, U.S.A. Small sections of phloem tissues were cut from the margins of discolored vasculature of a single wilt symptomatic branch with a sterile scalpel and surface sterilized following Parra et al. (2020). Surface sterilized wood chips were plated onto potato dextrose agar amended with antibiotics (PDA++) following Gazis et al. (2018). Three days after plating, we recovered a single fungal isolate from wood chips that when grown in ½ PDA resembled D. corticola, having irregular margins and white aerial mycelia that progressively turned greyish-black 15 days after sub-culturing (Alves et al., 2004). Total genomic DNA was extracted from the isolate following Gazis et al. (2018). The internal transcribed spacer (ITS) was then amplified using the ITS1 and ITS4 primers and the subsequent PCR product was sequenced. Resulting reads were assembled into a consensus sequence and identity was assigned using BLAST on the NCBI nucleotide database. The assembled sequence (accession OM716006) had a 100% identity match with D. corticola type culture CBS 112549 (accession NR_111152). To complete Koch's postulates and identify potential host range, 5 red oaks (Q. rubra; 2-3 yrs old; caliper 14.7 ± 2 mm) and 5 white oaks (Q. alba; 2-3 yrs old; caliper 22.8 ± 2.3 mm) were inoculated with D. corticola (isolate DC_2.5). Trees were inoculated 15 cm above the soil line in a greenhouse with a 3 mm diameter plug of a 10-day old culture of D. corticola grown on PDA following Sitz et al. (2017). As a negative control, 5 red and 5 white oaks were inoculated with a 3 mm diameter plug of PDA. For each species, trees were sampled when seepage was observed from D. corticola inoculated sites (15 days post-inoculation for red and white oaks). At time of sampling, bark adjacent to inoculation sites on each tree was removed and cankers were photographed. Using a sterile scalpel, four wood chips were cut from canker margins and placed onto PDA++. For all trees, canker areas were measured using ImageJ software (Rasband, 2012). Recovered isolate identities were confirmed by extracting total genomic DNA as described above (Gazis et al. 2018) and PCR amplification of the ITS, large ribosomal subunit (LSU), and elongation factor 1-α (ef1-α) following (Ferreira et al., 2021). Diplodia corticola was reisolated from wood chips of D. corticola inoculated red (5/5 trees) and white (5/5 trees) oaks and ITS (accession OM716954), LSU (accession OM716955), and ef1-α (accession OM752198) sequences matched D. corticola type culture 112549 ITS (100% identity), LSU (99.76%-100% identity; accession KF766323), and ef1-α (98%-98.9% identity; accession XM_020275852). All D. corticola inoculated trees exhibited seepage from inoculation sites with streaking present in vasculature. Cankers were significantly larger in D. corticola inoculated red (2.34 ± 1.36 cm; P=0.042) and white (2.96 ± 0.52 cm; P=0.00029) oaks compared to agar inoculated trees. To the best of our knowledge, this is the first report of D. corticola causing decline of oaks in Tennessee.

Assessment of Alternative Candidate Subcortical Insect Vectors From Walnut Crowns in Habitats Quarantined for Thousand Cankers Disease.

Thousand cankers disease (TCD) results from the combined activity of the fungal pathogen, Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat and its principle vector, Pityophthorus juglandis (Blackman) (Coleoptera: Curculionidae: Scolytinae) in Juglans L. spp. and Pterocarya Kunth spp. host plants. TCD has been reported from the eastern and western United States. To evaluate potential for other beetle species to vector the fungus in east Tennessee, specimens were collected using ethanol-baited traps that were suspended beneath crowns of TCD-symptomatic trees. Associations of G. morbida with insect species collected in traps were assessed in an unsuccessful, preliminary culture-based fungal assay, and then with a molecular-based detection method. For culture-based assays, rinsate from washed, individual insects was plated on nutrient media and growing colonies were subcultured to obtain axenic G. morbida cultures for identification. For the molecular-based method, G. morbida presence was detected by amplifying the previously developed, species-specific microsatellite locus GS004. Capillary electrophoresis was used to detect the amplified amplicons and representative reactions were validated using Sanger sequencing. Eleven beetle species were found to carry G. morbida, including Cnestus mutilatus (Blandford), Dryoxylon onoharaensum (Murayama), Hylocurus rudis (LeConte), Monarthrum fasciatum (Say), Monarthrum mali (Fitch), Xyleborinus saxesenii (Ratzeburg), Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford) (all Coleoptera: Curculionidae: Scolytinae), Stenomimus pallidus (Boheman) (Coleoptera: Curculionidae: Cossoninae), Oxoplatypus quadridentatus (Olivier) (Coleoptera: Curculionidae: Platypodinae), and Xylops basilaris (Say) (Coleoptera: Bostrichidae). These findings raise concerns that alternative subcortical insect species that already occur within quarantined habitats can sustain incidence of introduced G. morbida and contribute to spread within the native range of black walnut, Juglans nigra L., in the eastern United States.

Determining Spread Rate of Kudzu Bug (Hemiptera: Plataspidae) and Its Associations With Environmental Factors in a Heterogeneous Landscape.

By the end of 2017, kudzu bug was reported in 652 counties in the United States since it was first observed in Georgia in 2009. Modeling its invasion dynamics is valuable to guide management through early detection and prevention of further invasion. Herein, we initially estimated the spread rate of kudzu bug with county-level invasion records and then determined important spatial factors affecting its spread during years 2010-2016. As kudzu bug infests a large heterogeneous area and shows asymmetric spread, we first utilized spatially constrained clustering (SCC), an unsupervised machine learning method, to divide the infested area into eight spatially contiguous and environmentally homogenous neighborhoods. We then used distance regression and boundary displacement methods to estimate the spread rates in all neighborhoods. Finally, we applied multiple regression to determine spatial factors influencing the spread of kudzu bug. The average spread rate reached 76 km/yr by boundary displacement method; however, the rate varied largely among eight neighborhoods (45-144 km/yr). In the southern region of the infested area, host plant density and wind speed were positively associated with the spread rate, whereas mean annual temperature, precipitation in the fall, and elevation had inverse relationships. In the northern region, January minimum temperature, wind speed, and human population density showed positive relationships. This study increases the knowledge on the spread dynamics of kudzu bug. Our research highlights the utility of SCC to determine natural clustering in a large heterogeneous region for better modeling of local spread patterns and determining important factors affecting the invasions.

Short-term Detection of Imidacloprid in Streams after Applications in Forests.

Imidacloprid, a neonicotinoid insecticide, is a major component of hemlock woolly adelgid (HWA) [ (Annand)] management programs that are critical to protecting forest health in the eastern United States. However, the impact of imidacloprid soil applications in forests on some aquatic macroinvertebrate species by leaching into aquatic systems is uncertain. The time for residues from imidacloprid soil applications to migrate from treated hemlocks to nearby streams and the concentrations at which imidacloprid may occur after initial migration is unknown. The presence and concentration of imidacloprid in three streams adjacent to recently treated hemlock (soil drench >10 m from stream channels) were assessed in the Big South Fork National River and Recreation Area. Two standard water grabs were collected monthly for 1 yr from a location downstream from imidacloprid-treated areas. Samples were analyzed using liquid chromatography tandem mass spectrometry (limit of detection = 0.025 µg L). Imidacloprid was detected in all treatment streams during a single rain event that occurred 184 to 196 d after treatments, and concentrations ranged from 0.053 to 0.833 µg L. Imidacloprid was not detected on any other sampling date from treatment streams. All observed positive detections exceeded the USEPA freshwater invertebrate chronic endpoint (0.01 µg L). One stream sample exceeded the USEPA freshwater invertebrate acute endpoint (0.39 µg L). However, previous macroinvertebrate community assessments in streams with similar concentrations did not indicate negative effects to aquatic fauna. These findings help characterize the risk of imidacloprid treatments to stream macroinvertebrates within 1 yr of soil applications.

Risk assessment of imidacloprid use in forest settings on the aquatic macroinvertebrate community.

The isolated effects of a single insecticide can be difficult to assess in natural settings because of the presence of numerous pollutants in many watersheds. Imidacloprid use for suppressing hemlock woolly adelgid, Adelges tsugae (Annand) (Hemiptera: Adelgidae), in forests offers a rare opportunity to assess potential impacts on aquatic macroinvertebrates in relatively pristine landscapes. Aquatic macroinvertebrate communities were assessed in 9 streams in Great Smoky Mountains National Park (southern Appalachian Mountains, USA). The streams flow through hemlock conservation areas where imidacloprid soil drench treatments were applied for hemlock woolly adelgid suppression. Sites were located upstream and downstream of the imidacloprid treatments. Baseline species presence data (pre-imidacloprid treatment) were available from previous sample collections at downstream sites. Downstream and upstream sites did not vary in numerous community measures. Although comparisons of paired upstream and downstream sites showed differences in diversity in 7 streams, higher diversity was found more often in downstream sites. Macroinvertebrate functional feeding groups and life habits were similar between downstream and upstream sites. Downstream and baseline stream samples were similar. While some functional feeding group and life habit species richness categories varied, variations did not indicate poorer quality downstream communities. Imidacloprid treatments applied according to US Environmental Protection Agency federal restrictions did not result in negative effects to aquatic macroinvertebrate communities, which indicates that risks of imidacloprid use in forest settings are low. Environ Toxicol Chem 2017;36:3108-3119. © 2017 SETAC.

Emergence, Seasonality, and Hybridization of Laricobius nigrinus (Coleoptera: Derodontidae), an Introduced Predator of Hemlock Woolly Adelgid (Hemiptera: Adelgidae), in the Tennessee Appalachians.

From 2010 through 2013, adult emergence and seasonality of Laricobius nigrinus Fender, an introduced predatory species native to western North America, as well as hybridization with the native species Laricobius rubidus (LeConte), were evaluated using emergence traps and beat-sheet sampling in areas of previous release against hemlock woolly adelgid, Adelges tsugae Annand. The shortest emergence period of adult L. nigrinus was 7 wk beginning 22 October 2010, and the longest emergence was 15 wk beginning 17 October 2012. Native L. rubidus also were collected from emergence traps placed on the ground surface and beat-sheet samples all 3 yr, with emergence of L. rubidus initiating later than L. nigrinus each season. Seasonality of both Laricobius species was similar across a 44-mo study period. Adult L. nigrinus were present from October through April, and larvae of Laricobius spp. were collected from February to May. The average number of L. nigrinus from emergence traps was significantly greater than the average number of beetles collected from beat-sheet samples in 2010, while the converse was observed during 2012. Hybridization between L. nigrinus and L. rubidus was documented from 10.75% of specimens collected during 2010 and 2011, indicating periodic interbreeding between the introduced and native species. These findings suggest emergence trapping may be a useful method to assess establishment, population densities, and seasonality of Laricobius species in areas of release to enhance their use in management of A. tsuage.

Spatial and temporal distribution of residues of imidacloprid and its insecticidal 5-hydroxy and olefin and metabolites in eastern hemlock (Pinales: Pinaceae) in the southern Appalachians.

Widespread mortality of eastern hemlock, Tsuga canadensis (L.) Carrière, resulting from infestation by hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), has occurred throughout the native range of eastern hemlock within the eastern United States. Imidacloprid, a systemic insecticide, is one of the primary chemical compounds used to control hemlock woolly adelgid in both urban and, in a limited manner, in natural forest environments. The metabolism of imidacloprid in eastern hemlock produces 12 metabolites; two of these, imidacloprid 5-hydroxy and imidacloprid olefin, are considered toxicologically important metabolites. However, little is known about the persistence of these metabolites in eastern hemlock in the southern Appalachians. Concentrations ofimidacloprid, olefin, and 5-hydroxy were quantified by using HPLC/MS/MS techniques. Over the 3-yr study, concentrations of imidacloprid and consequent 5-hydroxy and olefin were highest in trees treated with a soil injection in the spring. Imidacloprid and 5-hydroxy concentrations in sap were highest at 12 mo posttreatment and in tissue at 15 mo posttreatment. Imidacloprid was detected through 36 mo posttreatment and 5-hydroxy was detected through 15 mo posttreatment. Olefin concentrations in both sap and tissue were highest at 36 mo posttreatment and were detected in high concentrations through 36 mo posttreatment. Concentrations of imidacloprid were highest in the bottom stratum of the canopy and lowest in the top stratum. Concentrations of olefin and 5-hydroxy were highest in the top stratum and lowest in the bottom stratum.

Spatial and temporal distribution of imidacloprid in eastern hemlock in the southern Appalachians.

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an exotic insect species dramatically reducing populations of eastern hemlock, Tsuga canadensis (L.) Carrieré, throughout the eastern United States. Systemic imidacloprid is one of the primary chemicals used to successfully control infestations of the hemlock woolly adelgid. The concentration levels for this systemic insecticide present in the sap of eastern hemlocks were evaluated from three strata within the canopy over a two year time span. Enzyme-linked immunosorbent assays were conducted every three months posttreatment to assess imidacloprid concentration within the sap. The effect of application timing (fall versus spring) and application method (soil drench, soil injection, and tree injection) on the translocation of imidacloprid throughout the canopy, and the quantity of imidacloprid translocated in the sap of eastern hemlock branches and the terminal twig and needle tissue was determined. Concentration levels progressively declined from the bottom strata to the top strata of the canopy. This trend was consistent in all chemically treated trees. Tree injections provided the lowest concentration and the most nonuniform distribution of imidacloprid throughout the canopy. The highest insecticide concentrations within the tree across all strata over the two year period were consistently associated with the soil drench method followed by the soil injection method. Imidacloprid concentrations peaked between month 9 and 12 posttreatment, and then declined; however, at two years posttreatment, soil drench and soil injected trees contained concentrations reported as being effective for control of the hemlock woolly adelgid.

Community response of insects associated with eastern hemlock to imidacloprid and horticultural oil treatments.

The hemlock woolly adelgid, Adelges tsugae Annand, is an invasive species reducing the populations of eastern hemlock, Tsuga canadensis L. Carrière, throughout the eastern United States. Systemic imidacloprid and horticultural oil are the primary chemicals used to control infestations of this invasive pest; however, the impact of these two chemicals on nontarget canopy insects is unknown. This study was initiated in November 2005 to assess the effects of (1) imidacloprid soil drench, (2) imidacloprid soil injection, (3) imidacloprid tree injections, and (4) horticultural oil applications on multiple levels of organization (composition, overall specimen abundance and species richness, guild specimen abundance and species richness, and individual species) within the phytophagous and transient canopy insect community. Community composition differed significantly among treatments based on analysis of similarity. Mean species richness and specimen abundance were significantly reduced by one or more treatments. Soil drench applications significantly reduced species richness for the detritivore and phytophaga guilds. Furthermore, specimen abundance for species in the detritivore, fungivore, phytophaga, scavenger, and transient phytophaga guilds was significantly lower in the soil drench treatment. This trend was consistent in all insect guilds examined, with the exception of the hematophaga guild that was not significantly lower than for species on the control trees. Of the 293 species documented to be associated with eastern hemlocks, 33 species were found to be directly effected by one or more of the chemical treatments.

Molecular and genomic basis of volatile-mediated indirect defense against insects in rice.

Rice plants fed on by fall armyworm (Spodoptera frugiperda, FAW) caterpillars emit a blend of volatiles dominated by terpenoids. These volatiles were highly attractive to females of the parasitoid Cotesia marginiventris. Microarray analysis identified 196 rice genes whose expression was significantly upregulated by FAW feeding, 18 of which encode metabolic enzymes potentially involved in volatile biosynthesis. Significant induction of expression of seven of the 11 terpene synthase (TPS) genes identified through the microarray experiments was confirmd using real-time RT-PCR. Enzymes encoded by three TPS genes, Os02g02930, Os08g07100 and Os08g04500, were biochemically characterized. Os02g02930 was found to encode a monoterpene synthase producing the single product S-linalool, which is the most abundant volatile emitted from FAW-damaged rice plants. Both Os08g07100 and Os08g04500 were found to encode sesquiterpene synthases, each producing multiple products. These three enzymes are responsible for production of the majority of the terpenes released from FAW-damaged rice plants. In addition to TPS genes, several key genes in the upstream terpenoid pathways were also found to be upregulated by FAW feeding. This paper provides a comprehensive analysis of FAW-induced volatiles and the corresponding volatile biosynthetic genes potentially involved in indirect defense in rice. Evolution of the genetic basis governing volatile terpenoid biosynthesis for indirect defense is discussed.

Elucidation of the genomic basis of indirect plant defense against insects.

Using airborne signals to attract herbivore predators and parasitoids is an important strategy that plants use in defense against herbivorous insects. The volatiles involved in this indirect plant defense are often chemically complex and variable across species. We recently established rice as a model for studying the molecular and genomic basis of volatile-mediated indirect plant defense. Rice plants when damaged by fall armyworm larvae become highly attractive to parasitic wasps. The volatiles potentially responsible for parasitoid attraction were determined to be a blend of compounds predominated by terpenoids, of which S-linalool is the most abundant. Racemic linalool alone was shown to be effective in attracting parasitic wasps. By combining volatile profiling and microarray analysis, a catalog of candidate genes for volatile biosynthesis was identified. Three genes encoding terpene synthases were biochemically characterized. They are responsible for the production of the majority of volatile terpenes released from insect-damaged rice plants. Additional candidate genes are being currently characterized for their role in production of other insect-induced volatiles from rice plants. Identification of a complete set of key genes for synthesizing herbivory-induced volatiles in rice will provide an important reference for comparative studies of this important defense trait across a variety of plant species.

Insect guild structure associated with eastern hemlock in the southern Appalachians.

An assessment of the insect guild structure associated with immature and mature eastern hemlock, Tsuga canadensis (L.) Carrière, at high and low elevations was made before the invasion by the hemlock woolly adelgid, Adelges tsugae (Annand), and elongate hemlock scale, Fiorinia externa Ferris. Guild dynamics were determined among 243 insect species associated with eastern hemlock in the southern Appalachians. Detritivore, hematophage, herbivore, fungivore, parasitoid, predator, scavenger, and transient guild assignments were made. The herbivore and transient guilds were subdivided into chewers, sapsuckers, flower feeders, pollen feeders, pollen/nectar/sap feeders, and seed feeders. A significant association was found between guilds on immature and mature hemlocks at low elevations, but no significant association was found at high elevations. There was also a strong association of phytophagous insects on immature hemlocks and scavengers on mature hemlocks. The observed transient, scavenger, and predator guilds had a significantly lower co-occurrence among species indicating segregation of the species within these guilds. The herbivore guild had a significantly higher co-occurrence among species than the simulated index indicating aggregation of species within this guild across sites. Detritivore, hematophage, and parasitoid guilds did not differ significantly from the simulated community. These results document a diverse and dynamic insect community on eastern hemlock before invasion by hemlock woolly adelgid and elongate hemlock scale.

Survival of Conidia of Discula destructiva in Frass of the Convergent Lady Beetle.

The survival of conidia of Discula destructiva in frass of convergent lady beetles (Hippodamia convergens) was investigated. D. destructiva was isolated from frass pellets of 76% of adult convergent lady beetles exposed to D. destructiva for 1 h. Of the beetles from which D. destructiva was initially isolated, more than 80, 32, 19, 13, and 12.5% retained viable conidia of D. destructiva internally for at least 12, 24, 48, 72, and 96 h, respectively. Numbers of conidia, estimated with a hemacytometer, ranged from 0 to 3.2 × 106 conidia per frass pellet.