Numerical Response and Impact of Laricobius nigrinus (Coleoptera: Derodontidae) on Adelges tsugae (Hemiptera: Adelgidae) in Their Native Range.

To determine if key attributes for a successful biological control agent are possessed by the predator, Laricobius nigrinus Fender, field studies were conducted in its native range of Seattle, WA. The relationship between adult and immature L. nigrinus abundance to different densities of its prey, Adelges tsugae Annand, were determined. In a second study, predator and prey densities, and survivorship of each sistens A. tsugae stage were determined to gauge the impact of predation. The predator strongly aggregated and increased its reproduction when prey density increased, the two mechanisms of a numerical response. Immature predator-prey ratios were high and average prey density was low in comparison with invaded areas of the eastern United States. Survivorship of aestivating first-instar sistens A. tsugae was low and survivorship of each instar (second, third, and fourth) and adults was high and increased with each stage. When pooled, however, the survivorship of sistens second instar-ovisac stages was low primarily owing to L. nigrinus larval consumption of ovisacs. In its native range, L. nigrinus has key attributes of a successful biological control agent, such as a strong numerical response, high predator-prey ratios, and an important larval impact on A. tsugae populations. Demographic data could serve as important benchmarks for future studies to determine if L. nigrinus and other predators can regulate densities of A. tsugae below eastern hemlock's physiological damage threshold in the eastern United States.

First Report of Verticillium Wilt of Ailanthus altissima in Virginia Caused by Verticillium nonalfalfae.

Ailanthus altissima (Mill.) Swingle, commonly known as tree-of-heaven, is an invasive tree species that has spread throughout the United States since its introduction in 1784 (2). During a survey in July 2009, approximately 1,100 A. altissima trees were observed at two locations in western Virginia (a roadside in Montgomery Co. and a wooded area adjacent to a railroad in Bedford Co.) exhibiting foliar wilt symptoms, defoliation, yellowish vascular discoloration, or death at an incidence of ~77%. Similar symptoms on A. altissima were reported in Roanoke, VA in the early 1930s and after 2005 in Pennsylvania, attributed to a Verticillium sp. (1,2). To identify the causal agent, discolored xylem tissue samples were excised from 10 symptomatic A. altissima trees at both locations, soaked in 1% NaOCl for 2 min, rinsed with sterilized distilled water for 5 min, and placed onto plum extract agar. Cultures were incubated in the dark at 22°C for 7 to 14 days. The resultant colonies (three to four per location) were subcultured and identified putatively as a Verticillium sp. closely related to Verticillium albo-atrum Reinke and Berthold (3), based on melanized, thick-walled, resting mycelia and phialides arranged in verticillate whorls that amassed round, oval-shaped conidia (5.1 ± 1.2 µm × 2.8 ± 0.4 µm, n = 100). Molecular identification of two fungal isolates (one per location) was determined by amplification of the protein coding genes elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS), using PCR primers developed recently for Verticillium (3). A BLAST search on the edited contigs revealed 100% sequence similarity for all three protein coding genes among the two isolates and reference sequences of isolates PD592 (GenBank Accessions JN188227, JN188163, and JN188035 for EF, GPD, and TS, respectively) and VnAaPA140 (KC307764, KC307766, and KC307768 for EF, GPD, and TS, respectively) of the newly-proposed species, V. nonalfalfae (formerly V. albo-atrum). Aligned sequences from one representative isolate, VnAaVA2 (Bedford Co.), were deposited into GenBank as KC307758 (EF), KC307759 (GPD), and KC307760 (TS). To confirm pathogenicity to A. altissima, the two molecularly characterized isolates (one per location) were inoculated into 18 10-week old A. altissima stems that were grown in an environmental chamber at 24°C, 60% RH, and a 12-h photoperiod from seeds collected in Blacksburg, VA. A conidial suspension of each isolate was injected into each stem (0.1 ml of 1 × 108 CFU/ml/stem). All 36 seedlings inoculated with the proposed V. nonalfalfae isolates developed wilting of leaflets within 2 weeks post-inoculation (WPI), defoliation of leaflets by 6 WPI, and were dead by 9 WPI. Eighteen control seedlings were inoculated similarly with distilled water, and remained asymptomatic. Fungi resembling the proposed species V. nonalfalfae were reisolated from all inoculated stems except the control plants, and the species confirmed morphologically as described above. V. nonalfalfae is a recently proposed species that can infect a variety of plant species (3). To our knowledge, this is the first report of this proposed species on A. altissima in Virginia. New state reports of this pathogen on A. altissima are important for regulatory issues associated with using this pathogen as a potential biological control agent. References: (1) G. F. Gravatt and R. B. Clapper. Plant Dis. Rep. 16:96, 1932. (2) M. J. Schall and D. D. Davis. Plant Dis. 93:747, 2009. (3) P. Inderbitzin et al. PLoS ONE, 6, e28341, 2011.

Host-range testing of Eucryptorrhynchus brandti (Coleoptera: Curculionidae), a candidate for biological control of tree-of-heaven, Ailanthus altissima.

Ailanthus altissima (Mill.) Swingle, tree-of-heaven, is an invasive species native to Asia. It first was introduced into the United States in the 1700 s and now is distributed throughout much of North America. Mechanical and chemical controls are current suppression tactics, however, implementation is costly. A weevil, Eucryptorrhynchus brandti (Harold), was identified in China and imported for quarantine testing in 2004 as a potential biological control agent. Host specificity tests on adult feeding, larval development, and oviposition of this weevil were conducted from 2007 to 2011 on A. altissima and 29 nontarget species. Eucryptorrhynchus brandti adults fed significantly more on A. altissima foliage when compared with all test species. Range of means for feeding on A. altissima was 32.5-106.5 mm(2)/adult/d. In no-choice tests, Simarouba glauca DC, Leitneria floridana Chapm., and Citrus limon (L.) Burm. F., had feeding rates of only 10, 49, and 10%, respectively, compared with the level of feeding on A. altissima. The mean range of adult feeding by E. brandti on all other test species was <7% of feeding on A. altissima (0.0-3.3 ± 5.0 mm(2)/adult/d). In the no-choice larval inoculation tests, larval development only occurred in two of 10 L. floridana seedlings compared with seven of 10 A. altissima seedlings. In the no-choice oviposition tests, oviposition and subsequent larval development did not occur in L. floridiana, whereas all seven A. altissima seedlings supported oviposition and subsequent larval development. The weevil did not appear to be a threat to L. floridana or any other nontarget species tested. Therefore, we conclude that Eucryptorrhynchus brandti is highly host specific to A. altissima.

Visual ability and searching behavior of adult Laricobius nigrinus, a hemlock woolly adelgid predator.

Very little is known about the searching behavior and sensory cues that Laricobius spp. (Coleoptera: Derodontidae) predators use to locate suitable habitats and prey, which limits our ability to collect and monitor them for classical biological control of adelgids (Hemiptera: Adelgidae). The aim of this study was to examine the visual ability and the searching behavior of newly emerged L. nigrinus Fender, a host-specific predator of the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Phylloxeroidea: Adelgidae). In a laboratory bioassay, individual adults attempting to locate an uninfested eastern hemlock seedling under either light or dark conditions were observed in an arena. In another bioassay, individual adults searching for prey on hemlock seedlings (infested or uninfested) were continuously video-recorded. Beetles located and began climbing the seedling stem in light significantly more than in dark, indicating that vision is an important sensory modality. Our primary finding was that searching behavior of L. nigrinus, as in most species, was related to food abundance. Beetles did not fly in the presence of high A. tsugae densities and flew when A. tsugae was absent, which agrees with observed aggregations of beetles on heavily infested trees in the field. At close range of prey, slow crawling and frequent turning suggest the use of non-visual cues such as olfaction and contact chemoreception. Based on the beetles' visual ability to locate tree stems and their climbing behavior, a bole trap may be an effective collection and monitoring tool.

Establishment of the hemlock woolly adelgid predator, Laricobius nigrinus (Coleoptera: Derodontidae), in the Eastern United States.

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), native to western North America and Asia, was accidentally introduced from Japan to the eastern United States. To potentially establish biological control of A. tsugae, we released a predator endemic to western North America, Laricobius nigrinus Fender (Coleoptera: Derodontidae), from 2003 to 2005, in 22 localities from Georgia to Massachusetts. Release sites spanned the invasive range of the adelgid across five United States Department of Agriculture plant hardiness zones (5a to 7a). Release sizes were 75, 150, 300, 600, or 1,200 adult L. nigrinus per site in the fall, winter, early spring, or sequentially (i.e., fall or winter and early spring). We monitored establishment by annual sampling for L. nigrinus adults with beat sheets and for L. nigrinus larvae by branch clipping. At the end of 3 yr, L. nigrinus was established in 13 of the 22 sites. The following variables were evaluated for their correlation with the numbers of L. nigrinus larvae and adults recovered and for their effect on establishment (scored as F(3) presence/absence): (1) Minimum winter temperature at the release site, (2) A. tsugae density at the time of release, (3) release size, and (4) release season. Only minimum winter temperature was correlated with larval recoveries and no variables were correlated with adult recoveries. Logistic regression modeling found that establishment was positively related to minimum winter temperature and release size. We recommend smaller release sizes in warm areas where establishment probability was high (i.e., zones 7a, 6b, and 6a) and larger release sizes in cold areas where establishment probability was low (i.e., zones 5b and 5a). Releases during fall-early spring and across the range of A. tsugae densities tested were successful.

Propagation, synchrony, and impact of introduced and native Laricobius spp. (Coleoptera: Derodontidae) on hemlock woolly adelgid in Virginia.

Synchrony and impact of the predators Laricobius nigrinus Fender and Laricobius rubidus LeConte, on hemlock woolly adelgid, Adelges tsugae Annand, were studied in an eastern hemlock field insectary in Virginia. First, a field insectary for propagation of the introduced L. nigrinus was established by planting hemlocks in 2001, infesting them with hemlock woolly adelgid in 2002 and 2003, followed by releasing 258 L. nigrinus in 2003. Initial sampling showed that the native L. rubidus was present in the area. Hemlock woolly adelgid and both Laricobius species populations increased annually, from which 305 F3 L. nigrinus adults were collected and redistributed to forests in 2007. Second, the phenology of hemlock woolly adelgid and Laricobius spp. life cycles were monitored in 2005 and 2006. Adult L. nigrinus (F2) and L. rubidus were active on hemlock from fall through mid-spring and overlapped with second-instar sistentes nymphs through progredientes eggs. The predators' eggs were oviposited and larvae developed (i.e., F3 L. nigrinus) from late winter to mid-spring on progredientes eggs, indicating synchrony with suitable prey life stages. Third, a predator exclusion experiment was used to examine the relationships between the predators and prey in 2005 and 2006. When exposed to L. nigrinus (F2 adults and F3 larvae) and L. rubidus, hemlock woolly adelgid survival and ovisac density were lower and ovisac disturbance was higher than hemlock woolly adelgid protected in cages. The establishment and production of L. nigrinus at a field insectary, synchronization with, and impacts on hemlock woolly adelgid after a small release 2 yr earlier makes it an important potential biological control agent of hemlock woolly adelgid.

Behavior and daily activity patterns of specialist and generalist predators of the hemlock woolly adelgid, Adelges tsugae.

The behavior and daily activity patterns of two specialist predators, Laricobius nigrinus Fender (Coleoptera: Derodontidae) and Sasajiscymnus tsugae, Sasaji and McClure (Coleoptera: Coccinellidae), and a generalist predator, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), of hemlock woolly adelgid, Adelges tsugae (Hemiptera: Adelgidae), were examined using digital video recording in the laboratory. The two specialists are part of a biological control program for A. tsugae, and it is not known if competitive interactions with previously established generalist predators will negatively impact their effectiveness. The behavior and daily activity patterns of adult females of each species were documented in single- and paired-predator assays under simulated spring and summer conditions. Behavior varied qualitatively and quantitatively by species, and did not appear to be highly coordinated temporally or spatially. All species exhibited continuous activity patterns that were punctuated by longer periods of rest. Extensive and intensive searching behavior occurred in all species, with intensive searching being highly variable. Specialist predators appeared to be more selective of feeding and oviposition sites, and rested at more concealed locations than the generalist species. In spring conditions, L. nigrinus had greater activity and a more even behavior distribution than S. tsugae or H. axyridis, which were skewed towards resting. In summer, the latter two species showed increased activity at higher temperatures. Conspecifics significantly altered the time allocated to specific behaviors for L. nigrinus and H. axyridis, resulting in reduced predator effectiveness by reducing time and energy expenditure on activities that directly impact the adelgids. In contrast, S. tsugae conspecifics and all heterospecific combinations showed non-interference. The activity of each species varied with time of day; L. nigrinus was more active at night, while S. tsugae and H. axyridis were more active during the day. All predator groupings maintained a high degree of spatial separation relative to assay size. The use of multiple-predator species combinations that include the specialist predators, is recommended over single-species for biological control of A. tsugae, as temporal and spatial patterns were not highly coordinated. Low-density releases may reduce the potential negative effects of intraspecific competition.