Optimizing Application Rates of Metarhizium brunneum (Hypocreales: Clavicipitaceae) Microsclerotia for Infecting the Invasive Asian Longhorned Beetle (Coleoptera: Cerambycidae).

The Asian longhorned beetle (Anoplophora glabripennis [Motschulsky]) is an invasive wood-boring beetle that threatens urban trees and forests in North America and Europe. The entomopathogenic fungus Metarhizium brunneum Petch strain F52 can infect and kill A. glabripennis adults. Products containing this fungus were available for commercial use in the United States but not registered for Asian longhorned beetle. This study tested different formulations and application rates of M. brunneum F52 microsclerotial granules for their potential development for management of A. glabripennis adults. Three application rates of M. brunneum microsclerotial granules relative to a 1× formulation from previous experiments (0.03 g/cm2; 2× = 0.06 g/cm2 and 3× = 0.09 g/cm2) were exposed on tree trunks for 4-wk periods during May-September. Increased application rates had better retention (% of initial g applied) than the 1× rate, rather than greater weathering loss. Microsclerotia at the 2× application produced 5.05 × 106 conidia/cm2, which was 18 times more conidia than the 1× application. Since A. glabripennis is under active eradication, bioassays with adult beetles were carried out in a quarantine laboratory, using the formulation samples from field exposures. The 2× application resulted in faster beetle mortality. The 3× and 2× rates were not significantly different in retention of the formulation, conidial production, or mortality, but 2× produced the most conidia per gram applied (3.92 × 109 conidia/g). An augmented formulation containing 70% M. brunneum by weight, rather than 50%, produced significantly more conidia and faster beetle mortality than the 50% formulation.

Asian longhorned beetle bioassays to evaluate formulation and dose-response effects of Metarhizium microsclerotia.

Asian longhorned beetles (ALB; Anoplophora glabripennis), are invasive wood borers susceptible to Metarhizium brunneum. This fungus can be prepared as dried microsclerotia which, after rehydration, produce infective conidia within weeks. Wood samples coated with formulated microsclerotia were attached to trees in the Ohio USA ALB-eradication zone and collected after 4-week periods. Adult ALB exposed to these samples had 100% mortality. In an experiment comparing formulations with or without humectant hydrogel, hydrogel did not significantly increase mortality of exposed ALB. In a dose-response experiment with 5 application rates, ALB survival decreased with increasing application rate and conidial density.

Microsclerotia of Metarhizium brunneum F52 Applied in Hydromulch for Control of Asian Longhorned Beetles (Coleoptera: Cerambycidae).

The entomopathogenic fungus Metarhizium brunneum (Petch) strain F52 (Hypocreales: Clavicipitaceae) is able to produce environmentally persistent microsclerotia (hyphal aggregates). Microsclerotia of strain F52 produced as granules and incorporated into hydromulch (hydro-seeding straw, water, and a natural glue) provides a novel mycoinsecticide that could be sprayed onto urban, forest, or orchard trees. We tested this formulation against adult Asian longhorned beetles (Anoplophora glabripennis (Motschulsky)) using three substrates (moistened bark, dry bark, absorbent bench liner) sprayed with a low rate (9 microsclerotia granules/cm2) of hydromulch. Median survival times of beetles continuously exposed to sprayed moist bark or absorbent liner were 17.5 and 19.5 d, respectively. Beetles exposed to sprayed dry bark, which had a lower measured water activity, lived significantly longer. When moist bark pieces were sprayed with increased rates of microsclerotia granules in hydromulch, 50% died by 12.5 d at the highest application rate, significantly sooner than beetles exposed to lower application rates (16.5-17.5 d). To measure fecundity effects, hydromulch with or without microsclerotia was sprayed onto small logs and pairs of beetles were exposed for a 2-wk oviposition period in containers with 98 or 66% relative humidity. At 98% humidity, oviposition in the logs was highest for controls (18.3±1.4 viable offspring per female) versus 3.9±0.8 for beetles exposed to microsclerotia. At 66% humidity, fecundities of controls and beetles exposed to microsclerotia were not significantly different. This article presents the first evaluation of M. brunneum microsclerotia in hydromulch applied for control of an arboreal insect pest.

The effect of time postexposure and sex on the horizontal transmission of Metarhizium brunneum conidia between Asian longhorned beetle (Coleoptera: Cerambycidae) mates.

A study using Metarhizium brunneum Petch fungal bands designed to improve delivery of conidia to adult Asian longhorned beetles, Anoplophora glabripennis (Motschulsky), was conducted to determine how a time delay between exposure to infective conidia and pairing of male and female beetles would affect the ability to successfully transfer lethal doses of conidia to a mate. We measured conidial load at the time of mate pairing (0, 4, 24, 48 h postexposure) and assessed its effect on beetle mortality. Conidial load per beetle decreased across the four sampling times, and there was no effect of beetle sex on conidial load. At all time periods postexposure, beetles that climbed across fungal bands carried enough conidia that at least some of their indirectly exposed mates died of mycosis. For indirectly exposed beetles, mortality decreased significantly as the time delay increased from 0 to 48 h, and this was independent of beetle sex. Median survival time was only 11.5 d for females indirectly exposed immediately after their mate had been exposed, but >3 wk when there was a 48-h delay before pairing. Generally, beetles exposed directly to fungal bands died faster than their indirectly exposed mates. In contrast to the pattern seen for indirectly exposed beetles, beetles exposed directly to fungal bands showed no change in survival times with a delay between exposure and pairing. Median survival times of exposed females and males were generally similar, at 10.5-12.5 d.

The within-season and between-tree distribution of imidacloprid trunk-injected into Acer platanoides (Sapindales: Sapindaceae).

Norway maple trees, Acer platanoides L. (Sapindales: Sapindaceae), that were trunk-injected with imidacloprid as part of an Asian longhorned beetle eradication program, were used to study the temporal and between-tree distribution of imidacloprid in twigs from June through September. The effect of injection time during spring on imidacloprid residues across the summer season and the distribution of imidacloprid in twig bark versus twig xylem were also investigated. Overall, we observed a significant decline in imidacloprid concentrations within each plant part sampled across the study period, although the 19 trees used in the study varied greatly in the pattern of imidacloprid residues over time. The concentration of imidacloprid in twig bark per dry mass was approximately two times higher than that of the twig xylem (means +/- SD of 1.21 +/- 2.16 ppm vs. 0.63 +/- 1.08 ppm imidacloprid, respectively). The majority (> 50%) of whole twig, twig bark and twig xylem samples from injected trees contained < 1 ppm imidacloprid and 37% of twig samples contained 0 ppm. Maximum values were 9 ppm for whole twigs from trunk-injected trees, 12 ppm imidacloprid for twig bark, and 5 ppm for twig xylem. Leaves, sampled only in September, had much higher imidacloprid residues than twigs collected at the same time; the majority (53%) of leaf samples contained > 5 ppm imidacloprid, with a maximum of 49 ppm. The concentrations ofimidacloprid in whole twigs, twig bark, and twig xylem were highly correlated, and levels in leaves were correlated with imidacloprid levels in whole twigs.

Conidial acquisition and survivorship of adult Asian longhorned beetles exposed to flat versus shaggy agar fungal bands.

Fungal bands can deliver lethal conidial doses to adult Asian longhorned beetles. Because higher doses result in shorter survival times, developing a method to deliver more conidia to beetles walking across the fungal bands is desirable. We compared fungal bands made using standard flat material to bands made using a shaggy, textured material. The median survival time of adult beetles exposed to shaggy bands was reduced to 10 d versus 18 d for beetles exposed to flat bands. Beetles climbing across shaggy bands acquired 1.83×10(6) conidia per beetle, which was 14.6 times greater than beetles exposed to flat bands.

Comparing fungal band formulations for Asian longhorned beetle biological control.

Experiments were conducted with the fungal entomopathogen Metarhizium brunneum to determine the feasibility of using agar-based fungal bands versus two new types of oil-formulated fungal bands for Asian longhorned beetle management. We investigated conidial retention and survival on three types of bands attached to trees in New York and Pennsylvania: standard polyester fiber agar-based bands containing fungal cultures, and two types of bands made by soaking either polyester fiber or jute burlap with oil-conidia suspensions. Fungal band formulation did not affect the number or viability of conidia on bands over the 2-month test period, although percentage conidial viability decreased significantly with time for all band types. In a laboratory experiment testing the effect of the three band formulations on conidial acquisition and beetle survival, traditional agar-based fungal bands delivered the most conidia to adult beetles and killed higher percentages of beetles significantly faster (median survival time of 27d) than the two oil-formulated materials (36-37d). We also tested the effect of band formulation on conidial acquisition by adult beetles kept individually in cages with a single band for 24h, and significantly more conidia (3-7times) were acquired by beetles from agar-based bands compared to the two oil formulations.

Efficacy of imidacloprid, trunk-injected into Acer platanoides, for control of adult Asian longhorned beetles (Coleoptera: Cerambycidae).

Feeding experiments with Asian longhorned beetles (Anoplophora glabripennis (Motschulsky)) in a quarantine laboratory were used to assess the effectiveness of imidacloprid in reducing adult fecundity and survival. The beetles were fed twigs and leaves cut between June-September 2010 from Norway maples (Acer platanoides L.) in the beetle-infested area of Worcester, MA. Treated trees had been trunk-injected once with imidacloprid in spring 2010 under the U.S. Department of Agriculture-Animal and Plant Health Inspection Service operational eradication program. The 21 d LC50 value for adult beetles feeding on twig bark from imidacloprid-injected trees was 1.3 ppm. Adult reproductive output and survival were significantly reduced when beetles fed on twig bark or leaves from treated trees. However, results varied widely, with many twig samples having no detectable imidacloprid and little effect on the beetles. When twigs with > 1 ppm imidacloprid in the bark were fed to mated beetles, the number of larvae produced was reduced by 94% and median adult survival was reduced to 14 d. For twigs with < 1 ppm imidacloprid, 68% of reproductively mature mated beetles survived 21 d and 56% of unmated recently eclosed beetles survived 42 d. For twigs with < 1 ppm, beetles ingested an average of 30 nanograms of imidacloprid per day. Bark consumption was reduced at higher imidacloprid levels (> 1 ppm). When given a choice of control twigs and twigs from injected trees, beetles did not show a strong preference.

The effect of exposure to imidacloprid on Asian longhorned beetle (Coleoptera: Cerambycidae) survival and reproduction.

The effect of imidacloprid delivery method and application rate on survival of adult Asian longhorned beetles, Anoplophora glabripennis (Motschulsky), was studied, along with the effect of repeated daily ingestion of imidacloprid on the survival and reproductive capacity of adult females. Beetles exposed repeatedly to 50 ppm imidacloprid died in < 2-3 wk, whether dosed orally each day, or through contact exposure. Beetles given 1 microl of 50 ppm imidacloprid daily for two, three, four, or five consecutive days died sooner with increasing consecutive days: the beetles treated for 5 d all died within 15 d, while 80% of beetles treated for only 2 d lived > 8 wk. For females given 1 microl daily, across a range of doses from 2 to 50 ppm imidacloprid, the total number of viable eggs laid was reduced with increasing dosage, but percentage egg viability was not affected. Survival of females at dosages of 10 or 30 ppm/d was not significantly reduced compared with controls but these females laid 23-38% fewer viable eggs, suggesting a sublethal effect of imidacloprid. Female beetles given 1 microl/d of 40 or 50 ppm imidacloprid died more quickly than controls and viable egg production was reduced 82-93%, because of a combination of lethal and sublethal effects of intoxication.

Seed dispersal by white-tailed deer: implications for long-distance dispersal, invasion, and migration of plants in eastern North America.

For many plant species in eastern North America, short observed seed dispersal distances (ranging up to a few tens of meters) fail to explain rapid rates of invasion and migration. This discrepancy points to a substantial gap in our knowledge of the mechanisms by which seeds are dispersed long distances. We investigated the potential for white-tailed deer ( Odocoileus virginianus Zimm.), the dominant large herbivore in much of eastern North America, to disperse seeds via endozoochory. This is the first comprehensive study of seed dispersal by white-tailed deer, despite a vast body of research on other aspects of their ecology. More than 70 plant species germinated from deer feces collected over a 1-year period in central New York State, USA. Viable seeds included native and alien herbs, shrubs, and trees, including several invasive introduced species, from the full range of habitat types in the local flora. A mean of >30 seeds germinated per fecal pellet group, and seeds were dispersed during all months of the year. A wide variety of presumed dispersal modes were represented (endo- and exozoochory, wind, ballistic, ant, and unassisted). The majority were species with small-seeded fruits having no obvious adaptations for dispersal, underscoring the difficulty of inferring dispersal ability from diaspore morphology. Due to their broad diet, wide-ranging movements, and relatively long gut retention times, white-tailed deer have tremendous potential for effecting long-distance seed dispersal via ingestion and defecation. We conclude that white-tailed deer represent a significant and previously unappreciated vector of seed dispersal across the North American landscape, probably contributing an important long-distance component to the seed shadows of hundreds of plant species, and providing a mechanism to help explain rapid rates of plant migration.