2005 Annual Report 4d.Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Cornell University, Ithaca, New York, USA. Additional details can be found in the report for the parent CRIS 1926-22000-019-00D, Invasion biology of invasive species: Biocontrol and related technologies for exotic insect pests, with emphasis on Asian Longhorned Beetle. The project objective is to develop methods for optimizing control of Asian Longhorned beetle using entomopathogenic fungi, especially focusing on the use of non-woven fiber bands for fungal delivery. 1.4.3. Fungal bands - FIELD STUDIES: During the 2005 field season we finally conducted a study including a treatment of fungal bands plus attractants in a location in China with a good beetle population. Studies were conducted in a planting of poplar trees (Populus simonii x Populus canadensis) in Friendship Village, northeast of Linhe (N 40o 43-44 x E107o 17-18; altitude = 1046 m), Inner Mongolia, People's Republic of China. Fungal bands were produced by the Anhui Agricultural University, who collaborated throughout this project. The fungal isolate used was Metarhizium anisopliae F-52, which has been registered with the EPA for use for control of ticks and lawn grubs in the US. The attractant that was used had two parts: a contact pheromone supplied by Dr. Aijun Zhang, USDA, ARS and a host plant attractant made by placing bark from twigs of Acer mono in dimethyl sulfoxide. Five plots of ca. 75-125 trees each (8 m wide containing 3 rows of trees x 100 m long) were created for each of three treatments: fungal bands alone, fungal bands plus attractant and controls with no fungal bands. The trees had been planted approximately 17-18 years before. Plots were separated from each other by 70 m of unbanded trees, which were not included in these studies and acted as a buffer zone between treatments. Bands were hung on 48 trees per plot in early-mid July. These trees were not very similar to trees in the northeastern U.S., because on many trees the canopy was not wide, foliage was very sparse and tops were dead. However, these were the trees available with a pre-existing ALB population. Before bands were hung and every 5 days afterward, up to 7 beetles were collected from each plot. More beetles than this were not collected because we did not want to deplete the population so that we would not have adults to collect over time. At each sampling date, the small bags with host plant extract were also refilled. Beetles were reared at constant temperature (25 C) for up to 70 days after collection and date of death was recorded as well as whether M. anisopliae subsequently grew out of cadavers. To evaluate the effect of fungal bands on oviposition and survival of eggs, in early August, 24 trees were cut down. All beetle exit holes were recorded in each tree but more importantly, new ovipositions were recorded and resulting eggs were maintained on wet filter paper under constant temperature to determine whether they hatched or not. For eggs that did not hatch, outgrowth of M. anisopliae was recorded. As background data, the density of adult beetles was recorded once every 5 days to document phenology and relative densities of ALB in different plots. These data were also used to assist when adults were collected. The experiment was terminated in mid-August. Some preliminary data was taken to study environmental contamination by this fungus from inoculated adults. Bark samples were taken after an inoculated adult was exposed to a fungal band and allowed to walk on tree trunks. Spore densities on bark samples were quantified using dodine plates at different dilutions from the washes from bark samples. At this time, the data from all studies have not yet been totally collected and have not been analyzed although computerization of some of the data will begin shortly. The data that were collected will be part of the M.S. thesis of a Cornell University graduate student who was part of the team collecting the data in Linhe. When this study was planned it was very difficult to find a location in a region that received summer rainfall similar to the northeastern U.S., but also provided a robust population of ALB, so that enough beetles would be present for this study. This is a difficulty in conducting such studies because naturally occurring beetle populations in China must be used. After extensive searching by both Drs. Smith and Li, the best study site that could be found was in a semi-desert region, where rainfall was questionable during the period of the study. The weather in the 2005 study area is not similar to normal weather in northeastern North America. While bands were hanging during this study, some rain fell occasionally but for the first two weeks after bands were hung, it was exceptionally hot and dry. Whether these weather conditions will affect the activity of fungal bands is not known although one could assume that this might have a negative impact. However, at present, it is too early to know the results from this study. LABORATORY STUDIES: (1) Fecundity studies: We completed the third replicate of a study of the effects of maternal exposure to M. anisopliae on (a) mates; (b) egg production; (c) egg hatch and infection; and (d) larval survival and infection. We have terrific data and a paper has been started; (2) Dose response: These studies have only begun. First priority over the past year was to complete the Fecundity studies so adult beetles were committed to that study first. Once we had adult beetles, we did not have good fungal bands and making bands took a few tries. Now, has offered fungal bands for out studies and this seems far preferable, to use bands made in the same facility where they have been produced for purchase by APHIS. Then, a method for quantifying the number of viable conidia had to be learned by the technician helping with this project. Thus far, we conducted one dose response replicate but the results are not very consistent with previous studies and, before continuing, we are going to conduct a complete review of the experimental procedures to understand why the results from this bioassay look so different from previous data.