Brief Summary of Minutes of Annual Meeting

Meeting called to order 9:00 - David Shapiro (Project Chair) S_Temp2582 is the interim project number that gives us 2 years to submit the proposal. David had forwarded an email originating from Bob Nowierski about grant opportunities. Patricia Stock - Local arrangements 24 paid and invited participants Information is available in the handouts about restaurants and maps to get around Tucson. Dawn Gouge is handling registration - fee = $65. A social gathering will be held at Gentle Bens Restaurant tonight at about 6:30. A sign-up sheet is passing around to identify the participants. Administrators Report: John Russin (Interim Vice-Chancellor and Director, LSU)) The revision has a temporary number that will allow this organization to continue. NIFA proposals: Many RFP releases with a broader umbrella for topics but with no additional money. There is a move in congress to remove congressionally mandated earmarks that will affect many USDA and university projects. Another proposal will push funding back to lower levels. Introductions by attendees: Patricia Stock - Entomology at University of Arizona (U of A), studying entomopathogenic nematodes (EPNs). Studying relationships and symbiotic organisms (Photorhabdus metabolites for pharmacological applications) and as model systems for interactions between pro and eukaryotes. Rousel Orozco - U of A, Anti -microbial compounds from EPN with Patricia Stock. Patricia Navarro - activity against Fusarium. Brian Federici - University of California, studying recombinant bacteria to kill mosquitoes. Lee Solter - University of Illinois, studying microsporidia of anything, classical control of gypsy moth and Diaprepes, and newer research with bumble bees. Lisa Chanbusarakum - Marrone Bio Innovations, testing microbes from various sources for insecticidal activity. Robin Stuart - University of Florida, Nematode lab for Diaprepes and working with food web interactions, and molecular ecology. Lerry Lacey - ARS Retired now IP International, working to control insect pests. Julie Graesch - Becker Underwood - working with researchers and sales to foster biological control products. Andrew Brown - Becker Underwood (UK), Oversees field trials and academic links. Nemat Keyhani - University of Florida, B. bassiana as a model system for molecular interactions and biorational approach to determine more suitable strains. Also studies Metarhizium. Jeremy Pierce - Becker Underwood (UK), down-stream processing and formulation. Drion Boucias - University of Florida, Studying a group of viruses that affect filth flies, basic biology studies. Also provides diagnostics at the university, no current applied projects. Ellie Groden - University of Maine, Studying pathogens of invasive ants including fungi and EPNs. Dawn Gouge - U of A, Urban entomologist consumed by consumer interest/concerns about bed bugs. Jarrod Leland - Novozyme, Metarhizium products (Met52), and Turfal is a company acquired to market products in Brazil. Novozyme is active in world enzyme market. Mark Boetel - North Dakota State University, Collaborates with Stefan Jaronski (not in attendance) and Bob Behle mostly on sugar beet root maggot, a pest of sugar beets in the red river valley. Dana Blackburn - Brigham Young University, Studying trait changes in EPNs. John Russin - Louisiana State University Ag Center, project advisor. E. Erin Morris - Cornell University, studying symbiosis of fungus with the Sirex woodwasp. Tatyana Stefanovsia - National University of Life and Environmental Science, Ukraine, microbiological agents in orchards and collaborates with UC Davis, Ed Lewis and Harry Kaya (not in attendance), and studying control of black vine weevil. Ed Lewis - University of California Davis, Studying the fate of EPNs in the field and when used in the nursery industry to apply basic knowledge to develop soil recipes. Jorge Pitton - Becker Underwood, Argentina, product manager for natural products to introduce biological product lines for Latin America (coordinates between research and field). Juan Diego Petri - Becker Underwood, Argentina Bruce Tabashnik - U of A (department head, guest speaker) Has post-doc positions with funding. $37,000 + benefits). Noha Abu Shadi (student with Patricia Stock, from Egypt) EPNs from Egypt David Shapiro-Ilan - ARS, Research on Pecan and Peach pest control specifically with EPNs and other microbes. The annual report not available yet because not all section reports have been submitted. The report will be circulated via e-mail when complete for review and approval. SEMINAR - Bruce Tabashnik on BT Resistance by insect pests (10:00 to 11:00 am) diamondback moth expressed resistance to Bt sprays and now have reports of resistance to GMO crops. However, thus far, proper implementation of refuges has prevented the development of resistance. Most of the presentation was directed at the pink bollworm program in Arizona. Announcements: Propose consideration of Florida, or Oregon, or other location for the 2012 meeting. Bacteria/Nematode- Symbiosis: Meeting before the Society of Nematologists meeting which is scheduled for July 17-21, 2011 in Corvallis, Oregon. Sub-Project 1 - Large Acreage Crops Bob Behle - Working with formulations of fungal agents produced in vitro. Storage of microsclerotia at room temperature has remained viable for liquid and granule formulations for over 8 weeks. Granule formulations may be used for a variety of pest and crop situations. Mark Boetel Met52 (Metarhizium brunneum) on corn meal granules to control sugar beet root maggot (SBRM) on sugar beets at NDSU. Fungal granules must compete against OP insecticides. The pest may cause $200/A in crop losses. Compared planting time granule applications. Showed significant yield increases by an integrated control program with seed treatments and Ma application. The experiment was conducted under moderate pest infestation levels. Fusarium solanii is still on research radar as a pathogen of SBRM. Need to check for pathogenicity of other plants associated with the typical sugar beet system. The SBRM is difficult to work with in the lab, can store larvae for up to 7 years in cold storage. Would like to find someone to work on basic research to maintain laboratory colonies. Becker Underwood may have trouble getting nematodes to the field in sufficient quantity for large crops, but improvements are being made in production. Treatments with nematodes typically focus on high value crops. Nematodes require refrigeration. (using nematodes for palm weevil in Europe) Nemat Keyhani - Beauveria research focuses on basic science with a background in genetics and molecular biology. Enzymes of Bb are important for infection of insects by Bb. The waxy layer on the cuticle is the first barrier to get through. Have identified lipases and cyt P450s with substrates including long chain fatty acids. Making (single and double) gene knock-outs showed a small phenotype change and suggest the lack of differences may be the result of redundant systems. Two approaches for applied virulence by expressing genes to increase toxicity to targeted hosts. First, focusing on host molecules as target of virulence, e.g., host hormones by disrupting hormone systems during infection process. Second, trying to develop a more environmentally tolerant fungal strain. Lerry Lacey - Potatoes - Grower became interested in granular virus for potato tuber moth (PTM). Infestations in storage are a separate problem. Producers want large potatoes for French fries. Wireworms (that can feed on potatoes) have a 2 to 3 year life cycle, but are attracted to bait balls with Met52. Zebra stripe in potatoes caused by the potato psyllid in Texas is a newer project and may be controlled using Isaria and Met52 and Lecanicillium. Sub-Project 2 - Trees, orchards, vegetables fruit and nuts. Robin Stuart - Nematology lab focusing on citrus. Most important citrus pest is currently citrus psyllid, which vectors the bacteria that causes citrus greening. Nematode labs are struggling to get funding. Raquel Campos Herrera developed quantitative PCR techniques to determine field populations of nematodes. Soils vary across Florida with central ridge and flatwoods. Flatwoods do not drain well. H. indica tends to be better in flatwoods or have higher densities than in the central ridge soils. There are differential susceptibilities of EPNs to nematophagous fungi. Ed Lewis - Research on citrus California is similar to Florida, focused on Diaprepes until the citrus psyllid was discovered. As yet, the greening has not been found in California. By sampling for nematodes (under pistachio trees), we found the earwigs to be susceptible to EPNs (Sc). Increases in predatory mites result from their feeding on the applied nematodes. Dawn Gouge questions about a bug Euschistus servus with nematodes in the nut trees. Dion Boucias - Psyllids, while looking for natural disease have identified Hirsutella citriformis as the most common pathogen, but with different phenotypes among the geographic regions sampled. Lee Solter - Continuing to work on microsporidia isolated from Diaprepes and having problems fitting it into genealogies. Dana Blackburn - Studying trait deterioration in EPNs and the impact of in vitro nutrition. Jarrod Leland - granule formulation of Met52 is being produced for nurseries for BVW, mites, and thrips in the European markets along with ticks. Trying to focus on these major pests for control. Patricia Stock - Working on H. sonorensis (EPNs) for vegetables to determine host ranges and results are encouraging because it (H. sonorensis) can tolerate low water and high temperatures while still causing pest mortality. (Roselle looking at new bacteria subspecies of P. temperata). Collaborating with Bruce Tabashnik to determine relationships of EPNs with Bt resistant pests. EPNs are more infective for Bt resistant insects indicating a fitness cost for insects with Bt resistance. For trophic level impacts, compared nutrients fed to insects for impact on EPNs. (Victoria Miranda) How does a current infection affect subsequent EPN infection attempts? Ming looked at co-evolution of nematodes with symbionts. Also have determined morphology of the nematode to be associated with bacterial association. David Shapiro-Ilan - Working toward IPM for Plum Curculio control by using improved pheromone to attract adults and then use nematodes to clean up larvae that drop with fruit (in collaboration with Tracy Leskey et al). For pecan weevil, fungi and nematodes must withstand fungicide applications for plant pathogens. Certain Bb strains have been identified to have fungicide resistance. Nematode applications were synergistic with insecticides (Carbaryl and Cypermethrin), and Bt applications were synergistic with Carbaryl and antagonistic with Cypermethrin. Strains of Beauveria and Metarhizium are being evaluated for resistance to fungicides and have observed enhanced resistance, but with trade-offs. For peaches, fire retardant chemical retains water and reduces dehydration of nematodes when applied to trees for control of peach tree borers. Sub-culturing of nematodes results in fitness decline with added culturing. Inbred lines decline less. Indian mealmoth control for stored pests, the nematodes will consume both parasitized and healthy hosts, preferring parasitized hosts. Trait characterization and mining for active chemistries are being explored. Sub-Project 3 - Natural Landscapes Ed Lewis - Model demonstrated the temporal pattern for infection of hosts is related to investment of stock on Wall Street such that the first to infect a host embarks on a risky situation but with potential of greater rewards. Selection for risk takers would show a genetic component. EPNs survive well until the lipid content decreases. The lipid is determined by a magnified photo to determine optical density. Dawn Gouge - interested in predators e.g., scorpions and black widow. Determine response of scorpions with nematodes (S. riobrave) and insecticides. Scorpions have structures to determine vibrations and movement and can determine the presence of nematodes, then begin defense behaviors. For widow spiders, nematodes have no effect. For infected prey, they do not consume the cadaver. Lee Solter - Two species of microsporidia have been released in 10 acre forest blocks to augment the natural enemy complex of gypsy moth. Mortality was high in treatments sites due to E. maimaiga, which may be out-competing the microsporidia. In a companion study in Bulgaria, microsporidia persisted for one year after release. A three-year national survey was completed to determine pathogen prevalence in bumble bees. Dominant pathogens were the microsporidium Nosema bombi and the trypanosome Crithidia bombi. SSU rDNA gene sequences have not determined if any of the isolates are invaders from Europe. Honey bees are being evaluated to address the interactions of microsporidia, viruses and temperature differences and their effects on bee health. Becker Underwood - Developing new technology for controlling slugs using EPN in a gel bait to attract the slugs and the EPNs will reproduce in the gel. Erin Morris - Studying Sirex wood wasp, a pest of pine. Wood wasp injects a fungus into the tree. A nematode has been found in the southern hemisphere where it is active against the wasps, but there are many mitigating factors in the north (alternative nematode hosts that are not pest, etc.). Some unidentified nematodes have been found in Sirex. Looking to do wasp behavior work relative to the presence of choice fungal strains. Drion Boucias - Studying chinch bug on St. Augustine grass to determine why the bug is resistant to most fungal pathogens and insecticides. Resistance appears to be a result of bacterial symbionts. Sub-Project 4 - Structural and Medical Brian Federici - Studying control of mosquito and black flies mainly using bacteria, more specifically by engineering Bacillus toxins. There is some engineering of Metarhizium for control of Dipteran pests . Some efforts directed at trying to kill or shorten the life span of adult mosquitoes. NIH renewal to look at the fitness costs of resistant insects. To be sensitive to the binary toxin, the insect needs to have the receptor to the binary toxin. The site A protein will effectively significantly delay resistance development. Also looking at some viruses, but believes viruses cannot compete with Bti. Bti is effective at about 0.25 lb per acre and at a cost of less than $1 fermentation ingredients. Work continues to reduce this cost by increasing production. For viruses, problems include the absence of an effective production system and the lab infection is poor at about 50%. Nemat Keyhani - He has strains with activity against adult mosquitoes and has a Bb strain that has the ability to produce an anti-trypanosomes enzyme. Drion Boucias - Continuing basic research of the house fly virus, which causes sterility of infected flies. The taxonomy has been accepted. Populations (world-wide) have the virus although some local populations do not have the virus. To infect a fly, oral ingestion yields 5 to 50% infection. The peritrophic membrane (PM) appears to be the major barrier that limits infection. Reducing agents used to disrupt the PM improves infection rates by the virus. Sprayed flies have higher rates of infection. Ellie Groden - Studying the invasive European ruby ant, a serious problem in Halifax and Newfoundland and some populations outside of Seattle, on the west coast. Comparing pathogens between native and invasive ranges. Entomophilic nematodes found for two years (are not Steinernema). Did not find nematodes in samples from England. Ants were not repelled by Beauveria conidia, so fungi were used with bait stations, which reduced density of ants in about 8 weeks. In the lab, there was no significant reduction in the size of ant colonies. Also mentioned David Ois work on fire ants in the written report. Dawn Gouge - Evaluating pesticide treatments for control of bed bugs, and using S. carpocapsae as a crack and crevice treatments provided poor control of bed bugs. Public surveys indicated an aversion to applying nematodes in homes. Jarrod Leland - Novozymes Metarhizium formulation is active against bed bugs and commercially available for control of ticks. David Oi - (not present) Submitted a written report on their work on Solenopsis to determine pathogens in both native and invasive ranges. Finding fewer pathogens in newly invaded areas. Variations among pathogens are being evaluated using DNA methods. Seminar by Patricia Stock. Use of molecular biology relative to insect pathogens Project Renewal Discussion - Led by David Shapiro-Ilan The project re-write is on track for timely completion. Generally, the text will be broad with med/vet and include pathogens of beneficial insects. People will have the opportunity to comment on the full proposal. The pre-proposal has been approved. Joint proposals. Related to biofuel crops (Ed Lewis), some insects are being recognized as potential problems. There is a diversity of crops, production systems and pests to select from. Proposals should go beyond pathogen surveys. It was suggested to focus on the importance of pests and to look at these environments as a haven for beneficial organisms. Check for specialty crop funds that are distributed to states (David Shapiro-Ilan). There may be funding to target specific formulation application considerations and persistence of beneficial pathogens (may include Stefan Jaronski). Potential outreach projects: Techniques Book revision was suggested? Web-based diagnostic (may be connected to SIP) as an ask the expert web site to identify diseased insects, Drion will look into potential for support by his IT department. For establishing a depository for nematode strains (Patricia Stock). Depositories for fungi and bacteria are largely covered. We may court a corporate sponsor or establish the service via University support. Site selection for 2012 meeting Nominations include Florida (Sanibel Island?), Oregon and Phoenix. Votes: Florida 11, Phoenix 1, Oregon 1 Theme: Biofuel pest management strategies proposed by Drion Boucias. Ed Lewis may help Steven Arthurs (not in attendance) with the program concerning the biofuel topic. Adjourn.

SUBPROJECT 1: Discovery of entomopathogens and their integration and safety in pest management programs for major acreage crops. [Co-Chairs: Mark Boetel and Bob Behle] Four research reports were received for Subproject 1, which addresses entomopathogen research on large-acreage crops (i.e., sugarbeet and potato). Results reported for Subproject 1 and involved the cooperation of four ARS locations (Peoria, IL; Sidney, MT; Weslaco, TX; Yakima, WA) and two universities (North Dakota State University; Texas A&M University). This work involved assessments of efficacy at controlling sugar beet root maggot, potato psyllid, and wireworms with Metarhizium anisopliae granules, control of potato tuber moth with a granulosis virus, and production by liquid fermentation and formulation of Metarhizium anisopliae microsclerotia. Sugarbeet The sugarbeet root maggot (SBRM), Tetanops myopaeformis (Röder), is a major economic insect pest of sugarbeet in the Red River Valley of North Dakota and Minnesota. It also is an annual threat to sugarbeet production in several western states and the Canadian province of Alberta. A field trial was conducted to evaluate a granular formulation containing Metarhizium anisopliae (MA1200) conidia for control of the SBRM. The site chosen for this experiment (Minto, ND) was selected a moderate SBRM infestation was expected in its vicinity, and a key objective of the experiment was to test the feasibility of integrated SBRM control programs under moderate SBRM pressure. MA1200 granules were tested using two application rates (i.e., 22.5 or 33.6 kg formulated product/ha), and four placement methods (i.e., band, in-furrow, modified in-furrow, and spoon). Additionally, integrated control programs, comprised of MA1200 granules combined with one of the following chemical seed treatment insecticides were also evaluated: 1) Poncho Beta (i.e., clothianidin + betacyfluthrin at 60 and 8 g a.i./100,000-seed unit) and Cruiser (i.e., thiamethoxam at 60 g a.i./seed unit). Significant reductions in SBRM root feeding injury were provided by the following alternative control tools when compared to untreated controls: 1) MA1200 granules, applied at 22.5 kg/ha using spoon placement; 2) Cruiser 5FS seed treatment; and 3) the integrated program consisting of Poncho Beta plus MA1200 granules. Combining Poncho Beta seed treatment with MA1200 granules appeared to have a slight additive benefit in root protection when compared to single applications of these tools; however, the difference was not statistically significant. Yield comparisons revealed significant increases in recoverable sucrose by using the following alternative treatments for SBRM control: 1) Cruiser 5FS seed treatment; 2) Poncho Beta seed treatment + MA1200 granules; and 3) Cruiser 5FS + MA1200 granules). All of these treatments, in addition to the stand-alone entry of Poncho Beta, also produced significant root tonnage increases over that of the untreated check. Combining MA1200 granules with Poncho Beta seed treatment produced an increase in gross revenue of $191 per acre when compared to plots protected solely by Poncho Beta seed treatment. Combining MA1200 granules with Cruiser seed treatment did not provide a significant increase in root protection, yield, or gross revenue when this integrated strategy was compared with either of its component control tools applied separately. (North Dakota State University, Fargo, ND and USDA-ARS, Sidney, MT) Potato Research was concluded on the larvicidal activity of the potato tubermoth granulovirus under field and storage conditions for control of potato tuber moth (PTM), Phthorimaea operculella, (Zeller). In storage, the virus killed a significant portion of larvae within tubers and prevented spread of PTM larvae from infested tubers to un-infested tubers. In Weslaco Texas, our team evaluated commercial products of Isaria fumosorosea and Metarhizium anisopliae for control of the potato psyllid [Paratrioza cockerelli (Sulc)], the vector of zebra chip disease in chipping potatoes. These products produced up to 77% mortality in psyllid nymphs, and significantly reduced plant damage. Research was concluded on the use of M. anisopliae in bait balls for control of wireworm pests of potato tubers in the field. The fungus killed a substantial portion of the larvae retrieved from bait balls. (USDA-ARS, Yakima, WA; USDA-ARS, Weslaco, TX; Texas A&M Agrilife Research and Extension Center, Weslaco, TX) Fermentation Production Microscleortial production has now been documented for Metarhizium species anisopliae, brunneum, robertsii, and pingshaense; M. acridum has not yet been tested. A microsclerotia is a melanized hyphal structure that is well suited for soil application where it can produce conidia to infect the target pest. Production of microsclerotia has been successfully scaled up to 100-Liter batch size. Shelf life of vacuum-packed granules is at least one year. Technology licensing agreements are being signed by two companies. (USDA-ARS, Peoria IL and USDA-ARS, Sidney MT) Formulation Additional formulations containing Metarhizium anisopliae microsclerotia are under evaluation for storage stability and conidia production. Both liquid and granular formulations are being tested that will allow for a wider variety of target pests and application environments. Fermentation broth, as a liquid formulation, has maintained viability for over 4 months when stored under refrigeration and produced > 5 x 108 conidia per ml of broth. Clay-based granules containing microsclerotia have remained viable for over 2 months when stored at room temperature and the formulations are capable of producing 5 x 109 conidia per gram of granules on water agar and 3.5 x 109 conidia when applied to potting soil. (USDA-ARS, Peoria IL) SUBPROJECT 2: Discovery of entomopathogens and their integration and safety in pest management programs of ornamental, vegetable, fruit and nut crops. [Co-Chairs: Robin Stuart and Patricia Stock] Reporting: Duncan, Lacey, Legrand, Shapiro-Ilan & Lewis. A number of successful efficacy trials indicate substantial potential for microbial control agents in peach and pecan orchards. In a pecan orchard, multiple applications of S. carpocapsae made to the pecan weevil, Curculio caryae, during the insects 2-yr life-cycle in the soil, reduced the pests survival to less than 1% (81% control relative to the untreated plots) [USDA-ARS, Byron, GA]. In a peach orchard, a two-year study indicated that a sprayable gel formulation (Barricade®) applied as a post-application covering to Steinernema carpocapsae, significantly enhanced control of the lesser peachtree borer, Synanthedon pictipes compared with nematodes applied alone; the combination resulted in 0 to 30% S. pictipes survival. The sprayable gel (normally used for fire protection) may have broad applicability in improving aboveground applications of entomopathogenic nematodes [USDA-ARS Byron, GA; University of Florida; University of Georgia]. A new method of formulating and packaging entomopathogenic nematode-infected cadavers was developed. The infected hosts (e.g., Tenebrio molitor) are automatically wrapped in tape using a specialized packaging machine. The tape formulation protects the cadavers from rupturing and provides ease-of-handling. The formulated cadavers provided high levels of control versus the small hive beetle Aethina tumida, and the Diaprepes root weevil, Diaprepes abbreviatus [USDA-ARS, Byron, GA; USDA-ARS Stoneville, MS; Southeastern Insectaries, Inc.]. The biofumigant fungus, Muscodor albus, was efficacious for control of codling moth (CM) larvae and adults in and on stored apples [USDA-ARS, Yakima, WA]. The fungus also produced deleterious effects in the western cherry fruit fly. The potato tubermoth (PTM) granulovirus was shown to effectively control PTM in stored tubers by killing larvae in infested tubers and limiting their dispersal in refrigerated warehouse storage [USDA-ARS, Yakima, WA]. Preliminary research on the insecticidal activity of entomopathogenic fungi for control of potato psyllid, Bactericera cockerelli, demonstrated sufficient insecticidal activity to encourage field trials for 2010 [USDA-ARS, Yakima, WA]. The research included development of bioassay techniques, effect of fungal species and stage of the psyllid on insecticidal activity. Infective juveniles of Steinernema feltiae and S. carpocapsae formulated in wood flour foam or fire retardant gel were more effective for control of overwintering CM than aqueous suspensions of IJs increasing the utility of nematodes by orchardists [USDA-ARS, Yakima, WA]. Laboratory experiments tested the pathogenicity of Metarhizium anisopliae on adult Japanese beetles and adult Oriental beetles. The F52 strain was obtained from Novozymes Biologicals, Inc., Virginia. The results indicated pathogenic activity against Japanese beetle adults. Percent mortality of Japanese beetles at 11 days after exposure was 67, 81, 47, 86 and 83% for the 0.5, 1, 2.5, 5, and 10mg/100 beetles dose respectively. Oriental beetles did not exhibit similar results but mycosis was confirmed in a few Oriental beetle specimens [University of Connecticut]. Laboratory experiments were conducted in sand columns and on water agar in Petri dishes to study the attraction or repellency of three species of nematophagous fungi (NF; Gamsylella gephyropaga, Myzocytium sp. and Catenaria sp.) to five species of entomopathogenic nematodes (EPNs; Steinernema diaprepesi, Steinernema sp. glaserigroup, S. riobrave, Heterorhabditis zealandica and H. indica). EPN species were generally repelled by the trapping NF G. gephyropaga in sand, whereas on agar only S. riobrave was repelled while S. diaprepesi, Steinernema sp. glaserigroup, H. zealandica and H. indica were attracted to the fungus. With the exception of S. riobrave, the endoparasitic NF Myzocytium sp. consistently attracted EPN in sand and on agar. A second endoparasite, Catenaria sp., also tended to attract EPN in either medium. There was no evidence that EPN species which differ in susceptibility to a given fungal species also differ in whether they are attracted or repelled by the fungus. Furthermore, the different results in sand column versus agar plate assays indicate the potential importance of conducting such assays under the most naturalistic conditions possible [University of Florida]. Quantitative realtime PCR (qPCR) assays were developed to detect and quantify 6 species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, S. riobrave, Heterorhabditis indica, H. zealandica, H. floridensis, and an undescribed species in the S. glaserigroup) and an exotic species, S. glaseri. A survey of an 8ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae and to data obtained from the same site in 20002001 by baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The spatial patterns of EPN in the orchard were remarkably stable over nearly a decade. The qPCR assay was more effective than the Galleria baiting method for detecting EPN species composition in population mixtures and offers great potential for studying the ecology of EPN [University of Florida]. Greenhouse experiments were conducted to measure the persistence and efficacy of three species of entomopathogenic nematodes (EPN) against the citrus root weevil, Diaprepes abbreviatus, in soils of different textures. Analysis of plant root and top weights showed highly significant main effects of soil and nematode species, and a highly significant interaction between soil type and nematode species. Soil porosity was inversely related to plant damage by weevils, whether or not EPN were present; and porosity was directly related to plant protection by EPN. Steinernema riobrave afforded protection to citrus root systems in all 3 soils and the plants often had significantly higher root weights than other EPN treatments. Root weights were greater than positive controls in pots treated with S. diaprepesi in coarse and fine sand but not in sandy loam; and Heterorhabditis indica protected root systems significantly only in coarse sand. A second experiment produced similar results except that plant protection by S. diaprepesi was greater than that by S. riobrave. The results of these experiments confirmed the hypothesis that EPN would provide greater protection of seedlings against DRW in coarse textured soil than in soils of finer texture, a pattern previously observed in nature but the mechanism for this interaction was unresolved because there was no evidence that EPN killed weevils more effectively in any soil type. Rather, less vigorous growth of the negative control seedlings in the two finer textured soils suggests that unidentified factors stressing seedlings in those soils also impaired the ability of seedlings to tolerate weevil herbivory [University of Florida]. Tests of entomopathogenic nematodes (EPNs) on the European earwig suggest that the nematodes may be an effective biological control agent for this invasive arthropod pest (Univ CA, Davis). In the laboratory, the LC50 for F. auricularia was 226 S. carpocapsae /earwig and the reproductive potential was 123.54 infective juvenile nematodes/mg tissue. In a study of host recognition behavior, S. carpocapsae infective juveniles responded to earwig cuticle as strongly as they did to Galleria mellonella cuticle. We also found that earwigs exposed to S. carpocapsae cleaned and scratched their front, middle and back legs significantly more than controls (P<0.001). Coupled with previous field data, these findings lead us to suggest that F. auricularia may be a natural host for S. carpocapsae. The influence of insect cadaver desiccation on the virulence and production of insect cadavers infected with EPNs was investigated for common natural enemies of many soil-dwelling insects (Univ CA, Davis). Desiccation studies were conducted using the factitious host, Galleria mellonella and three EPN species (Heterorhabditis bacteriophora, Steinernema carpocapsae and S. riobrave). We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. From EPN-infected insect cadavers, we observed differences in the desiccation rate among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than -2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. Additionally a specific objective was to survey for the presence of native EPNs in California citrus groves and identify soil parameters that would allow us to predict EPN efficacy (Univ CA, Davis). We collected soils from 36 sites in California where citrus is produced commercially, sampled them for native nematode presence and tested the foraging efficacy (movement and host finding) of S. riobrave in these soils at three soil depths. Soil textures and physical characteristics were analyzed. We found five isolates of endemic EPNs (Heterorhabditis spp.) from southern California. Three were identified as Heterorhabditis bacteriophora, and two as Heterorhabditis sonorensis. S. riobraves ability to infect hosts varied among soil types and depths. We also found significant correlations between soil characteristics (texture and physical characteristics) and S. riobrave foraging efficacy. Our results and analyses can play a role in the prediction of S. riobrave success in various soil types. Perhaps more importantly, we suggest an approach to predict biological control potential based on soil characters that might be used with other insect pathogens. While soil texture is the main factor that affects EPN efficacy, we found that other parameters like organic matter level and dissolved solids are also important. SUBPROJECT 3 Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural landscapes. [Chair: Ed Lewis] Tree Defoliators Gypsy moth/Entomophaga maimaiga: Long-term studies were conducted investigating the activity of the gypsy moth fungal pathogen Entomophaga maimaiga in central New York State (2010 was the 19th year of sampling). Improvements to methods for counting E. maimaiga resting spores in soil were developed. Temperature and moisture levels associated with E. maimaiga resting spore germination were investigated during field studies. (Cornell) Monitoring of coleopteran colonies (Sasajiscymnus tsugae, Scymnus ningshanensis, S. sinuanodulus, Laricobius nigrinus, and L. osakensis) being reared for biological control of Adelges tsugae (hemlock woolly adelgid) continues. It appears that three species of microsporidia infect S. tsugae, one of which is being described. Field collections where beetles have been released will determine whether the microsporidia have infected other species predatory on A. tsugae. (Univ. of IL, INHS; USDA-FS-Morgantown; Virginia Polytech. State Univ.; Univ. Tenn.; Univ. Kentucky; CT Ag. Expt. Stn.;Univ. Georgia; North Georgia College and State Univ.) A 2008 field release of two species of microsporidia, Vairimorpha disparis and Nosema lymantriae, was followed in 2010 with a second release in gypsy moth, Lymantria dispar, populations in two sites in Northern Illinois. Entomophaga maimaiga, a fungal pathogen, killed most of the late stage L. dispar larvae in 2008 and 2009, and was again present in 2010. A late stage microsporidia-infected larva was recovered in one site in 2010. Final monitoring for establishment of the microsporidia will take place in June 2011. (Univ. of IL, INHS; USDA FS, Hamden; Illinois DNR and DOA). Borers Asian longhorned beetle/Metarhizium brunneum (formerly M. anisopliae): We conducted field studies in the Asian longhorned beetle-infested area in Worcester, Massachusetts with fungal bands and attractant volatiles. We also investigated a new band formulation, developed by collaborator Nina Jenkins (Penn State). A graduate student is investigating whether adult Asian longhorned beetles regulate their temperature in response to fungal infection, using our quarantine colony of beetles. An undergraduate is testing whether Asian longhorned beetle adults are attracted or repelled by fungal bands and whether this effect is influenced by a volatile + pheromone combination that is known to attract Asian longhorned beetle adults. (Cornell) Sirex noctilio/Deladenus siricidicola: Growth of this nematode, used for biological control of Sirex noctilio where it has been introduced throughout the Southern Hemisphere, is being evaluated on different strains of the white rot fungus (Amylostereum areolatum) eaten by the mycophagous form; we are investigating strains of this fungus that we have isolated from North America. We have been collecting parasitic nematodes from native siricids and are developing a molecular method for differentiating between D. siricidicola that is mass produced for control of S. noctilio and other strains and species of nematodes parasitizing species of Sirex in North America. (Cornell, University of Arizona) Entomopathogens of agriculturally important beneficial insects A nation-wide evaluation of Bombus spp. populations for presence of the microsporidium Nosema bombi was completed in 2010. Bombus pensylvanicus was the most frequently infected host species in the Midwest and B. occidentalis and B. mixtus were most frequently infected in the West. A consistently recovered single nucleotide polymorphic allele, most commonly recovered from N. bombi infecting B. pensylvanicus but also recovered from other Bombus species, suggests that a native strain of N. bombi is present in US bumble bee populations. Bombus populations were also evaluated for the presence of the protozoan, Crithidia bombi; distribution of the pathogen does not suggest introduction of an exotic species (Univ. of IL, INHS; USDA-ARS-Logan, UT) Combinations of viruses and microsporidia under different temperature regimes were investigated for their role in decline of the honey bee, Apis mellifera. Results are currently being analyzed. Evaluation of the progression of Nosema apis and Nosema ceranae disease in individual host insects showed that infection only occurs in midgut tissues. (Univ. of IL, INHS; Pennsylvania State Univ.; Michigan State Univ.; USDA, Weslaco; Kentucky State University; USDA ARS, Beltsville, MD) Range Insects Metarhizium brunneum (F52) and M. robertsii (DWR346) were evaluated against a mixed population of grasshoppers in replicated 0.9-hectare plots of Wyoming rangeland. Both fungi were used as emulsifiable concentrates, at the rate of 6.25x1012 conidia/ha. There were no significant treatment effects until 21 days after application at which time F52 caused a 54% reduction and DWR346 a 52% reduction in grasshopper numbers. (USDA ARS, Sidney MT and University of Wyoming). The following fungi were evaluated against Anabrus simplex (Mormon cricket) nymphs in small outdoor cages in an ungrazed pasture situation: Beauveria bassiana GHA, Metarhizium brunneum F52, and M. robertsii DWR346 and DWR356. Fungus conidia were applied ULV in vegetable oil using a novel, highly portable, simulated ULV sprayer developed by APHIS CPHST. Only DWR346 provided significant mortality (63%) and only at the rate of 3.7x1013 conidia/ha and only 28 days after treatment. In contrast, Mormon crickets moved to the lab two days after treatment suffered 50-100% mortality from mycosis by the different fungi after 14 days at 28° C.; DWR346 caused 95% kill within 6 days in the lab. These results were worse than in previous years and were associated with lower than normal temperatures (USDA ARS, Sidney MT). SUBPROJECT 4 Development, evaluation and safety of entomopathogens for veterinary and structural arthropod pests [Chair: David Oi, ARS-Gainesville] Cooperating Agencies: AZ, CA, FL, ARS-Gainesville FL, ME). Reports submitted by Drion Boucias, Brian Federici, Dawn Gouge, Eleanor Groden, David Oi and Steven Valles. Summary Entomopathogens of veterinary and structural arthropod pest included viruses, bacteria, fungi (including microsporidia), and nematodes. For veterinary pests, research on the Hytrosavirus group has established a framework to incorporate this insect sterilizing agent into a bait for fly management. Studies have established a framework for the development of novel RNAi technologies targeting both host and viral genes via an oral delivery system. Functional studies on selected open reading frames (ORFs) of the Musca domestica salivary gland hypertrophy virus (MdSGHV) will allow examination of viral morphogenesis. In addition, the replication of the MdSGHV in adult stable flies, Stomoxys calcitrans, sterilizes female flies. Resistance to the Bin toxin of Bacillus sphaericus in Culex quinquefasciatus was reduced significantly by constructing recombinants in which this toxin was engineered into strains of Bacillus thuringiensis subsp. israelensis. For structural pests, analysis of the molecular diversity at the 16S ribosomal gene of the fire ant pathogen Kneallhazia solenopsae from the several fire ant species indicated an expanded host range. Surveys of six microbes infecting fire ants from recently established S. invicta populations in several countries supported the general prediction that invasive species lose many of their natural enemies during invasion. Phylogenetic analysis of nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) implicated the US as the source for the recent introduction of S. invicta into Taiwan. A year-long survey of was conducted to determine the seasonality and co-occurrence of four pathogens and a group of parasitic flies in colonies of S. invicta, in north-central Florida. Behavioral response and subsequent mortality of European fire ant, Myrmica rubra, colonies were assessed after soil inoculation with Beauveria bassiana. Three novel strains of nematodes were compared for virulence to three species of termites. Veterinary Pests A major research effort on the Hytrosavirus group has established a framework to incorporate this insect sterilizing agent into a bait for fly management. Assays will be conducted in the spring/summer 2011 to test various virus/bait formulations. Secondly, our molecular studies have served as a framework for the development of novel RNAi technologies targeting both host and viral genes via an oral delivery system. This work is being conducted in collaboration with scientists at the IAEA in Seibersdorf, Austria. Functional studies on selected open reading frames (ORFs) of the MdSGHV conducted in collaboration with scientists at Wuhan University, China, have provided a framework to examine the details of viral morphogenesis. For example, ORF 86, a major structural peptide of the Musca domestica salivary gland hypertrophy virus (MdSGHV), appears to be a matrix protein that mediates the egress of nucleocapsids through the nuclear membrane and later serves as a template for acquisition of viral envelopes. Working with USDA scientists we have characterized the replication of the MdSGHV in adult Stomoxys calcitrans. This work has shown that this virus although not causing gland hypertrophy, does sterilize female flies. (FL). Resistance to the Bin toxin of Bacillus sphaericus in Culex quinquefasciatus was reduced significantly by constructing recombinants in which this toxin was engineered into strains of Bacillus thuringiensis subsp. israelensis. Selection to strains containing this mixture, which included the Bin toxin and Cry11A, Cry4A, Cry4B, and Cyt1A, showed no significant resistance after 20 generations of selection. Tests of the selected mosquito strain with B. sphaericus in the absence of the B. thuringiensis subsp. israelensis showed high levels of resistance, indicating the latter strain phenotypically masked resistance to B. sphaericus. (CA). Structural Pests Analysis of the molecular diversity at the 16S ribosomal gene of the fire ant pathogen Kneallhazia solenopsae in the fire ants Solenopsis invicta, S. richteri, S. geminata, and S. geminata/S. xyloni hybrids from North America, Argentina and Brazil revealed a total of twenty-two 16S haplotypes, one of which, (WD_1), was identical between S. invicta from the USA and S. geminata from southern Mexico. The 16S phylogenetic reconstructions revealed that K. solenopsae genetic lineages formed two main clades differentiated by p-distance values of 2 to 3%. This is the first empirical evidence for K. solenopsae infection of S. geminata, the tropical fire ant, and the S. geminata x S. xyloni hybrids which are in the geminata species taxonomic group and not the saevissima group where infections have typically been detected. Interestingly, infections were only found in the tropical fire ants collected from southwest North America (Texas and Mexico), and not in Florida. (ARS-FL) Extensive surveys for the presence of six microbes were conducted in recently established S. invicta populations located in California, Australia, New Zealand, Taiwan, and China. These microbes include Wolbachia, two microsporidia (K. solenopsae and Vairimorpha invictae) and three RNA viruses (SINV-1, -2 and -3), all of which previously have been reported in native South American populations of S. invicta. The total number of enemy species is lower in the recently invaded areas compared with both South American and USA populations. The results support the general prediction that invasive species lose many of their natural enemies during invasion. (ARS-FL) The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/ richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. (ARS-FL). A year-long survey of was conducted to determine the seasonality and co-occurrence of four pathogens and a group of parasites in colonies of S. invicta, in north-central Florida. S. invicta colonies were examined for the presence of parasitic flies (Pseudacteon. curvatus, P. tricuspis, P. obtusus), a microsporidian pathogen (Kneallhazia solenopsae) and 3 Solenopsis invicta viruses (SINV-1, SINV-2, and SINV-3) SINV-1 and K. solenopsae infections were most prevalent among colonies during warmer periods of the year, while SINV-3 was most prevalent during the cooler periods. Pathogens were found more commonly in polygyne colonies than in monogyne colonies. The majority of monogyne colonies (>80%) were either uninfected or infected with only a single pathogen/parasite while the majority of polygyne colonies (>55%) were infected with 2 or more pathogens/parasites simultaneously. Higher pathogen/parasite prevalence among polygyne colonies is attributed to lower genetic relatedness among nestmates, increased colony longevity, and the proclivity of polygyne colonies to exchange resources. Pathogens and fly parasites were usually independently distributed among host colonies. The overall colony infection rate among the 360 colonies sampled, regardless of social form, was 60.3% (SINV-1), 8.9% (SINV-2), 10.8% (SINV-3), 22.5% (K. solenopsae), and 8.1% (Pseudacteon flies). (ARS-FL) In laboratory bioassays, we assessed the behavioral response and subsequent mortality of European fire ant, Myrmica rubra, colonies offered food surrounded by soil inoculated with Beauveria bassiana (lab grown and formulated GHA strain). Worker ants readily crossed and tunneled through fungal treated soil in order to acquire food, and colonies exposed to lab grown GHA experienced significantly greater mortality than with the formulated product, which was significantly greater than the control. Field deployment of food bait stations placed in the center of 1 m diameter Mycotrol treated areas did result in reductions in activity of ants at 8 weeks post treatment (ME). Research continues with S.P. Stock on the identification and virulence of nematodes found infecting M. rubra in Maine (ME, AZ.) Under laboratory conditions we compared three novel strains of Steinernema riobrave (3-8b, 7-12, and TP) with the 355 strain for virulence to the termites Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus workers. H. aureus was very susceptible to all the S. riobrave strains, and termites in all nematode treatments were dead after 4 d. The TP strain of S. riobrave caused greater mortality in R. flavipes and C. formosanus compared to the other nematode strains. Specifically, the TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively, which was more than 300% and 70% higher than the mortality caused by other strains. (AZ)

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