Duration: 10/01/2006 to 09/30/2011

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Project's Primary Website is at http://cipm.ncsu.edu/S301/ (direct link can be found under LINKS)

There is an urgent need to accelerate the development and implementation of cost-effective, environmentally safe alternatives to chemical pesticides for insect control. Changes in pest management programs, such as the reduction in organophosphate use dictated by the Food Quality Protection Act (FQPA), necessitate the development of new management tactics that are environmentally sound and compatible with current production practices, including biological control. Most stakeholder groups involved in setting research priorities and development of strategic plans for integrated pest management (IPM) for agricultural and forest commodities throughout the U.S. have identified this as a major research need. Several have specifically identified greater use of entomopathogens as a priority. Numerous listings by commodities, states, and pests can be found on Regional IPM Center websites, e.g. NE IPM Priorities for IPM in Apples (http://nepmc.org/priority/index.cfm) lists the development of biological alternatives, including nematodes and insect diseases for management of apple maggot and plum curculio. This project is critical to the advancement of biological control and IPM in general. The Experiment Station Committee on Organization and Policy (ESCOP) has established the development of pest management strategies as one of its highest-priority initiatives and has identified biological control, including the use of entomopathogens as a priority research objective. The discovery and development of entomopathogens and other biologically based pest management technologies have further been identified in the Southern Strategic Research Plan as requiring more focused effort within the Southern Region (http://www.cals.ncsu.edu:8050/saaesd/5yrplan.htm). With the use of newer targeted technologies for primary pests, such as transgenic crops and many of the newer chemistries, we are seeing natural enemy populations increase in managed systems. The use of entomopathogens can provide another targeted tool for primary or secondary pests that will compliment the build-up of endemic natural enemies.


Several criteria were used in selecting pests as targets for the development of microbial control agents under the proposed project. Selecting criteria included: a) invasiveness of the pest insect; b) pest importance and economic impact in the host crop; c) suitability of the pest as a model for development of entomopathogens; d) lack of economically and environmentally viable control methods for these pests. Although the research proposed herein focus on a relatively small number of pests, the information generated is expected to have applications beyond the boundaries of the pest/host systems discussed in this proposal. This proposal has been organized into four commodity-based subprojects to facilitate both the research and the future implementation of technologies. Special focus has been placed on invasive species, especially new invasive pests, which require urgent attention.


Control of invasive organisms has also been designated a priority by a number of environmental organizations (http://nature.org/initiatives/invasivespecies/) and State and National regulatory agencies (National Invasive Species Council, 2001. Meeting the Invasive Species Management Plan, 80 pp.; Clinton, W.J. 1999. Presidential Executive Order 13112 of February 3, 1999: Invasive Species, Federal Register Doc. 99-3184, Vol. 64 No. 25, Feb. 8, 1999, pp. 6183-6186; invasivespecies.gov). Many invasive species have achieved major pest status in their introduced range because they have escaped components of their native ecosystems, including natural enemies that frequently kept their populations in check. Entomopathogens as a group represent an important component of a natural enemy complex whose potential role in the regulation of invasive species has frequently been overlooked in comparison to parasitoids and predators. Further exploration, discovery and development of entomopathogens for introduced pests will likely lead to new tools for their suppression.


The discovery, implementation and commercial development of entomopathogens have had a major impact on agricultural insect pest management for fifty years. The commercialization of Bacillus thuringiensis (Bt) products, including Bt-transgenic plants, is probably the most notable and commercially significant. New scientific tools, including molecular markers, in vitro production techniques, and others allow for discovery, identification, and development of entomopathogens previously overlooked. Although progress has been made, entomopathogens still represent a relatively under-developed and under-utilized resource in the insect pest management arsenal. Opportunities for the incorporation of biopesticides into new pest management programs exist. Transgenic crops and newer, targeted chemistries both have resulted in reductions in pesticide loads in agricultural crops. Biopesticides can be used for minor pests whose status is elevated when not regulated by pesticides traditionally used against major pest species. This project proposes to pursue opportunities to discover entomopathogens with novel or increased activity, evaluate their safety, and explore ways of integrating them into pest management programs. Meetings and output from the project will facilitate interactions between participants, pest management researchers, extension personnel, and end users to identify opportunities for further development and integration of entomopathogen-based pest management tools.



Microbial insecticides and transgenic plants are registered for crop protection across state lines. This requires tests of efficacy, persistence, safety, resistance management and other parameters under different sets of environmental conditions. Therefore, the development of entomopathogens for pest management systems requires multi-state cooperative research among State Agricultural Experiment Stations, USDA research groups and industry to be successful in fulfilling the objectives of this project proposal. Multistate research is essential to the development of new management strategies for insect pests. Entomopathogens and their host pest insects are not limited by artificial boundaries. There must be a mechanism in place to facilitate the exchange of entomopathogens among scientists for optimal development. Protocols must be developed and standardized for the diverse types of research being proposed which can best be accomplished through multistate cooperation.


Further development and implementation of entomopathogens for biological control of insects will directly benefit farmers, consumers and the environment. Use of entomopathogens as applied microbial insecticides or as classical biological control agents will lessen reliance on chemical pesticides and therein reduce potential environmental and human health hazards, and pollution of soil and groundwater. The proposed research will further contribute to the greater implementation of entomopathogens as biological control agents for noxious insect pests and invasive species throughout the US. The work will increase our fundamental knowledge of physiological and ecological relationships among entomopathogens, their toxins and host insect populations including virulence, pathogenicity, transmission mechanisms, persistence, safety, and host resistance.

Related, Current and Previous Work

Over the past five years of this project, significant multistate collaborations have been established (or continued) addressing the development of entomopathogens for biological control of arthropod pests. These efforts have resulted in over 100 scientific publications per year, four patents, and production of educational material for professional and end users.

SUBPROJECT 1. Development, evaluation and safety of entomopathogens for leaf feeding insect defoliators. (Coordinator: Robert Behle, USDA-ARS, Peoria, IL)
Studies on the effect of transgenic Bt cotton have shown no adverse effects on non-target organisms and no change in tolerance or resistance after 8 years of commercial use (AL), documenting the environmental safety of this pest control technique. Similarly, studies comparing transgenic baculovirus with native baculovirus demonstrate significant differences in the biology and ecology such that the transgenic virus does not persist in agroecsystems (LA), directly addressing concerns raised by the greater scientific community and public groups. Researchers at 11 state and federal research institutions in the U.S. and collaborating scientists from four nations (Slovakia, Austria, Hungry, Germany) conducted studies on fungal, microsporidian and viral pathogens for control of primary lepidopteran and coleopteran pests (CT; IL; NY; ME; USDA-FS, Hampden, CT; USDA-FS, E. Lansing, MI; USDA-ARS, Ithaca, NY; USDA-ARS, Yakima, WA; USDA-ARS, Fresno, CA). Improved methods for production of Entomophaga maimaiga resting spores and identification of microsporidia that infect gypsy moth (Lymantria dispar) and browntail moth (Euproctis chrysorrhoea) support augmentative and classical microbial control projects for lepidopteran pests in environmentally sensitive forest, park, and urban landscape habitats. Applications of commercially available Beauveria bassiana and Bacillis thurengiensis have been demonstrated to effectively control Colorado potato beetle (ME; USDA-ARS, Ithaca, NY; USDA-ARS, Yakima, WA).

SUBPROJECT 2: Development, evaluation and safety of entomopathogens for homopteran and other piercing-sucking insects. (Coordinators: Michael Brownbridge, VT and Rosalind James, USDA, ARS-Logan) Research on entomogenous fungi for microbial control of mites, aphids, thrips, whiteflies, stink bugs, lygus bugs, and glassy-winged sharpshooter was carried out across a diverse range of agricultural crops, including cotton, soybeans, rice, avocado, vegetables and ornamentals. Their use was also assessed for control of honey bee parasites. Monitoring for Neozygites fresenii, which controls cotton aphids (AR), has saved cotton growers thousands of dollars by eliminating the need for pesticide sprays, concurrently reducing environmental and human health risks. Efforts to introduce the fungus into Californian cotton fields have so far not been successful (AL; AR; CA; GA; LA; MS; SC). Efficacious strains of the predominant natural pathogen of lygus bugs, Beauveria bassiana, were selected for biopesticide development; work is now focusing on novel encapsulated formulations to enhance field stability and efficacy (CA; IL; MS). B. bassiana and Metarhizium anisopliae were the main pathogens recovered from avocado orchard soils in CA. Virulent isolates have been identified for potential use against avocado thrips. M. anisopliae selectively killed varroa mite, a parasite of honey bees, with minimal to no harmful side-effects on bee brood survival or honey production. Hirsutella thompsonii isolates also showed activity against the varroa mite, and metabolites produced by sporulating cultures inhibited oviposition in two-spotted spider mite.

SUBPROJECT 3: Development, evaluation and safety of entomopathogens in cryptic and soil habitats. (Coordinators: Parwinder Grewal, OH, and Edwin Lewis, CA)
Studies on control of the Asian longhorned beetle with Beauveria brongniartii, B. bassiana and M. anisopliae and the entomopathogenic nematodes Heterorhabditis marelatus, Steinernema carpocapsae, and S. feltiae have yielded promising results (NY; USDA-FS, Hampden, CT; USDA-FS, E. Lansing, MI; Integrated Biocontrol Systems-IN; IL). Interactions between several combinations of entomopathogenic nematodes and plant-parasitic nematodes have been investigated in several cropping systems (CA; NJ; HI; OH; VA; USDA-ARS, Byron, GA). The susceptibility of larval instars of the root weevil Diaprepes abbreviatus and the codling moth to entomopathogenic nematodes under various formulation and application strategies have been tested and control of these pests has been improved (FL; USDA-ARS, Yakima, WA). Persistence, longevity and microbial control efficacy studies are advancing with several species of fungi and entomopathogenic nematodes for suppression of various key pests such as the black vine weevil Otiorhynchus sulcatus, plum curculio Conotrachelus nenuphar, pecan weevil, Curculio caryae, and white grubs (Coleoptera: Scarabaeidae) (GA; LA; OH; NJ; VA; USDA-ARS Bryon, GA; USDA-ARS Corvallis, OR). Safety of entomopathogenic nematodes to the soil nematode community was demonstrated and the on going work on the microbial community and soil ecosystem processes is encouraging.

SUBPROJECT 4: Development, evaluation and safety of entomopathogens for veterinary and structural arthropod pests (Coordinators: James J. Becnel, David Oi, USDA-ARS, Gainesville, FL)
Two multi-state IPM projects (Southern Region IPM Grant [1999-2001; USDA-ARS Areawide Pest Management Grant [2001-present]) for control of fire ants involving the protozoan Thelohania solenopsae were established among 11 states (AL; AR; FL; GA; LA; MS; NC; OK; SC; TN; and TX) and coordinated by ARS Gainesville. Recombinant strains of Bacillus thuringiensis israelensis and B. sphaericus have been engineered to increase virulence and reduce resistance development for more effective mosquito control (CA). A recombinant strain of Bti has been developed that produces large amounts of the Bs binary toxin. This strain is eight-fold more potent than either Bti or Bs against Culex mosquitoes, the primary vectors of West Nile Virus (CA). A new mosquito pathogenic baculovirus (CuniNPV) was discovered in Culex mosquitoes from Florida. When combined with magnesium, this virus infects and kills all instars of Culex larvae and may be useful in controlling vectors of West Nile Virus and other types of encephalitis. (USDA-ARS, Gainesville, FL; CT).

Objectives

1. Discovery of entomopathogens and their integration and safety in pest management programs for major acreage crops.
   
2. Discovery of entomopathogens and their integration and safety in pest management programs for ornamental, vegetable, fruit and nut crops.
   
3. Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural landscapes.
   
4. Discovery of entomopathogens and their integration and safety in pest management programs for medical, veterinary, and structural pests.
   

Methods

Procedural Plan: The proposed re-organization of this project is to address the needs of commodities and reflects the project's previous progress on discovery, expansion of our fundamental understanding of entomopathogens and their hosts, and development of entomopathogens for pest management programs. Although this project proposes to continue to address basic issues relevant to invasive and emerging pests and changing pest management programs, we also propose to incorporate studies that move towards greater implementation and integration of entomopathogens into current pest management systems. In addition to conducting information exchange and coordination of collaborative projects between participants, annual project meetings will be structured to involve maximum participation of colleagues working in extension IPM, and involvement of representative growers of the commodities targeted in each project objective. Successive meetings over the duration of the project will address commodities under one selected objective per meeting. During the meeting, a facilitated workshop will be conducted to explore potential integration of entomopathogens into pest management programs for the selected commodities. Presentation of the results of the workshop may be made in a symposium at the Annual Meeting of the Entomological Society of America, or other similar forums, and will be presented in a report on the project website(http://cipm.ncsu.edu/S301/). The safety of entomopathogens in pest management programs will be addressed through continued research on the impacts of these organisms on non-target species. SUBPROJECT 1. Discovery of entomopathogens and their integration and safety in pest management programs for major acreage crops. Sub-objectives: 1. Identification of virulent strains of microbial agents; 2. Ecological characterization of the microbial control system; 3. Development of selected pathogens as biopesticides as part of an integrated pest control system; 4. Verification of environmental safety of microbial control agents. Experimental Methods: Planned projects will encompass one or more of these categories over the time frame of this regional project. Initial efforts will focus on control of three specific pests: a) soybean aphid on soybeans, b) Lygus spp. on cotton and alfalfa, and c) sugar beet root maggot on sugar beets, and d) tobacco budworm and cotton bollworm on Bt cotton. Projects will emphasize development of methods for integrated use of these microbial control agents. Soybean aphid is a new invasive pest in the midwest (Hartman et al. 2001) and eastern states (Venette and Ragsdale 2004), and is susceptible to infection by insect pathogenic fungi. Over the next 5 years, the species of fungi infecting soybean aphid, and spatial and temporal ecology of host/pathogen interactions will be investigated. Efforts will focus on the development of conservation biological control through manipulation of soybean cultivar (Hill et al. 2004) and cropping practices to enhance infection by insect pathogenic fungi while protecting soybean yield. Because the invasive soybean rust is predicted to arrive soon in these areas (Anonymous 2004), rust control pesticides will be screened to identify those that will kill soybean rust, but not insect pathogenic fungi. Sugarbeet root maggot (SBRM) is the primary insect pest in sugar beets (Wozniak et al. 1993), potentially affecting 1,200,000 acres in the U.S. Management strategies for sugarbeet root maggot will be developed that integrate microbial control with other IPM tools. Combination of Metarhizium with cover crops can provide acceptable yield protection in the face of moderate to heavy SBRM pressure (Dregseth et al. 2003). This strategy will be evaluated in field experiments in two distinctly different areas: two sites on leased commercial land in Pembina/Walsh Counties, North Dakota, and two at the Montana State University Eastern Ag Research Center (EARC), Sidney. The locations represent different insect pressures, agronomic practices, soil types, and irrigation methods. Integrated pest control practices will also explore the combination of Metarhizium with resistant beet hybrids (Campbell et al. 2000a, 2000b) and reduced rates of pesticides. A variety of formulation technologies will be explored to improve efficacy through development of a more accurate delivery system. It has been hypothesized that Metarhizium will colonize sugarbeet rhizosphere and thereby protect the developing root system from attack by SBRM. Green fluorescent protein transformants of Metarhizium anisopliae F52, MA1200, and Beauveria bassiana TM28 will be transformed with pEGFP-CP, using established protocols to serve as a means of determining the extent and ecological limitations of colonization. Efficacy of colonization events against SBRM will be assessed by infesting appropriately treated sugar beets in greenhouse assays. Lygus spp. are primary pests of cotton across the United States. No specific control exists for Lygus spp. and application of broad-spectrum chemicals can lead to secondary pest outbreaks. New isolates of the entomopathogenic fungus, Beauveria bassiana have been discovered (McGuire 2002) with activity against Lygus spp. and the new isolates can grow at relatively high temperatures. Multi-location efforts will focus on characterizing the bioactivity of these new isolates against target pest insects and non-target beneficial insects. Techniques for mass production, formulation and field persistence will be studied in an effort to develop a biological pesticide of selected new isolates. A novel application technique of treating field margins before cotton emerges to specifically reduce the initial infestation will be tested (Snodgrass et al. 2000). This technique emphasizes the pathogens host specificity to target the pest in these non-crop areas (Craig and Luttrell 1997) with minimal impact on beneficial insects. Field tests will determine the efficacy of B. bassiana against Lygus spp. on cotton, alfalfa, and wild host plants (field borders) in efforts to reduce the adverse impact of this pest by either crop damage or applications of broad-spectrum pesticide. Tobacco budworm (TBW) and cotton bollworm (CBW) are two of the three major lepidopteran pests of cotton in the US and targets of Bacillus thuringiensis (Bt) transgenic cotton. The development of resistance to these transgenic Bt crops by these two pests is a primary scientific concern. TBW and CBW will be monitored for potential resistance in AL by collection of eggs and larvae from Bt and non-Bt plants, and adults in light and pheromone traps. F1 larvae will be assayed for susceptibility to Bt. A colony of CBW is being selected for resistance to Bt in order to characterize resistance so that appropriate resistance management decisions can be made. Collaboration: Auburn University, AL; Illinois Natural History Survey; Cornell University, NY; PA St. Univ.; Univ. of DE - Extension; USDA-ARS-Ithaca, NY; USDA-ARS-Shafter, CA; USDA-ARS-Stoneville, MS; USDA-ARS-Peoria, IL; USDA-ARS-Sidney, MT. SUBPROJECT 2. Discovery of entomopathogens and their integration and safety in pest management programs for ornamental, vegetable, fruits and nut crops. Sub-objectives: 1. Identification of novel strains of pathogens with improved field efficacy and persistence; 2. Improvement of preservation and production techniques, and; 3. Development of enhanced methods of use and delivery. Experimental Methods: Various pathogen screening, development, ecological, and genetic studies will be conducted to further microbial control in vegetable, fruit, nut, and ornamental systems. Soil and insect surveys (e.g., based on Galleria mellonella baiting) will yield novel strains of entomopathogenic nematodes and fungi (or other pathogens) to test for improved lab and field efficacy against various pests, including pecan weevil and plum curculio. Pathogen efficacy will be enhanced through genetic improvement methods (e.g., artificial selection). Improved efficacy and conservation techniques for entomopathogenic nematodes and fungi will also be pursued through manipulation of the agroecosystem (e.g., ground cover, soil amendments, etc). Also, to improve efficacy, the nature of genetic trait deterioration in entomopathogenic nematodes during culture will be characterized; methods to overcome this problem will be elucidated. To date, rhizosphere-competence has been reported for only two isolates of entomopathogenic fungi, and on only two plant species. To determine if this trait is common to all M. anisopliae and B. bassiana strains, systematic studies will be done to determine rhizosphere-competence for a range of representative isolates on several ornamental plant species (Bruck 2005). Studies will also include an evaluation of efficacy of rhizosphere-competent isolates against root-feeding insects, characterization of the spatial and temporal distribution of these isolates on the plant root system, and an assessment of compatibly with other rhizosphere colonizers. Factors influencing establishment of entomopathogenic fungi in bulk potting media and the rhizosphere will also be elucidated. To facilitate early detection of thrips in commercial greenhouses and more timely intervention with mycoinsecticides, indicator (trap) plants will be assessed for attractiveness to thrips; use practices will be refined to allow ready transfer of this technology to growers. The daily activity of western flower thrips on spring bedding plants will be documented at three stages of crop development; this information will be used to optimize pesticide applications to coincide with periods of peak thrips exposure. Interactions between entomopathogenic fungi and spray additives will be assessed as a means of increasing efficacy. Finally, the efficacy of novel whey-based sprayable and granular fungal formulations will be evaluated for control of thrips on foliage and in soil; compatibility with predatory mites will be determined. Collaboration: Univ. of GA; Univ. of FL; Rutgers Univ., NJ; Univ. of VT; Univ. of NH; Brigham-Young Univ., UT; USDA-ARS-Peoria, IL; USDA-ARS-Byron, GA; USDA-ARS-Corvallis, OR; USDA-ARS-Yakima, WA. SUBPROJECT 3. Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural landscapes. Sub-objectives: 1. Development of novel methods for application of pathogens against invasive pests of ornamental trees.; 2. Evaluations and release of introduced pathogens for control of gypsy moth; 3. Development of entomopathogen-based management of landscape pests that contribute to pest levels in food crops; 4. Development of methods for timely application of entomopathogenic nematode infected insect cadavers in the field; 5. Development of conservation methods for maintenance of high populations of entomopathogens in the field, and; 6. Development of molecular methods for study and improvement of entomopathogenic nematodes as control insect agents. Experimental Methods: Studies with the fungal pathogen Entomophaga maimaiga and its host gypsy moth, Lymantria dispar (L.), will be continued to investigate the genotypic and phenotypic variability in host and pathogen. Long-term studies of spatial population structure of this virulent fungal pathogen infecting gypsy moth will be initiated. These studies will address questions of the potential for decreasing virulence or persistence of this fungal pathogen, with resulting increases in the frequency of defoliation events in North America. Several experiments will also be conducted to evaluate three species of microsporidia as potential biological control agents of the gypsy moth in North America. These efforts are expected to culminate in a 2005 North American Plant Protection Organization proposal via USDA-APHIS-PPQ to release the microsporidia in <10-acre plots in Illinois in 2005 or 2006. Variability of several isolates of microsporidian pathogens of the gypsy moth will be evaluated using PCR and proteomics techniques. More than 20 isolates represent 3 or 4 species. At least one species description will result, as well as a taxonomic re-evaluation of the species currently described. Field studies will continue to test the effects on non-target Lepidoptera of releasing microsporidia via ULV sprays as a method for inoculating a gypsy moth population (Goertz et al. 2004). The studies are conducted in Slovakia, the aboriginal sites of the pathogen and host. Transmission of a Nosema microsporidian isolate in simulated natural environments (caged trees) will be studied as a mechanism of establishment and spread in a release site. Horizontal transmission will be evaluated as a mechanism of competition between different microsporidian pathogens of the gypsy moth. Immune responses of gypsy moth to several natural enemies will be investigated using proteomics techniques to distinguish potential differences. Microsporidia attacking different internal organs, two species of viruses, and several species of parasitoids will be evaluated. Nosema microsporidia, isolated from the Eastern tent caterpillar, Malacosoma americanum in Kentucky and Illinois, will be evaluated for use as augmentative biological control agents during outbreaks in the Southern U.S. With an overall goal of developing application methods of a registered strain of Metarhizium anisopliae in the field for Asian longhorned beetle control, studies will be continued to develop a novel application strategy. Dose/response associations for Asian longhorned beetle adults will be established for insects exposed to non-woven fiber bands containing cultures of this fungus--the application strategy marketed in Japan to control a closely related longhorned beetle (Dubois et al. 2004). Studies will also include in-depth investigations of cuticular penetration by this fungal pathogen. Field studies in China will merge attractants with fungal bands to see if this merger enhances beetle mortality. In order to predict the population structure of entomopathogenic nematodes in field situations, the infection dynamics of these nematodes will be studied, based upon how infective juveniles assess hosts in various infection states. The attractiveness, acceptability and quality of hosts will be determined using a series of behavioral tests (Griffin et al. unpublished data). This knowledge will assist in understanding the dynamics of endemic entomopathogenic nematode populations and in predicting the fate of applications of them for biological control. A microsporidium from the black vine weevil, Otiorhynchus sulcatus (F.) will be studied and described. Larvae of the weevil attack roots of nursery stock, particularly yew, hemlock and rhododendron. The microsporidium causes cessation of feeding and death of host larvae at late stages of infection. The impact of Wolbachia infection on development and reproduction of the black vine weevil will be studied. Preliminary results showed that the parthenogenetic reproduction of the black vine weevil is not due to Wolbachia infection ant that tetracycline reduces the level of infection, but does not cure weevils. This work will be expanded to determine the more subtle impact of Wolbachia infection on the black vine weevil. A culture of Wolbachia-free weevil culture will be developed to allow further studies on the infection. The potential of entomopathogenic nematodes to manage olive fruit fly will be assessed. This insect is an invasive pest of olives in California that pupates in the soil, where the insect is potentially vulnerable to infection. Preliminary data show that entomopathogenic nematodes infect and kill the insect. Our objective is to develop entomopathogenic nematode based management for olive fruit flies that are associated with landscape plantings of olives, which contribute substantially to the olive fruit fly problem by serving as a constant source of flies that move into commercial olive orchards. Several methods of application, and the timing of these applications of entomopathogenic nematodes will be investigated in order to enhance field efficacy. Recent studies with entomopathogenic nematodes have shown that the application of the nematodes still inside their host cadavers is superior to the control achieved using entomopathogenic nematodes applied in an aqueous carrier. With the objective of fine-tuning the applications of cadavers to optimize timing, research will focus on developing mathematical models for both weevil phenology and the timing of emergence of infective juvenile entomopathogenic nematodes from cadavers. In order to develop conservation strategies that will preserve high levels of entomopathogenic nematodes in the field, the dynamics of natural nematode populations, including the influence of management practices on the survival and activity of the entomopathogenic nematodes, will be studied in turfgrass and vegetable landscapes in Ohio. Factors affecting nematode population dynamics will be studied using a variety of statistical methods including principal component analysis. These studies will expand our understanding of the population level processes of entomopathogenic nematodes and allow formulation of conservation strategies. To improve the efficacy of the entomopathogenic nematode/bacterium complex with tools from molecular biology and genetics, we will modify laboratory techniques that were originally established for genetic manipulation of Caenorhabditis elegans, and adapt them to study Heterorhabditis bacteriophora as an entomopathogenic nematode model. We will develop an Entomopathogenic Nematode Growth Medium in which the growing nematodes can be individually and clearly observed under a stereomicroscope. We will isolate mutants of the symbiotic bacterium Photorhabdus luminescens that do not overgrow the nematodes on the medium. Homozygous inbred lines of H. bacteriophora will be established and procedures for inducing morphological and drug resistant mutants will be isolated. Molecular biology studies will be conducted to identify important genes involved in the infection and survival biology of entomopathogenic nematodes and determine sequence similarities of expressed genome between Heterothabditis bacteriophora and Caenorhabditis elegans. A pilot scale cDNA sequencing project will provide the first foray into the gene diversity and gene discovery of H. bacteriophora nematodes. As the infective stage, which is similar to the dauer stage of C. elegans in ontogeny, serves as the basis for parasitism and symbiosis in H. bacteriophora, studies will focus on the expressed genome of this stage. Collaboration: Univ. of CA-Davis; Cornell Univ., NY; Univ. of KY; Illinois Natural History Survey; Univ. IL; Ohio St. Univ.; USDA-ARS-Stoneville, MS; USDA-ARS-Corvallis, OR; USDA-ARS-Bryon, GA; USDA-ARS-Wooster, OH; USDA-FS, CT; Bulgaria Academy of Science; Fach. Eberswalde, Germany; Charles Univ.-Prague, Czech Republic; Forestry Inst. Slovakia; CT Ag. Expt. Stn.; BOKU-Vienna, Austria; Forestry Inst., Hungary). SUBPROJECT 4. Development, evaluation and safety of entomopathogens for veterinary and structural arthropod pests 4a. Entomopathogens for Ant Pests Sub-objectives: 1. Identification of pathogens of the European fire ant (Myrmica rubra) and assessment of their potential for biological management of this invasive species; 2. Identification and development of pathogens for imported fire ant (Solenopsis spp.) control, and; 3. Development of biologically-based IPM programs for imported fire ant control in the field with emphasis on microbial agents. Experimental Methods: Cadavers and live ants collected from within M. rubra's native and introduced range will be examined for pathogens (Groden and Drummond 2004). Cadaver samples will be surface sterilized and incubated or squashed and examined microscopically for evidence of fungal or protozoan pathogens or entomopathogenic nematode. In collaborations with other project members, pathogens will be identified by microscopic examination and/or molecular analyses. If unknown, modes of infection will be investigated and factors influencing infection rates will be explored. Promising agents for biological control will be further studied for specificity and environmental safety, and implementation strategies that may enhance the likelihood of triggering epizootics. Aspects of ant defense against pathogens will also be researched. Traditional and molecular-based methods (EST-based arrays) will be used to identify new pathogens of the imported fire ants. Searches for viruses and other pathogens will be conducted in South and North America. Utilization of molecular techniques will initially focus on the recently discovered viruses (Valles et al. 2004). Search methods can be extended for use in the identification of other pathogenic organisms in fire ant populations. Bioassays for evaluating pathogens (e.g., viruses, microsporidia, fungi, etc) as possible control agents for imported fire ants will be developed. Pathogen inocula will be applied to individuals or groups of ants to determine optimal conditions for disease establishment. Inoculation methods and evaluations will be developed to meet nuances of individual pathogens. Colony growth and survivorship will be assessed by monitoring brood levels, the number of workers, and queen survivorship. Field impact of selected native pathogens on fire ants will be determined by comparing ant populations in plots with and without infected colonies. Ant populations and infection levels per plot will be monitored periodically. To test pathogen-based IPM programs, field studies will be conducted to compare chemical-only treatments versus microbial control regimes (Pereira 2004). Monitoring will be conducted over several years to determine long-term effect of pathogen applications, the potential reduction of fire ant populations, and the prevention of fire ant reinvasions provided by the pathogen presence. Basic biology of fire ant pathogens will be investigated to enhance their use in microbial control. Pathogen life cycles will be elucidated to enhance transmission, including the functions of microsporidian spore types. Factors affecting epizootics of pathogens in these ants will be studied, particularly with respect to different social forms of the host, which greatly affects the success of at least one microbial control agent. Environmental factors that hinder short-term microbial control will be investigated to improve entomopathogen efficacy. 4b. Entomopathogens for Veterinary Pests Sub-objective: Development of the baculovirus from Culex nigripalpus (CuniNPV) as a biopesticide for control of Culex mosquitoes that vector West Nile Virus and other mosquito vectored encephalitis. Experimental Methods: Studies will focus on improving in vivo production system for CuniNPV. An alternative approach will be to investigate the development of an in vitro production system for virus production in a mosquito cell. Formulation development would entail micro-encapsulation of CuniNPV together with magnesium into a particle that could be delivered to the larval mosquito. Requirements are for a particle of 10-30 microns that would only release the virus and magnesium in the alkaline (pH 9.5-10) conditions of the mosquito midgut. Formulated products will be screened with laboratory bioassays and promising candidates will be evaluated under field conditions against Culex mosquitoes. Collaboration: Univ. of ME; LA St. Univ. AgCenter; Univ. of AZ; TX A&M Univ.; CT Agric. Exp. St.: USDA-ARS-Laramie, WY; USDA-ARS-Beltsville, MD; USDA-ARS-Gainesville, FL; USDA-ARS-Ft. Pierce, FL

Measurement of Progress and Results

Outputs

• Annual reporting will include the summary of research achievements, publication lists, tabulation of extension related activities (including videos, web-based resources, factsheets and presentations) related to this project.
• The project will maintain a website (http://cipm.ncsu.edu/S301/) which, in addition to the Annual Reports and meeting information, will publish the specific recommendations from workshops addressing opportunities for integration of microbials into pest management program.

Outcomes or Projected Impacts

• Cross-project: Extension-related presentations and workshops and scientific symposia, Printed material, videos and CDs on new developed techniques, and Tabulated hits at the project and related websites will be used as outcome indicators for this project.
• Subproject 1: Increase the specific knowledge base in the topic areas, represented by the publication of scientific manuscripts on strain discovery and efficacy, microbe/host ecology, microbe production techniques, formulation and processing, and safety of microbes for non-target insects and resistance, Additional information for growers will be made available through extension publications, and Information and technology suitable for commercial development of new microbial pesticides by future industrial partners.
• Subproject 2: Novel strains of pathogens for use against orchard pests (pecan weevil, plum curculio, black vine weevil, white grubs, etc.), Effective use practice recommendations, New approaches to the management of root-feeding insects, e.g., black vine weevil, Otiorhynchus sulcatus, Rhizosphere-competent isolates that can fully colonize roots, persist for extended periods, and effectively control the target insect, and Effective use of mycoinsecticides and other reduced-risk compounds, and novel formulations that promote efficient delivery of fungal inoculum to thrips pests.
• Subproject 3: Novel methods for application of pathogens against invasive pests of ornamental trees, Release of introduced pathogens for control of gypsy moth, Entomopathogen-based management methods for landscape pests, Methods for application of entomopathogenic nematodes in host cadavers, Methods for conservation of entomopathogenic nematodes in the field, and Molecular methods for improvement of entomopathogenic nematodes.
• Subproject 4: Potential biological control agent(s) for the invasive European fire ant, New potential microbial control agents for the imported fire ants, Molecular techniques for discovery, identification, and enhancement of pathogens for imported fire ant control,IPM methods for integration of pathogens in field control of fire ant populations, and A cost-benefit analysis of CuniNPV as a biopesticide for mosquito control.

Milestones

(0): attached SDC314Milestones.doc

(0):JECTED PARTICIPATION Appendix E as of April 20, 2005 shows 12 State researchers from AZ, CT, FL, GA, IL, LA, MN, NJ, ND, NY, and PA, plus 7 USDA researchers from CA, FL, IL, KS, MT, MD, and OR. However, projected participation based on contributors the project proposal, and past participation in project S-301, far exceeds these figures.

Projected Participation

View Appendix E: Participation

Outreach Plan

Educational Plan:

This project will provide educational material for: a) Entomology colleagues via symposia at national meetings, and research publications; b) Extension personnel and growers via participation in annual project meetings, presentations, and production of published and web-based resources on the use of entomopathogens in pest management. Products from this project have applications for both conventional and organic producers, resource managers (e.g. forests), and urban clientele. Organic growers in particular have typically been under-served by research and extension activities, which have tended to focus on development of chemically-based pest management programs. Today, research and outreach efforts emphasize increasing the implementation of IPM strategies on farms, forests and urban landscapes, with biological controls forming the first line of defense against pests. This project enhances our ability to achieve this goal on a broad range of agricultural commodities and other managed ecosystems.

Organization/Governance

Governance (Officers):

Chair: Roberto Pereira, USDA-ARS CMAVE, and Gainesville, FL

Secretary: Parwinder Grewal, Ohio State University, Wooster, OH

Member at Large: Edwin Lewis, Virginia Polytechnic Institute and State University, Blacksburg, VA

Literature Cited

Anonymous. 2004. Soybean Rust USDA-APHIS-PPQ, http://www.aphis.usda.gov/ppq/
ep/soybean_rust/UreMelPp502.pdf.

Bruck, D.J. 2005. Ecology of Metarhizium anisopliae in soilless potting media and the rhizosphere: implications for pest management. Biol. Control 32: 155-163.

Campbell, L. G., J. D. Eide, L. J. Smith, and G. A. Smith. 2000a. Control of sugarbeet root maggot with the fungus Metarhizium anisopliae. J. Sugar Beet Res. 37: 57-69.

Campbell, L. G., A. W. Anderson, and R. J. Dregseth. 2000. Registration of F1015 and F1016 sugarbeet germplasm with resitance to the sugarbeet root maggot. Crop Sci. 40: 867-868.

Craig, C., and R. G. Luttrell. 1997. Host plant preferences of tarnished plant but: a foundation for trap crops in cotton. Proc. Beltwide Cotton Conf. 2: 1176-1181.

Dregseth, R. J., M. A. Boetel, A. J. Schroeder, R. B. Carlson, and J. S. Armstrong. 2003. Oat cover cropping and soil insecticides in an integrated surgarbeet root maggot (Diptera: Otitdae) management program. J. Econ. Entomol. 96: 1426-1432.

Dubois, T., Z. Li, H. Jiafu and A.E. Hajek. 2004. Efficacy of fiber bands impregnated with Beauveria brongniartii cultures against the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae). Biol. Contr. 31: 320-328.

Goertz, D., D. Pilarska, M. Kereselidze, L. Solter, and A. Linde. 2004. Studies on the impact of two Nosema isolates from Bulgaria on the gypsy moth (Lymantria dispar L.). J. Invertebr. Path. 87: 105-113.

Griffin, C., N. Boemare and E.E. Lewis. Biology and Behaviour. In: Nematodes As Biocontrol Agents (Grewal, Ehlers and Shapiro-Ilan, eds.) CABI. In Preparation.

Groden, E., S. Yan, F.A. Drummond. 2004. Pathogens associated with the ant, Myrmica rubra, in its introduced and native range. (Abstract) Proceedings of the 2004 Annual Meeting of the Society of Invertebrate Pathology, August 1 - 6, 2004, Helsinki, Finland, p. 65.

Hartman, G. L., L. L. Domier, L. M. Wax, C. G. Helm, D. W. Onstad, J. T. Shaw, L. F. Solter, D. J. Voegtlin, C. J. DArcy, M. E. Gray, et al. 2001. Occurrence and distribution of Aphis glycines on soybeans in Illinois in 2000 and its potential control. http://planthelthprogress.org/current/briefs/aphisglycines/article.htm.

Hill, C. B., Y. Li, and G. L. Hartman. 2004. Resistance to the soybean aphid in soybean germplasm. Crop Sci. 44: 98-106.

McGuire, M. R. 2002. Prevealence and distribution of naturally occurring Beauveria bassiana in San Joaquin Valley populations of Lygus hesperut (Heteroptera: Miridae). South Carolina Entomol. Soc. 19: 237-246.

Pereira, R. M. 2004. Areawide Suppression of Fire Ant Populations in Pastures: Project Update. J. Agric. Urban Entomol. 20:123-130.

Valles, S. M., C. A. Strong, P. M. Dang, W. B. Hunter, R. M. Pereira, D. H. Oi, A. M. Shapiro, and D. F. Williams. 2004. A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization. Virology 328: 151-157.

Vinette, R. C., and D. W. Ragsdale. 2004. Assessing the invasion by soybean aphid (Homoptera: Aphididae): Where will it end? Ann. Entomol. Soc. Amer. 97: 219-226.

Wozniak C. A., G. A. Smith, D. T. Kaplan, W. J. Schroeder, and L. G. Campbell. 1993. Mortality and aberrant development of the sugarbeet root maggot (Diptera: Otitidae) after exposure to steinernematid nematodes. Biol. Control. 3: 221-225.

Attachments

Land Grant Participating States/Institutions

AL, AR, AZ, CA, CT, FL, GA, IL, ME, MN, MS, ND, NJ, NY, OH, PA, VT

Non Land Grant Participating States/Institutions

Northern Plains Area, USDA, USDA ARS, USDA-ARS, USDA-ARS/Maryland, USDA-ARS/MT, USDA/ARS Grain Marketing and Production Research Center