Duration: 10/01/2022 to 09/30/2027

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Broad-spectrum chemical insecticides continue to be the mainstay for the control of arthropod pests in most agricultural systems as well as natural and urban landscapes. While several chemical pesticides are capable of rapidly killing various pests, heavy reliance on their use has generated significant problems including safety risks to human and environmental health, negative impact on beneficial arthropods, outbreaks of secondary pests which are normally held in check by natural enemies, decrease in biodiversity, and increased risk of insecticide resistance. Several bacteria, fungi, nematodes, and viruses attack a variety of arthropod pests and multiple species of entomopathogens are commercially available as biopesticides (Lacey, 2016). In the last two decades or so the market has also improved and currently, there are about 400 registered biopesticides (derived from natural materials including animals, plants, microbials, and some minerals) in the U.S. (Arthurs and Dara, 2019).

Recent studies have also indicated that entomopathogenic microbials manage arthropod pests and improve soil and enable plant pathogen management. These microbials also play disease-antagonizing roles and improve overall plant growth and health (Dara, 2019). Although, these are majorly employed in the food production system, lately, the employment of entomopathogenic microbials is being expanded to manage veterinary arthropods as well (Ebani and Mancianti, 2021).

Changes in pest management programs, such as the reduction in organophosphate use dictated by the Food Quality Protection Act (FQPA), and proposed legislation by the EPA regulating the use of neonicotinoids to protect pollinators (Suryanarayanan, 2015) necessitate the development of new management tactics that are environmentally sound and compatible with current production and integrated pest management (IPM) practices. One viable alternative to chemical insecticides is the use of microbial control agents. In contrast to chemical insecticides, microbial control agents are generally not harmful to humans or the environment, and have minimal or negligible potential to cause resistance or harm non-target organisms. This project focuses on the development and advancement of entomopathogens for biological pest suppression.

Development of microbial control tactics using entomopathogens is of great importance to US agriculture. The Experiment Station Committee on Organization and Policy (ESCOP), and National Association of State Universities and Land-Grant Colleges (NASULGC) have identified environmental stewardship, including the need to decrease chemical pesticide use, as a primary agricultural challenge in the US. Furthermore, most stakeholder groups developing strategic plans for pest management throughout the U.S. have identified biological control as a major research need. Many have specifically identified the use of entomopathogens as a priority. Some examples that list the development of entomopathogens as a priority include pecan, peaches, apples, grapes, blackberry, tomato, and coffee (http://www.ipmcenters.org/).

Microbial control research and application has had major impacts on IPM during the past fifty years. The commercialization of Bacillus thuringiensis (Bt) products, including Bt-transgenic plants, is probably the most notable and commercially significant. New discoveries of suitable entomopathogens and advances in their production have facilitated the commercialization of numerous products. Although there has been a significant increase in biopesticide sales and research, microbial control is not considered as a major choice of pest management in many cropping systems, especially in conventional production (Leng et al., 2011; Sinha, 2012; Lacey, 2016). However, recent estimates suggest that biopesticides will become the fastest-growing crop protection market sector globally, with growth greatly exceeding conventional chemical insecticides (Glare et al. 2012). For example, in the USA biopesticides are projected to achieve a compound annual growth rate of 14.7% from 2020 to 2025, with sales reaching $8.5 billion (Markets and Markets). Thus, there is an urgent need to expand microbial control research and incorporate microbial control options into integrated pest management (IPM) strategies in order to maintain global competitiveness of US agriculture. Therefore, additional research is required to expand and complement the expected increase in demand and use of entomopathogens as vital components of IPM. New scientific tools, including molecular markers, genomics, and in vitro production techniques allow for novel discovery, identification, and development of entomopathogens previously overlooked or out of reach. The key challenges that limit microbial control for arthropod pests will be addressed in this project including: enhancing efficacy through strain discovery and improvement, advancing production and delivery, integration with existing management techniques, conservation of endemic entomopathogens, and gaining greater understanding of fundamental entomopathogen biology and ecology to further improve applied pest management. The project objectives will address pest issues in large acreage crops, orchards, small fruits and vegetables, and urban areas, landscapes, and nurseries. The consequences of not doing the proposed research include increased chemical pesticides in the environment (risking the health of humans and other nontargets) and increased crop losses due to endemic and invasive pests.

In addition to numerous endemic pests, several invasive pests such as the Asian citrus psyllid (Diaphorina citri), Bagrada bug (Bagrada hilaris), brown marmorated stink bug (Halyomarpha halys), polyphagous shot hole borer (Euwallacea fornicatus), spotted wing drosophila (Drosophila suzukii), redbay ambrosia beetle (Xyleborus glabratus), red imported fire ant (Solenopsis invicta) and spotted lanternfly (Lycorma delicatula) pose a serious threat to several important hosts in agriculture, orchard, urban, landscape, and nursery systems. Entomopathogens will also be of significant importance in non-agricultural situations where chemical pesticide use is undesirable and poses a higher human or environmental risk.

Given that the research needs indicated above are common to numerous commodities across the US, a cooperative multi-state approach is demanded to provide broad impact solutions that are widely applicable. Entomopathogens and their pest insect hosts are not limited by artificial boundaries. Therefore, tests of efficacy, persistence, safety, resistance management and other parameters must be conducted under different sets of environmental conditions across state lines. Protocols must be developed and standardized for the diverse types of research being proposed. Thus, to be successful in fulfilling the objectives of this project proposal, multi-state cooperative research among universities, USDA, and industry partners is required.

We anticipate that the project will produce substantial benefits for both producer and consumer stakeholder groups. Stakeholders will include farmers, pest control advisors, biopesticide industry, the scientific community, and the general public. Experiments will be conducted in numerous cropping systems; and, given the broad nature of the research, we anticipate significant knowledge transfer to additional crops during the project period. Foremost, the proposed research will facilitate transition away from the reliance on chemical insecticide usage by providing effective and environmentally friendly alternatives. Further development of insect pathogens for use in pest management programs will fill vital gaps in pest management caused by removal of broad-spectrum chemicals. This is of particular importance in specialty crops that have few remaining pest management options. Furthermore, as new pest assemblages arise, novel microbial control tactics will contribute substantially to the development of innovative IPM programs. Additionally, microbial control plays an important role in several market sectors, including organic crops, and in the control of invasive pests. The proposed project will improve quality of life by providing farmers tools to manage arthropod pests without risk of poisoning, and by providing the public with food containing less chemical residue. Economic opportunities will be created by enhancing the biological control industry and improving the productivity of various crops. The broad and unique expertise represented by our working group will enable us to achieve our ambitious objectives. This project strongly addresses several US agriculture and SAAESD Priority Areas, particularly Priorities 1, 4, and 5, i.e., Developing greater harmony between agriculture and the environment; Establishing an agricultural system that is highly competitive in the global economy, and Enhanced economic opportunity and quality of life for Americans.

Related, Current and Previous Work

The proposed project is unique. There is no significant overlap with other multi-state projects or individual CRIS projects. Our project focuses on microbial control of a wide range of arthropod pests involving researchers from California to Maine. This is the only multistate project that focuses on microbial control of arthropod pests. Our project also complements other multistate projects focusing on environmentally sustainable pest management practices, including the S1058: Biological Control of Arthropod Pests and Weeds, https://www.nimss.org/projects/view/mrp/outline/14496.

A CRIS search (using keywords such as microbial control, insect, entomopathogenic, as well as individual genus names of interest) reveals that the vast majority of research on applied microbial control of arthropod pests in the US is being conducted by our project participants and collaborators, who are world class scientists. Although some level of research with entomopathogens is being conducted at several institutions outside of our project participation the specific objectives of these projects do not overlap directly with those of our project. We will continue to encourage all scientists working on microbial control of arthropod pests to join our project as participants or collaborators; we will ensure that these scientists are aware of our project by advertising through professional societies (e.g., Entomological Society of America (ESA), Society of Invertebrate Pathology) and other channels. We will continue to organize the annual meeting of our working group at ESA annual meetings, the largest venue for entomologists worldwide.

The proposed project builds strongly upon decades of research on invertebrate pathology and microbial control. The breadth of research on insect pathology and microbial control is reviewed in various texts such as Tanada and Kaya (1993), Metz (2003), Grewal et al. (2005), Ekesi and Maniania (2007), Vega and Kaya (2012), and Lacey (2016). 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 400 scientific publications. The following section lists examples of significant accomplishments the project has made over the past five years. Advancements were made in the discovery, production, formulation and efficacy of microbial control agents used in biological pest suppression. Additionally, significant advances were made in studying the basic biology of microbial control agents. Research conducted under this project has led to the initiation of microbial control, or expanded microbial control, for dozens of arthropod pests in diverse systems. In the proposed project, we will expand microbial control efforts against these targets as well as others. Thus, the accomplishment examples listed below serve as a basis for future research during the next phase of the project.

Accomplishments from prior S1070 subproject objectives:

SUBPROJECT 1: Discovery of entomopathogens and their integration and safety in pest management programs for major acreage crops.

Anamika Sharma (Virginia Tech) worked with USAID, IPM innovation lab, at Virginia Tech. In Tanzania, currently using Metarhizium and Beauveria against stem borers, Trichoderma, and Bacillus sp. against rice blasts. Real IPM (private sector) in supplying all the microbial agents. In the future they also plan to use Trichoderma as foliar application for rice. For managing fall armyworm, planning to use entomopathogenic fungi (EPF) along with egg and larval parasitoids. Strains of entomopathogenic fungus (Metarhizium and Beauveria) were tested in the field conditions to manage wireworms (Coleoptera: Elateridae) in wheat and barley crops in Montana. These fungus formulations were obtained from Dr. Stefan Jaronski and were formulated as granules to improve the efficacy of EPFs in managing soil-dwelling arthropods. Commercially available biorational products were also evaluated to manage flea beetles, (on canola), pea leaf weevil (on pulses), and alfa-alfa weevil (on alfa-alfa crop) in Montana.

 Robert Behle (USDA-ARS) in cooperation with scientists from the University of Georgia and ARS (Byron, GA), Crop Bioprotection (CBP) scientist (Robert Behle-ARS, Peoria, IL) studied methods for liquid culture production of the Wf GA 17 strain of Cordyceps javanica blastospores along with processing using a spray dryer to create a wettable powder formulation using skim milk powder. Media with greater nitrogen concentrations produced more spores although media costs increased. Production of Wf GA 17 was less than that of the commercial Apopka-97 strain of C. javanica (formerly C. fumosorosea) when produced under identical culture conditions. In addition, CBP scientist Patrick Dowd continues to study the impact of host plant resistance genes in corn that target plant fungal pathogens for their direct impact on insect pests and EPF used to control these same insect pests in an evert to understand interactions among pest control strategies and pest damage.

Researchers at University of Georgia and USDA-ARS (Byron, GA) collaborated with USDA-ARS scientists (Louela Castrillo, Ithaca, NY; Robert Behle -ARS, Peoria, IL) to identify and isolate the Wf GA17 strain of C. javanica from field epizootics among whitefly populations in southern GA and tested the environmental tolerance of the new strain compared to commercial strains (Wu et al., 2020, 2021). They also conducted field trials of the new fungal strain for managing whiteflies in cotton and evaluated persistence of blastospores post application.

In collaboration with EMBRAPA, Brazil and Ismailia Agricultural Research Station, Egypt, scientists at USDA-ARS, Peoria, IL developed a biopesticide with Bacillus thuringiensis and Beauveria bassiana, which had synergistic insecticidal activity against the cabbage looper, Trichoplusia ni.

Scientists at USDA-ARS, Sidney, MT developed a prototype loop-mediated isothermal amplification (LAMP) method for identifying B. pseudobassiana and generic Beauveria in plants and other places in an effort to simplify molecular characterization. They also evaluated the efficacy of B. bassiana encapsulated in alginate microbeads against chewing pests and endophytic potential of five B. pseudobassiana isolates from wheat stem sawfly, Cephus cinctus in 15 varieties of spring, winter, and durum wheat. Additional studies included survey of the seasonal occurrence of Zoophthora phytonomi and Beauveria spp. in alfalfa weevil, Hypera postica in alfalfa, and field evaluation of B. thuringiensis subsp. gallariae-based biopesticide with Cry 8Da protein against larvae of H. postica.

Researchers at Montana State University, Conrad, MT evaluated several strategies for controlling wheat midge, Sitodiplosis mosellana with entomopathogenic nematodes, entomopathogenic fungi, and other options and the impact on secondary pests. In Montana, entomopathogenic nematodes were also evaluated to manage soil-dwelling wireworms. This task was accomplished by a Ph.D. student (Ramandeep Kaur Sandhi, supervised by Dr. GVP Reddy) with the collaboration of Dr. David Shapiro-Ilan.

SUBPROJECT 2 and 3: Discovery of entomopathogens and their integration and safety in pest management programs for ornamental, vegetable, fruit (orchards)and nut crops

Stefan Jaronski (USDA-ARS, Retired; Virginia Tech) spotted lanternfly has spread to many locations including Virginia. He has a graduate student, Jason Bielski, working on management of Lanternflies at Virginia Tech, while Eric Clifton at Cornell has been looking at various native Beauveria for Lanternfly management. Clifton screened the virulence of different commercial fungi and presented the work in ESA 2020. His bioassays targeted 1^st through 4th instars, and adults, and found differential results varying with stages. For 1^st instar, Velifer (B. bassiana strain from BASF) was more effective than the BioCeres Beauveria strain (ANT-03), Beauveria GHA strain, Beauveria ATCC74040 (Naturalis®) or C. javanica Apopka 97 (PFR-97®). Against 2^nd to 4^th instars, many of the differences among fungi disappeared; overall, Apopka97 was inferior to all Beauveria strains.

Pasco Avery (University of Florida) isolated an EPF from whitefly; it could be Cordyceps. They have sent the samples to USDA-ARS in Peoria, IL to get them identified. They will evaluate if this can be used to manage whiteflies. Bemisia tabaci has been a pest of vegetables in Quincy, Florida. A fungal epizootic was found among B. tabaci and the isolate was identified as C. javanica. Same species was identified on B. tabaci from an epizootic in Gainesville, Florida. Earlier it was also found in Georgia (Shaohui Wu). They are trying to identify if the strain is the same. Dr. Martini is putting together a grant to work on this.

Adam Chun-Nin Wong (University of Florida) continuing their basic research on the symbiotic gut microbiome associated with Drosophila suzukii and Mediterranean fruit fly (with colleagues from Israel). A new automated video tracking system has been established in my lab to study microbial impacts on insect foraging behavior. They discovered that the microbiome influences suzukii foraging activity in a sex-specific manner, due to differences between male and female reproduction response to microbiome manipulation. They also developed a hybrid genome assembly method integrating short-read (Illumina) and long-read (nanopore) sequencing to resolve the complete genome of fly gut symbionts.

David Shapiro-Ilan (USDA-ARS) involved in PEER project in South Africa and Benin, on sweet potato weevils with EPNs in various formulations. It’s known that nematodes may lose virulence and other beneficial traits after several passages, and there are some work on inbred lines in South Africa. In conjunction with Shaohui Wu’s work on whitefly management in cotton, they are also doing the test with vegetables. Also, he is involved in the whitefly project in collaboration with Dr. George Mbata at Fort Valley State University and his postdoc Yinping Li for using EPNs against whiteflies and screening for the best strains. They also did a screening test on EPNs and EPF and their combinations against tobacco thrips, conducted by the Ph.D. visiting students from Pakistan, and saw some positive results.

Albrecht M. Koppenhofer (Rutgers University) continued a study on the use of EPNs for the control of plum curculio (PC), Conotrachelus nenuphar, in highbush blueberries. Laboratory and field studies were conducted to determine the persistence of S. riobrave, S. carpocapsae, S. feltiae, and Heterorhabditis bacteriophora in acidic blueberry soil; compare the virulence of these EPNs to PC larvae and pupae; and compare the efficacy of these EPN species to control this pest in blueberry fields. The greatest persistence in blueberry soil was exhibited by S. riobrave followed by S. carpocapsae. Superior virulence was observed in S. riobrave against PC larvae and pupae. In the field, S. riobrave provided significantly higher levels of PC suppression (90%) than the others EPNs (all at 50 IJs per cm² = 1.67 billion IJs per ha total blueberry hectarage). In 2021, the field efficacy of S. riobrave against PC at low (25 IJs per cm²) and high rate (50 IJs per cm²) was confirmed. S. riobrave has the potential to become an important component in the management of PC in highbush blueberry.

Anamika Sharma (Virginia Tech): In Kenya, using Trichoderma harzianum, T. asperellum (Real Trichoderma), and T. harzianum + T. asperellum to manage root knot nematodes and bacterial wilt in cauliflower, kale and tomato crops. Farmers have found that use of T. asperellum was able to reduce the root knot nematodes populations by 75%. For cauliflower, the seedlings were raised in an insect-proof nursery. The IPM package including rice straw mulching, application of biological controls Trichoderma viride, B. bassiana, Pseudomonas fluorescens, neem oil, yellow sticky traps, and sweet bait made from cabbage and molasses to trap moths were found to be highly successful.

Stefan Jaronski (USDA-ARS, Retired; Virginia Tech) collaborated with APHIS for control of Asian citrus psyllids, a pest problem in South Texas, with EPF. They did spray type bioassays using all the U.S. commercial strains of fungi plus several noncommercial strains, as conidia and, where appropriate, as blastospores. NOFLY^® and BioCeres^® WP were far superior to PFR-97™.  The manuscript has been accepted recently. The work was conducted by the staff of Dr. Dan Flores at the APHIS Mission TX lab.

David Shapiro-Ilan (USDA-ARS) conducted collaborative research at USDA-ARS, Byron, GA addressed multiple issues that included the following: fungal endophytes in pecan, looking at different cultivars and inoculation methods, impact on pecan aphids and also plant growth; impact of the host plants on the efficacy of entomopathogenic nematodes; optimization of artificial media components for producing Steinernema feltiae (Leite et al., 2016); following; following previous report of antibiosis (e.g., to entomopathogenic fungi) in an insect pupal cell in pecan weevil, Curculio caryae (Shapiro-Ilan and Mizell, 2015), confirmed antibiosis of pupal cells in three other weevils and identified a bacterium suppressive to insect and plant pathogenic fungi (Wu et al., 2021); discovered that the bacterial-based product from Chromobacterium subtsugae can provide equal levels of control for pecan weevil relative to the standard chemical (carbaryl or pyrethroids) (Shapiro-Ilan et al., 2013, 2017, Shapiro-Ilan and Wells, 2021); protecting entomopathogenic nematodes from environmental damage (e.g., UV radiation and desiccation) with low concentrations of a specialized fire gel formulation, Barricade against some greenhouse pests and the lesser peachtree borer, Synanthedon pictipes (Shapiro-Ilan et al., 2015); and effective control of the peachtree borer, Synanthedon exitiosa through preventative or curative spring-time applications of S. carpocapsae (control of the pest was equal to standard chemical application, chlorpyrifos) (Shapiro-Ilan et al., 2016); enhanced entomopathogenic nematode movement, infectivity and biocontrol efficacy was discovered upon exposure to ascaroside nematode pheromones; exposure of entomopathogenic nematodes to pheromones led to increased control of pecan weevil and is expected to be applicable to other insect pests as well (Oliveira-Hofman et al., 2019; Shapiro-Ilan et al., 2019).

Scientists at Cornell, Ithaca, NY, Illinois Natural History Survey, Urbana, IL, and USDA-ARS, Gainesville, FL have been working on a microsporidian pathogen infective to the invasive brown marmorated stink bug, Halyomorpha halys and other stink bugs.

SUBPROJECT 4: Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural landscapes.

Stefan Jaronski (USDA-ARS, Retired; Virginia Tech) has ongoing collaboration on grasshopper microbial control, a program at APHIS in Phoenix, Arizona, continuing research he accomplished while in ARS. The work focus in the past few years has been to develop a bait carrier for fungus for control of grasshoppers. Jaronski has been working on tick biocontrol since Met 52 disappeared from the marketplace at the end of 2020. He conducted a preliminary screening bioassay spraying fungus spores at the equivalent of 2*10^13 conidia/acre using the strains GHA (Certis Biologicals), PPRI 5339 (BASF), ANT-03 (Anatis Bioprotection), the Naturalis L strain (Lallemand), and Apopka-97 (Certis Biologicals) against adults of two of the tick species present in Virginia. Jaronski reported collaborative work with EMBRAPA in Brazil, whereby microsclerotial granules of Metarhizium robertsii could serve as a biocontrol tool of the cattle tick, Rhipicephalus microplus, under semi-field conditions.

Robert Behle (USDA-ARS) cooperated with USDA-APHIS personnel and assisted with studies on Ovavesicula popilliae, a host-specific microsporidia for control of Japanese beetle. Beetles trapped around Peoria will be evaluated for the presence of this organism using specific PCR primers. CBP Post-Doc (Kristin Duffield – ARS, Peoria, IL) administrated and participated in the Insect Meal Grand Challenge, a project exploring the use of insect cultures as livestock to provide a new source of feed and food. Her research focus was on identifying and understanding the impact of insect pathogens on these cultures and has recently published results of initial studies on viruses infecting cricket colonies. CBP scientist (Jose Ramirez – ARS, Peoria, IL) evaluated entomopathogenic microbes for control of mosquitoes and the impact of pathogenic and endemic microbes on the vector competence of mosquitoes.

Navneet Kaur (Oregon State University), working on using grass endophytes to manage insect pests, surveyed and determined what native EPN species occur in grass seed fields and tested if they can offer effective control against sod webworms. Soil samples were collected during a field survey at biweekly intervals during March-May 2021 in 22 commercial grass seed fields in western Oregon. Out of 88 composite samples (440 single point samples); nematodes were recovered from 22 samples. Three different EPN isolates were identified using molecular tools. BLASTn analyses indicated that the most predominated Oregon EPN isolate (Oregon_WV-3) were conspecific to an unidentified EPN isolate N6734 from Nebraska Cornfields (GenBank accession: MK754228) with up to 100% identity of the partial sequence of the cytochrome oxidase subunit 1 (cox1) gene and other two isolates being conspecific to Steinernema and Oschieus sp. Laboratory infectivity tests using native strains identified in this study are in progress.

Albrecht M. Koppenhofer (Rutgers University) tested whether inoculative applications of native EPN strains adapted to persist in the local conditions can effectively suppress turfgrass pest populations for several years. They surveyed fairways and roughs at two golf courses in central New Jersey for such EPNs. The majority of EPNs collected were H. bacteriophora and S. carpocapsae. EPN populations were determined 1 week before application and again 1, 4, 6, 13, and 16 months after application. Higher EPN numbers were detected in the rough vs. the fairway. EPN numbers also tended to be higher in the treated plots than the untreated plots for the species the plots were treated with. A third species, likely S. cubanum, was also found regularly in many plots. Annual Bluegrass Weevil (ABW) populations were determined in mid-June 2020 and 2021. In both years, numbers in the fairway were significantly lower in the plots treated with both EPN species than in the untreated plots and the ones treated with S. carpocapsae only. The only other insects found in significant numbers during the ABW sampling were larvae of the black turfgrass ataenius (BTA), numbers of which were not significantly affected by EPN treatments. Surface- active insect populations were determined in July and early September of 2020. Soap extraction revealed many insects but only adults of ABW and especially BTA were found in number high enough for meaningful analysis. ABW numbers in the fairway were lower in the plots treated with carpocapsae and the species combination than in the untreated plots. BTA numbers in the fairway were lower in all EPN treatments than in the untreated plots. White grub populations were determined in late September 2020 and 2021. In the rough, densities were significantly lower in the plots treated with H. bacteriophora than in the untreated plots. BTA larvae were not affected by EPN treatments.

David Oi (USDA, ARS-Gainesville, FL) and Steven Valles continued the search for viral pathogens for the biocontrol of the little fire ant, Wasmannia auropunctata, an invasive, stinging ant. Transcriptome sequencing of little fire ant samples from Florida, Hawaii, and Argentina collected in 2020 revealed six sequences that appear to be of viral origin across several families. Some of the virus sequences were only found in the Argentine samples. Samples collected in 2021 from Florida, Hawaii, and Australia confirmed this result. These represent the first viruses from the little fire ant. Surveys to track the spread of Solenopsis invicta virus 3 (SINV-3) in fire ants at an inoculation site located in the Coachella Valley of California (Palm Springs area) in 2021 were postponed to 2022 due to COVID-19. Inoculations were initially conducted in 2014 resulting in very localized spread.

Pasco Avery (University of Florida) quantified the effect of plant extracts from croton on the viability of EPF. Excised leaves used in the experiment were categorized as either young (newly formed leaves at the bottom of the plants, with purple and green coloring only, ~65 days old) or mature (variegated colors, ~105 days old). After 15 days, extracts from the young and the mature croton leaves incorporated in the agar had a significant stimulatory effect on the fungal hyphal growth of C. javanica Apopka strain and B. bassiana GHA strain in vitro compared to the control with no extract added. For both fungi, there was no significant difference in the stimulatory effect on in vitro fungal hyphal growth between leaf extracts of young or mature croton leaves incorporated into the agar. Identification of biochemicals by using gas chromatography-mass spectrophotometry analysis is still in progress.

David Shapiro-Ilan (USDA-ARS) collaborated with John Goolsby on using EPNs to control cattle fever tick (a big problem in Texas) in rangeland. The application was made by smart spraying when the animals come to feed. It was found that EPNs were viable on the grass ground in the spraying application, and the cattle or antelope can pick up the nematodes. In lab and outdoor tests, Barricade^® gel improved nematode survival on cow hide, but the Barricade test has not been conducted on the animals yet. A screening test on EPN efficacy for control of the small hive beetle, a serious pest of bee hives, was conducted by targeting the pupal stage under the ground, and S. carpocapsae was most effective. They have a new project in collaboration with Fort Valley State University, and a Master student has worked on screening the nematodes for their persistence in soil for prolonged control of the small hive beetle using different formulations.

Anamika Sharma (Florida A&M University) has been working on the management of urban pests, including fire ants, stored product pests, and termites. Implying registered microbial pesticides (strains of Metarhizium and Beauveria) to manage fire ants (Solenopsis invicta) and working on the attract and kill traps. 

Objectives

1. To establish, collaborate and promote research on entomopathogens and the use of microbial control options in IPM strategies for large acreage crops (alfalfa, corn, dry beans, potatoes, and small grains).
   
2. To establish, collaborate and promote research on entomopathogens and the use of microbial control options in IPM strategies for orchard systems (fruits and nuts)
   
3. To establish, collaborate and promote research on entomopathogens and the use of microbial control options in IPM strategies for small fruits and vegetables (blackberries, blueberries, raspberries, strawberries, and vegetables).
   
4. To establish, collaborate and promote research on entomopathogens and the use of microbial control options in IPM strategies for urban and natural landscapes, rangelands, and nurseries.
   

Methods

General Methods & Approach Across Subprojects:

Building upon the accomplishments from the previous project, research will target enhanced implementation of microbial control in IPM systems. Research will also elucidate the basic biology and ecology of entomopathogens and their multipurpose use in crop production and protection.

Following general scientific research procedures, laboratory, greenhouse, and field research will be conducted to evaluate the potential of entomopathogens as effective alternatives to chemical pesticides, important options in IPM programs, and enhancers of plant growth and health. There is a need for operational scale field evaluation of entomopathogens to determine how they can be integrated into IPM programs that are acceptable to growers. Studies to evaluate the efficacy of entomopathogens individually and in combination with each other or botanical and chemical pesticides will be conducted against endemic and invasive pests.

Enhanced implementation of microbial control in diverse systems will be achieved in a multi- faceted approach. Entomopathogen strain improvement techniques will be implemented to enhance efficacy. Efficacy will also be improved by developing novel application techniques and optimizing parameters such as application rates and timing. Efforts will include conservation, classical introduction, and inoculative or inundative approaches to biocontrol.

Research will also be directed toward understanding fundamental entomopathogen biology and ecology and avenues to improve their efficacy by modifying agricultural practices. For example, recent studies suggest that entomopathogenic fungi antagonize plant pathogens, impact arthropod pests through endophytic colonization, and promote plant growth forming a mycorrhiza-like relationship with plant roots (Parsa et al. 2013). Entomopathogen-based metabolites also appear to have a potential as biopesticides (Orozco et al. 2016; Sbaraini et al. 2016). Among the subprojects, the role of entomopathogens as pathogens of arthropods, antagonists of plant pathogens, plant growth promoters, endophytes, or producers of toxic metabolites will be evaluated. Basic research will study the interactions of insect immune system response when exposed to entomopathogens as well as alterations to the insect’s gut microbiota.

The following subprojects will identify some key pests and develop strategies to improve microbial control integration in IPM programs.

Subproject I: Large acreage crops

Subproject I Collaborating Institutions: USDA-ARS, Byron, GA, and Peoria, IL; University of Georgia, Tifton, GA.

Research on microbial control of insect pests infesting major acreage crops has culminated in the development of new and significant pest control technologies. For this proposal, we define major acreage crops to generally include commodity grain crops and forage crops along with many common food crops under commercial agricultural production, such as potatoes and dry beans. Two of the most significant historical technologies were the development of transgenic Bt-crops for pest control and discovery of new pesticides from microbial fermentation, both of which provide wide-spread control of arthropod pests of major acreage crops and other pest control situations. Successful implementation of microbial control for major acreage commodities offers substantial rewards as a result in the reduction of chemical pesticide applications. Unfortunately, large scale agricultural production imposes economic restrictions on costs for pest control, often precluding applications of relatively expensive biological pesticides when compared with less expensive chemical insecticides. As a result, a significant research effort focuses on reducing costs of production and formulation of microbial biopesticides. Successful implementation of microbial control against large acreage crops continues to require innovative and specialized technology advancement and application.

Discoveries of improved strains of microbial agents (Wu et al. 2020), more efficient production techniques (Behle et al. 2022), and effective applications within integrated pest management continue to provide opportunity for adoption of microbial-based biological control for large acreage crops. New technologies include: fermentation techniques that produce novel fungal structures [microsclerotia (Jaronski and Jackson 2008) or blastospores (Muscarin et al. 2016)] formulations such as alginate beads or hydro-mulch containing natural products to protect microbes from environmental degradation, discovery of new pathogens and isolates of known pathogens with better virulence to native and exotic insect pests, and effective integration of pathogens into cropping systems for targeted pest control.

As specific research projects, insects with piercing and sucking mouthparts remain a primary target for control using entomopathogenic fungi. Whiteflies infesting cotton and stink bugs infesting soybeans represent two situations where successful research will support implementation of microbial pest control. Several weevil pests have been identified as targets for microbial control. Weevils are a large and diverse group of insects, which includes many economically important pest species. The alfalfa weevil (Hyper postica) causes damage to alfalfa beginning early in the growing season. A variety of biorational pesticides are active against the damage-causing larval stage when evaluated under laboratory conditions (Reddy et al. 2016) and may also be susceptible to relatively traditional foliar spray applications in the field. By contrast, the sweet potato weevil (Cylas formicarius) is a world-wide pest that damages sweet potatoes in the soil environment. Although susceptible to a variety of bacterial, fungal, and nematode agents, specialized applications may be required to control the pest and prevent crop damage. Like the sweet potato weevil, the Cowpea weevil (Callosobruchus maculatus) adds a twist to crop damage, causing plant damage in the field followed by the potential to cause additional damage for harvested seeds while in storage. Thus, controlling field populations provides added benefits as reduced damaged during storage. Potential control strategies for cowpea weevil in the field will focus on applications of entomopathogenic fungi and nematodes. Beyond these weevils, diapausing larvae of the wheat stem sawfly (Cephus cinctus) are susceptible to infection by endophytic strains of entomopathgenic Metarhizium and Beauveria fungi. Basic and applied research are expected to elucidate plant/microbe/insect interactions for the development of these endophytes to provide plant protection. Wireworms (Coleoptera: Elateridae) continue to damage small grains and potatoes grown in the western high plains. Planting time applications of entomopathogens as granules or seed coatings will be evaluated for economics of pest control under field conditions.

Additionally, basic research on entomopathogens continues in the form of studying the interactions among trophic levels of crop production. For example, implementing host plant resistance for control of fungal plant diseases can impact efficacy of fungal insect pathogens applied as bioinsecticides. Further, efforts continue to complete genome sequences for entomopathogenic microbes that are housed in culture collections to verify proper taxonomy and also for discovery of specific genes providing beneficial traits to those organisms. Once known, these genes and traits can be manipulated to enhance those characteristics needed for effective pest control. These basic studies will support further development of entomopathogenic microbes as pest control agents in all the subprojects of this proposal.

Subproject II: Orchard systems

Subproject II Collaborating Institutions: USDA-ARS, Byron, GA, Kearneysville, WV; University of Georgia, Tifton, GA; University of Florida; Tennessee State University; Michigan State University.

A number of key orchard pests have been identified as targets for microbial control and varying levels of farm-scale adoption have been accomplished against these pests. The pending project will conduct advanced studies for improving microbial control against these important pests.

In stone fruits, the peachtree borer is a highly attractive target for entomopathogenic nematodes, particularly due to the pending removal of chlorpyrifos (the standard chemical used against this pest). The pest attacks trees at the base and girdles down into the roots. Research to-date indicates nematodes (S. carpocapsae) can control peachtree borer curatively or preventatively in an economically feasible approach (e.g., approximately $10.00 per acre). Research will be implemented to optimize nematode application rates and timing for peachtree borer control and also to determine other simultaneous benefits of nematode application such suppression of root- feeding weevils and plant parasitic nematodes residing in stone fruit orchards. This research has been bolstered by a USDA-SARE grant (ending 2022) and a USDA-CPPM grant (ending 2025).

Another key pest in stone fruits is the lesser peachtree borer, which attacks the tree aboveground. The use of entomopathogenic nematodes aboveground is generally not feasible due to environmental sensitivity of the nematodes; yet use of a fire-gel (Barricade) formulation allows for effective application of the nematodes for lesser peachtree borer control (equivalent levels of control compared with chlorpyrifos). Additional research will be focused on optimizing Barricade or other formulations for economically feasible control of lesser peachtree borer; significant promise has been discovered in some low-cost natural formulation compounds. The method will also be applied to other borer insects of economic importance such as dogwood borer, shot hole borers, roundheaded appletree borer.

Plum curculio, Conotrachelus nenuphar, is a key pest of stone and pome fruits. The insects attacks fruit directly. Entomopathogenic nematodes have been shown to be highly effective in killing ground-dwelling stages of the pest (before adults emerge). An integrated trap-tree system has been developed to attract weevils to several trees per hectare; an adulticide is sprayed on the canopy and then nematodes are used to kill off remaining plum curculio below ground to prevent spread into the interior of the orchard. Research will be implemented to optimize application parameters in the novel sentinel tree approach and expand the method to other crops (so far it has only been tested in apples yet it has potential in other crops such as peaches, plums, pears, etc.). Additionally, border sprays, which will also entail reduced costs, will be explored for microbial control efficacy.

Research will be expanded to develop microbial control programs for pecan weevil (a key pecan pest). Grandevo® (C. subtsugae) application rates and timing will be optimized and integrated use of entomopathogenic nematodes and fungi for weevil hotspots will be incorporated. This research has been provided a boost from a funded USDA-NIFA-Organic Transitions (ORG) grant.

Ascaroside pheromones from entomopathogenic nematodes have been found to enhance nematode movement, infection and efficacy in biocontrol in greenhouse and small field trials. The ability to use these pheromones in larger field trials against pecan weevil, peachtree borers and other orchard pests (e.g., ambrosia beetles and flatheaded borers).

Endophytic fungi such as Beauveria bassiana have been shown to suppress insect pest populations. Recently, B. bassiana has been successfully established as an endophyte in pecan trees. Additional research will explore suppression of pecan pests such as ambrosia beetles and pecan aphids. As time allows the approach will also be expanded to other pests and orchard cropping systems.

The citrus weevil, Diaprepes abbreviatus, is a major pest of Florida citrus; the insect feeds on roots and can kill trees. Florida citrus is threatened due to greening disease and D. abbreviatus exacerbates the situation. The pest has also spread to citrus in Texas and California. Entomopathogenic nematodes can provide high levels of control against D. abbreviatus. Research will be implemented to optimize nematode species effects and efficacy while integrating into new management plans that have been initiated to counter the spread of greening disease. Research will also be conducted to develop the use of entomopathogenic nematodes for D. abbreviatus control in new crops that are expanding in Florida due to the loss of citrus (e.g., peaches).

Subproject III: Small fruits and vegetables

Subproject III Collaborating Institutions: Coastal California Counties; Driscoll’s, Coastal and Central California Counties.

A variety of small fruits and vegetables are important commercial crops in California with a crop value of several billion dollars. Several arthropod pests attack these crops causing significant yield losses. Except in a few situations where biological control is possible by the release of commercially available natural enemies, growers typically rely on chemical pesticides. Significant quantities of several insecticides and acaricides are applied annually (CDPR, 2016). Some important pests include masked spotted lantern flies (Lycorma delicatula), chafer white grubs (Cyclocephala hirta and C. longula), spotted wing drosophila (Drosophila suzukii), and citrus thrips (Scirtothrips citri) in blueberries; spotted wing drosophila, twospotted spider mite (Tetranychus urticae), western flower thrips (Frankliniella occidentalis), lygus bugs (Lygus hesperus and L. lineolaris), red berry mite (Acalitus essigi), and broad mite (Polyphagotarsonemus latus) in caneberries; the western tarnished plant bug (Lygus hesperus), greenhouse whitefly (Trialeurodes vaporariorum), two-spotted spider mite, and multiple species of Lepidopteran larvae on strawberry; green peach aphid (Myzus persicae), cabbage aphid (Brevicoryne brassicae), cabbage maggot (Delia radicum) on cole crops; western flower thrips on lettuce. While these pests are susceptible to a variety of entomopathogens, microbial control is not widely considered a choice, especially in conventional agriculture. Several studies conducted in California or elsewhere show the potential of microbial control in small fruits and vegetables (Dara, 2016; Klick and Seagraves, 2015, 2016). Follow-up surveys conducted after the extension meetings and feedback received for publications related to microbial control indicate a significant interest of the growers in such control options (Dara, unpublished). The preference of consumers for organic and sustainably produced fruits and vegetables also emphasizes the need for non-chemical pest management options such as microbial control. California supplies a major portion of fruits and vegetables to the United States and has a significant impact on agriculture. Promoting microbial control in California is essential for promoting sustainable crop production. Ann Hajek (Cornell University) and her team have been working on establishing bioassays with Beauveria bassiana and Batkoa major to manage spotted lanternflies.

Additionally, studies conducted in California indicate a positive impact of entomopathogenic fungi in promoting plant growth and health as well as antagonizing plant pathogens (Dara, 2013; Dara et al. 2016a & b). More studies are needed to understand the non-traditional role of entomopathogens, which can enhance their use in agriculture.

Multiple studies will be conducted on different crops:

1. Evaluating the efficacy of entomopathogens alone and in combination with other control options against different arthropod pests to identify avenues to incorporate microbial control in small fruit and vegetable IPM in organic and conventional agriculture in California.
2. Extending training and guidance in microbial control to facilitate grower adoption.

Subproject IV: Urban and natural landscapes, rangelands, and nurseries

Subproject IV Collaborating Institutions: Cornell University, Ithaca, NY; Oregon State University, Corvallis, OR; USDA-ARS, Gainesville, FL; Rutgers, New Brunswick, NJ; Texas A&M, College Station, TX; USDA-ARS, Byron, GA; USDA-ARS, Peoria, IL; Virginia Tech, Blacksburg, VA.

Invasive ants are a growing concern throughout the world, because of the broad diversity of habitats they can establish, thrive, and eventually dominate. Often, they have cryptic, smoldering populations that slowly increase, until their populations explode. By this time, the invaders are too widespread to eradicate and too expensive or logistically impossible to regionally suppress in natural or conservation lands, even in urban landscapes. Release from natural enemies is hypothesized to be a major factor in the dominance of invasive ant species (Porter et al. 1997). Biological control with entomopathogens is considered to be one of the few, if not the only, sustainable strategy for the regional suppression of invasive ants. Advances in metagenomics and sequencing have facilitated the discovery of pathogens in invasive ants such as the red imported fire ant, Solenopsis invicta, the tawny crazy ant, Nylanderia fulva, and the Argentine ant, Linepithema humile (Valles et al. 2004, 2012; Sébastien et al. 2015). We will continue to search for entomopathogens such as viruses to control invasive ants.

Turfgrass areas cover about 20 million ha in the USA and the size of the turfgrass industry is estimated at $40 billion per year. Homeowner lawns represent the largest part of the total turf area (66%). Golf courses only represent about 2% of the total area covered by turfgrasses in the USA, but, due to their much higher maintenance and use intensity, contribute about 20% to the total economic impact. Many different types of insect pests can cause damage to different turfgrass areas including white grubs (Coleoptera: Scarabaeidae), mole crickets, lepidopteran larvae (cutworms, armyworms, sod webworms), weevils (billbugs, annual bluegrass weevil), crane flies, and many others. Various entomopathogens have provided satisfactory control of several of these pests (Koppenhöfer et al., 2015, 2020, 2022; Koppenhöfer and Wu, 2017), but to increase the use of entomopathogens their efficacy and ease of use needs to be further improved and their potential for long-term pest suppression further exploited. Changing attitudes in consumers (e.g., increasing demand for organic lawn care) and local or statewide legislation (e.g., School-IPM programs) will also increase the need for effective entomopathogen-based products. The widespread resistance to multiple types of insecticides in the annual bluegrass weevil for example (McGraw and Koppenhöfer, 2017), should increase the demand for alternatives to synthetic insecticides on golf courses. We will evaluate new pathogen species/strains against white grubs, lepidopteran larvae, and annual bluegrass weevil adults and larvae. For example, the entomopathogenic nematode Steinernema riobrave has shown great potential for the control of several weevil species in other commodities. We will test it in turfgrass. Several bacterial species (e.g., Yersinia entomophaga; Hurst et al. 2011) have shown potential for insect control. We will test them for the control of annual bluegrass weevil, black cutworm, and white grubs in turfgrass. To optimize the performance of promising control agents we will test them in split applications, species combinations, and potentially synergistic combinations with other control agents (Koppenhöfer et al., 2020, 2022). Promising agents will also be tested for long-term pest suppression.

Rangelands are a uniquely diverse habitat that can benefit greatly through increased productivity from targeted applications of microbial control agents. Novel formulations that provide UV light protections to fungal spores show promise for improving the microbial control of rangeland grasshoppers. Additional studies will be conducted on managing rangeland grasshoppers with microbial control. We will also continue research projects on tick management with microbial agents.

Bark and ambrosia beetles (Coleoptera: Curculionidae: Scolytinae), especially the invasive redbay ambrosia beetle (Xyleborus glabratus), are destructive to many woody ornamentals and nursery plants (Urvois et al., 2022). The beetles vector the pathogenic fungus Raffaela, the causing agent of the laurel wilt disease, which has killed hundreds of millions of trees in the Lauraceae family since it was first detected in the USA in 2002. The susceptible tree species include redbay, Persea borbonia L. Spreng., and other native Persea species such as avocado, Persea americana Mill., pecan, and Carya illinoinensis (Wangenh.) K. Koch, and three critically endangered natives of the southeastern USA, Lindera melissifolia (Walter) Blume, Litsea aestivalis (L.) Fernald and Licaria triandra (Sw.) Kosterm (Hughes et al., 2017; Monterrosa et al., 2021). A multi-state project supported by Specialty Crop Research Initiative (SCRI) was recently initiated to explore strategies for the management of ambrosia beetles and diseases caused by their mutualistic pathogens. As part of the project, we will investigate the potential of using entomopathogens to control these pests.

Within all of these systems, the sustained presence of entomopathogens can contribute to the IPM of difficult-to-control pests.

Ongoing and future projects toward the microbial control of pests in urban and natural landscapes, and nurseries include:

1. Search for new entomopathogens of native, naturalized, and invasive pests.
2. Determine host susceptibility of native, naturalized, and invasive pests to entomopathogens.
3. Evaluate novel formulations and/or application techniques of microbial biopesticides.
4. Incorporate microbial biocontrol agents or microbial insecticides into IPM programs. 

Measurement of Progress and Results

Outputs

• Improved regional collaboration leading to new and enhanced microbial control tools, application strategies, and delivery mechanisms. Comments:
• Scientific publications including peer-reviewed journal articles, books, or book chapters.
• Extension meetings and publications to improve the knowledge and implementation of microbial control.
• Annual meeting of the workgroup members in conjunction with the Entomological Society of America (ESA) annual meetings.
• Microbial control symposium at ESA annual meetings organized by the workgroup members.
• Annual reporting that includes the summary of research achievements, publication lists, and tabulation of extension-related activities related to this project.
• Website to publish specific recommendations from workshops.

Outcomes or Projected Impacts

• Reduced impact on non-target organisms and the environment, and protection of human health due to decreased use of chemical pesticides.
• Improved use of microbial control options in conventional agriculture.
• Increased food security due to the development of alternative pest management tools that be applied to increase productivity and protect against new threats (e.g., invasive pests).
• An Increased market for biopesticides based on microbial control agents.

Milestones

(0):See attachment for Milestones.

Projected Participation

View Appendix E: Participation

Outreach Plan

This project will provide educational material for a) Entomology colleagues via symposia at national meetings, and refereed research publications, books, and book chapters; and b) Extension personnel and growers via participation in annual project meetings, trade shows, field days, presentations, trade journal articles and factsheets, 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. Additionally, outreach will also extend to numerous institutions some of the project participants have ties to these colleges (e.g., through adjunct professorships) and populations sectors they serve. 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. During COVID we all have learned and realized the importance of online resources. This project will build online resources for researchers and students to learn about production, formulations, calculations, and experiments involving microbials in arthropod management. Short videos of the annual microbial course (a weeklong insect pathology short course led by Ann Hajek at Cornell University), could be produced for outreach activity. Other members will also collaborate to create some educational videos to promote microbials.

Organization/Governance

Organization: Officers include Chair, Vice-Chair, Member-at-Large, and Secretary. Each position runs for a two-year term, at which time a new Member-at-Large and a new Secretary are elected at the annual meeting, the retiring the Member-at-Large replaces the Vice-Chair, and the retiring Vice-Chair replaces the Chair. The Chair is in charge of running all aspects of the project, and the Vice-Chair and Member-at-Large assist the Chair and serve in his/her place when the Chair is unavailable. The Chair can appoint other ad-hoc officers as needed. The Secretary’s primary responsibility is to compile annual reports and meeting minutes.

Current Officers:

Chair: Stefan Jaronski, MycoSystems Consulting, VA Vice-Chair: Julie Graesch, BioWorks, NY

Member-at-Large: Anamika Sharma, Florida A & M University, FL Secretary: Shaohui Wu, University of Georgia, GA

Administrative Advisor: Paula Agudelo, Clemson University, SC NIFA Representative: Vijay K. Nandula, REE-NIFA

Subproject Chairs:

1. Large Acreage Crops: Robert Behle, USDA-ARS-Peoria, IL

2.  Orchard Systems: David Shapiro-Ilan, USDA-ARS

3.  Small fruits and vegetables: Pasco Avery, University of Florida & Jimmy Klick, Driscoll’s, Oxnard, CA

4.  Urban and natural landscapes, rangelands, and nurseries: Albrecht Koppenhöfer, Rutgers & David Oi, USDA-ARS, Gainesville, FL

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Bioworks, Brigham Young University, USDA ARS, USDA-ARS