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ARS Home Midwest Area Peoria, Illinois National Center for Agricultural Utilization Research Crop Bioprotection Research Research





- Muturi, Ephantus
- Dowd, Patrick
- Dunlap, Christopher
- Ramirez, Jose
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Discovery and Production of Beneficial Microbes for Control of Agricultural Pests through Integration into Sustainable Agricultural Production Systems **Location:
                            Crop Bioprotection Research** 2023 Annual Report **Objectives**
                                

Objective 1: Develop effective entomopathogenic fungi for implementation as augmentative biological controls to support integrated pest management systems.

Objective 2: Expand fundamental knowledge of biological interactions between the beneficial pathogens(s), target host pest and crop environment to enhance the production, formulation, and application of beneficial microbial products for sustainable pest management. **Approach**
                                

The commercial use of microbial pathogens as biopesticides to manage crop pests continues to be constrained not only by expensive production methods, limited shelf-life, and variable pest control efficacy, but also by a lack of understanding of how basic fungal metabolism affects liquid-culture production in the factory and pest control efficacy in the field. This research project focuses on developing beneficial microbes (predominantly entomopathogenic fungi) as biopesticides and follows a vertical research path from understanding microbe metabolism during liquid culture production through practical formulation processing and integrative application into pest management systems. Although we have empirical data supporting efficient production of beneficial fungi, we still lack a basic understanding of the interaction between physical and nutritional conditions of liquid culture and the basic metabolisms of these organisms. To fill this void, effective microbial biopesticides will be developed by uncovering at the molecular level how entomopathogenic microbes interact with nutritional and environmental conditions present during the production, formulation, and application processes. Gaining this understanding is critical given that these processes likely affect fungal differentiation, biopesticide yield, product stability, and pest control efficacy. Post-production, research will evaluate specific processing and formulation technologies to create a usable product that retains physical characteristics suited for application against targeted pests and is expected to focus on product storage and handling characteristics for sprayable (yeast-like blastospores) and granular (microsclerotia-based) fungal biopesticides. Following application, the host plant environment will be studied to identify interactions among a variety of pest control practices (e.g. crop genetics providing host plant resistance to fungal pathogens) within specific cropping systems. Microbial biopesticides represent an additional tool for the management of crop pests. Non-chemical pest control tools such as these are particularly important for organic, chemically sensitive, and natural environments where few pest control measures are available, and to avoid the development of pesticide resistance to current chemical insecticides and transgenic crops used for pest control. The strategic development of microbial biocontrol agents will enhance the nation’s ability to effectively control pests and support increasingly sustainable crop production. **Progress Report**
                                

Objective 1.
In support of Sub-objective 1.1, the entomopathogenic fungus, Cordyceps javanica, has been used to create a biopesticide and was field tested for control of whiteflies infesting cotton and vegetable crops. This fungal isolate was recently patented, being more stable than commercial isolates, when exposed to hot weather that is typical in summer crop fields. ARS researchers in Peoria, Illinois, produced high quantities of the infective blastospore in liquid culture, which were processed by spray drying to form a wettable powder and was largely comparable to an identical formulation made with a commercial isolate of beneficial fungus for spore viability and insecticidal efficacy. When applied to field grown plants in collaboration with ARS researchers in Byron, Georgia, this treatment caused significant infections and mortality of whiteflies. This progress supports the use of beneficial microorganisms as biopesticides to control crop pests, especially to reduce chemical residues in vegetable crops. 
 In support of Sub-objective 1.2, ARS researchers in Peoria, Illinois, evaluated the respiration pathways of an important entomopathogenic fungi, Beauveria bassiana to establish how this fungus converts fermentation components into energy for growth. The results confirmed the alternative oxidase pathway is the primary respiration pathway in standard production conditions. In addition, we completed a gene expression study of Beauveria bassiana in the presence of different respiration pathway inhibitors. This data will be used to understand how growth conditions change the development of the fungus. This data is currently being analyzed and will help us understand the metabolic state of the fungi during liquid culture production. Understanding the role and function of these primary metabolic systems is key to the long-term development of entomopathogenic fungi for biological pest control. In recent years, the development and applications of such knowledge has enabled manipulations of physical and nutritional conditions resulting in improved biomass and secondary metabolite production from liquid culture.
Objective 2.
In support of Sub-objective 2.1, ARS researchers in Peoria, Illinois, have completed long read DNA sequencing for eight strains of entomopathogenic fungi to develop high-quality drafts of the genome. This information will allow us to better understand how entomopathogenic fungi have evolved to kill insects. This information will help guide us in improving the efficacy of these fungi in crop protection applications. In collaboration with ARS’s microbial culture collection (NRRL), a project to sequence the genomes of microbes associated with bees and bee environments continues to produce data. This project supports the agency wide initiative to improve pollinator health. A second set of 80 microbes were sequenced and the draft genomes have been assembled. The data are currently undergoing quality control, and it is anticipated that most of the data will be publicly released by the end of the calendar year. Currently, 64 genomes and associated data have been made available on GenBank. All raw data will be released to the GenBank sequence read archive and all processed data to the whole-genome sequencing portal on GenBank. The project will provide reference data and new information to better understand the tripartite interactions (microbe-insect-plant) that impact the life cycle of these important pollinators.
In support of Sub-objective 2.2, ARS researchers in Peoria, Illinois, in collaboration with cricket production farms across the United States, Canada, and Mexico, are creating the first catalog of viruses infecting commercially produced crickets in North America. Sixteen farms provided samples of their crickets to ARS for metagenomic sequencing to identify both known and unknown virus pathogens of these insects being grown as a commodity resource. Following robust screening of viruses, several known and many unknown pathogens were identified in these populations. This information is critical to the health of the reared insect industry as very little is known about the specific pathogens that result in production loss. Proper identification of these pathogens is the first step towards developing effective control strategies that will ultimately improve production of crickets as a viable agricultural commodity for use as human food and animal feed. 
Using funds awarded through the Office of Technology Transfer (OTT), ARS researchers are quantifying the global activation of genes as well as microbiome analysis across two populations of reared crickets: one that is infected with a highly pathogenic cricket iridovirus and the other that has an asymptomatic infection of the same iridovirus. These results identify the biological and molecular make up that determines infection outcomes in these insects and can be used as a model to understand viral asymptomatic infections in other insect production systems of agricultural importance (such as bees and black-soldier flies).
In support of Sub-objective 2.3, corn plants with transgenically introduced antifungal proteins for control of plant fungal diseases were evaluated by ARS researchers for the impact of these proteins on the efficacy of beneficial fungi applied as a biopesticide to control insect pests. Fewer insects that fed on leaves from some transformants treated with the insect fungal disease were killed over the same time period compared to those that did not contain the introduced gene. Identifying harmful interactions as observed here is necessary for effective pest management when integrating biological control measures for multiple crop pests. This information will be of value to regulators determining what nontarget studies should be performed prior to registration of transgenic plants with genes introduced to control fungal plant pathogens. 
As a new initiative, ARS researchers in Peoria, Illinois, collected potential biological control agents of the invasive tar spot of corn disease from infected field grown plants. Hundreds of samples were taken from corn fields and microorganisms infecting the tar spots were isolated and are being identified using genetic methods. Two bacterial isolates were applied as a seed coating to corn and planted in experimental plots. Although neither bacterium was re-isolated from the plants, disease evaluations indicated that the seed treatments lowered the levels of tar spot disease. Insects were also collected from corn fields with tar spot disease and are currently being evaluated for their potential to vector tar spot among corn plants. Identification of effective biological control organisms, once commercialized, should add an important component for an integrated control program for tar spot, resulting in improved corn production and lower prices for corn users and consumers. **Accomplishments**
                                

1. Discovered a gene in corn that codes for a protein that inhibits insect and fungal pests of corn. Insects and fungal diseases can damage corn with losses estimated in millions of dollars. Genes from corn that inhibit these pests are a useful way of reducing damage. For a second time in two years, ARS researchers in Peoria, Illinois, isolated a gene from corn that codes for a quinone oxidoreductase protein that slows the growth of plant pests: two insects and a disease fungus. When inserted in corn cells so the protective protein would always be produced, insect larvae feeding on these cells were smaller (up to 40%) and fungal pathogens grew slower (up to 47%). Continuous expression of this dual-purpose gene using gene editing in corn could reduce pest damage, resulting in a cheaper and higher quality product for consumers. 2. Identified food related additives that make biological control agents more effective. Use of biological control organisms is desirable because they are renewable and less hazardous to use compared to pesticides, but acceptance has been slow due to factors that cause variability in effectiveness. ARS researchers in Peoria, Illinois, identified food grade additives that enhanced the toxicity of two commercial formulations of biocontrol fungi against insect pests, increasing insect mortality rates by more than 100% in some cases. Adding these compounds to commercial formulations of biocontrol organisms will result in more predictable and effective control, increasing their rate of adoption and acceptability by growers. 3. Identified Cricket Iridovirus (CrIV) and other viruses that decimate mass-reared crickets. Due to their high feed conversion ratio and ability to remediate waste, mass-reared insects, including edible crickets, offer a sustainable solution for agriculture feed. While insect agriculture is rapidly expanding globally, including within the U.S., microbial pathogens pose one of the biggest hurdles for insect production farms. Therefore, the proper identification and characterizations of pathogens in insect farming is imperative. ARS researchers in Peoria, Illinois, studied multiple farms across the United States, Mexico, and Canada to catalogue known cricket viral pathogens including Cricket Iridovirus (CrIV), Acheta domesticus densovirus (AdDNV), and Cricket Paralysis Virus (CrPV), which cause deadly diseases in mass-reared crickets. Beyond known viruses, this team has also discovered novel and uncharacterized potential viral pathogens. This catalog of new viruses facilitates further research to understand their impact on insect farming and provides growers with information to devise control measures to prevent disease spread and limit production losses. 4. Sequenced reference genome of Beauveria bassiana, a fungus with important insect control properties. Bioinsecticides are a one-billion-dollar market in North America and additional improvements in their efficacy are needed to meet the needs of growers. Beauveria bassiana ARSEF 6444 is currently registered for use in crop protection in the United States to control insect pests on numerous crops. ARS researchers in Peoria, Illinois, completed sequencing a high-quality genome of Beauveria bassiana ARSEF 6444. The high-quality genome provides researchers with a blueprint of all the genes in the fungus. Researchers can use this information to determine how the fungus kills insect pests. It also allows researchers to study how this strain is different from other fungi that can kill insects. This high-quality genome will allow researchers to understand how this fungus kills insect pests. **Review Publications**
                            *Duffield, K.R., Foquet, B., Stasko, J.A., Hunt, J., Sadd, B.M., Sakaluk, S.K., Ramirez, J.L. 2022. Induction of multiple immune signaling pathways in Gryllodes sigillatus crickets during overt viral infections. Viruses. 14(12). Article 2712. https://doi.org/10.3390/v14122712.*
                                *Dowd, P.F., Naumann, T.A., Johnson, E.T. 2023. Potential role of a maize metallothionein gene in pest resistance. Plant Gene. 34. Article 100409. https://doi.org/10.1016/j.plgene.2023.100409.*
                                *Baati, H., Siala, M., Azri, C., Ammar, E., Dunlap, C.A., Trigui, M. 2022. Genomic analysis of heavy metal-resistant Halobacterium salinarum isolated from Sfax solar saltern sediments. Extremophiles. 26. Article 25. https://doi.org/10.1007/s00792-022-01273-0.*
                                *Silva, L.G., Camargo, R.C., Mascarin, G.M., De Oliveira Nunes, P., Dunlap, C.A., Bettiol, W. 2022. Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants. Frontiers in Plant Science. 13. Article 983127. https://doi.org/10.3389/fpls.2022.983127.*
                                *Ramirez-Carino, H.F., Morales, I., Guadarrama-Mendoza, P.C., Gonzalez-Terreros, E., Martinez-Gutierrez, G.A., Dunlap, C.A., Valadez-Blanco, R. 2023. Biofertilizing effect of putative plant growth promoting rhizobacteria in vitro and in tomatillo seedlings (Physalis ixocarpa Brot.). Scientia Horticulturae. 308. Article 111567. https://doi.org/10.1016/j.scienta.2022.111567.*
                                *Pantoja-Guerra, M., Burkett-Cadena, M., Cadena, J., Dunlap, C.A., Ramirez, C.A. 2023. Lysinibacillus spp.: An IAA-producing endospore forming-bacteria that promotes plant growth. Antonie Van Leeuwenhoek. 116: 615-630. https://doi.org/10.1007/s10482-023-01828-x.*


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