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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** 2022 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.  Some microorganisms are known to cause diseases that are specific to insects and can be used as biological pest control agents. Our goal is to understand how to best select, produce, and formulate beneficial microorganisms for use as biological insecticides.  Unfortunately, commercial production techniques can select for undesirable microbe characteristics like poor insecticidal activity. ARS scientists in Peoria, Illinois, continue to study liquid culture production of insect killing fungi. ARS scientists completed 60 continuous cycles of liquid culture with six species of beneficial fungi that represent fungal species used as biological insecticides. The experiment was designed to cause extreme selection pressure by this artificial culture technique. While some fungi showed little change, others lost the ability to produce the spores that are needed to infect insect pests. Repeated culture cycles also caused changes in the ability of the fungi to produce hyphae, consume glucose and mediate the pH of their cultures. When grown on standard nutrient agar, continuously cultured fungus had different morphology when compared with the original (pre-culture) fungus, which suggests some level of genetic selection away from the parent strain. This research has established samples for future genetic analysis to identify specific genes associated with altered traits and will eventually support commercial production of entomopathogenic fungi for use as biopesticides.
ARS scientists also studied formulation additives and processing techniques with a goal to improve pest control efficacy and product handling of prototype biopesticides. Several food grade additives were mixed with commercial biopesticides and significantly improved the effectiveness of the biocontrol fungus in killing target pest insects when applied to corn leaves. These results demonstrated that insecticidal activity can be improved with the proper formulation. Also, simple processing to concentrate fungal spores in formulation feed stock resulted in higher spore concentrations in the end product, without degrading spore viability or insecticidal activity. Increasing product concentration reduces production costs of biopesticides, making them more attractive to consumers, resulting in safer and more effective control of insect pests, benefitting both crop producers and consumers.
Relative to integrated pest control, it was demonstrated that sweet corn varieties can impact the effectiveness of biocontrol fungi. Two commercial biopesticides were applied to several varieties of sweet corn. Control of caterpillar pests ranged from 12% to 64% and was dependent on combination of corn variety and biopesticide. This information helps explain variable pest control sometimes reported for biopesticide treatments. Selecting compatible combinations of crop varieties and biocontrol treatments will lead to more predictable pest control for producers.
In cooperation with industry scientists, processing methodologies were updated to successfully spray dry double stranded RNA (dsRNA) as a highly specific biopesticide. dsRNA samples were provided by the cooperator and were dried using a wide range of temperatures and varied ingredients to create powdered formulations. These prototype formulations retained dsRNA molecules. Combining industry production technologies with ARS processing techniques provides an opportunity to develop dsRNA as a biological pesticide for crop protection.
In collaboration with cricket production farms and industry partners across North America, we are creating the first catalog of insect pathogenic viruses infecting commercially produced crickets across 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 that were grown as a commodity resource. Proper identification of these pathogens is the first step toward developing effective control strategies that will ultimately improve production of crickets as a viable agricultural commodity for use as human food and animal feed. 

Objective 2.  Biopesticide companies have successfully marketed a few insect killing microorganisms as commercial products. These few organisms were selected from a vast number of candidate organisms based primarily on empirical observations to identify commercially desirable physical characteristics. ARS scientists in Peoria, Illinois, continue to study fundamental biological interactions among beneficial pathogens and their environments. One goal is to correlate beneficial traits with specific genetics to facilitate rapid selection of useful microbial organisms for pest control agents. Scientists have successfully completed genomic sequencing of approximately 800 entomopathogenic fungal strains that were collected from around the world. A collaborative project is underway to annotate the genomes. Taxonomy for all strains has been confirmed based on the genomic data. These data are currently undergoing quality control and it is anticipated that the majority of the data to be publicly released by the end of the 2022 calendar year. ARS scientists will release all raw data to the GenBank sequence read archive and all processed data to the whole-genome sequencing portal on GenBank. These sequences provide fundamental information for accurate taxonomy and insights into the ecological evolution of these fungi. 
Although some biopesticides are commercially successful, specific understanding is often lacking about why some insects are susceptible to infection while others are resistant. To study the interaction between pathogens and their insect hosts, ARS scientists in Peoria, Illinois, have quantified the immune gene expression of insects when infected with insect-specific disease pathogens. Scientist studied two diverse insect/pathogen systems to better understand the universal and specific insect immune responses. For example, a pest caterpillar infected with pathogenic fungi upregulated multiple genes across multiple immune defense pathways. Additionally, crickets infected with iridovirus not only upregulated the same pathways, but also upregulated pathways for specific defense against viral infections. Understanding these basic insect immune responses to their disease pathogens is necessary to render insect pests susceptible to diseases by interfering with their immune system during a biological control strategy or to prevent insect disease by promoting immune responses in insects grown commercially as a feed or food commodity.
In collaboration with ARS’s microbial culture collection (NRRL), a project was initiated to sequence the genomes of microbes associated with bees and bee environments. The initial set of 80 microbes was sequenced and the draft genomes have been assembled. The data is currently undergoing quality control and it is anticipating that the majority data to be publicly released by the end of the calendar year. 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 collaboration with Illinois State University, ARS researchers studied the immune response of bumblebees for its impact on the bumblebee’s natural gut microbiota. Alterations of the microbiota can impact bee health. A metagenomic microbial analysis of more than 123 bumble bee gut samples across different microbial infections was conducted. This study analyzed the bacterial diversity, richness and core microbiome to elucidate infection-related effects on the bumblebee microbiome. Knowledge of these interactions will provide the understanding needed to retain healthy bumblebee populations as a natural pollination resource. 
Insect and mold damage to corn causes millions of dollars in damage. ARS scientists in Peoria, Illinois, isolated a gene from corn that inhibit pests by slowing growth of caterpillars and a plant disease fungus. The gene was transformed into corn cells so the protein would always be produced. Cells with this protein were grown as callus tissue that was fed to caterpillars and exposed to a plant disease fungus. Insects were up to 90% smaller than control insects and fungal growth was reduced by 65%. Breeding this gene in corn should reduce pest damage without the need for chemical pesticide applications. **Accomplishments**
                                

1. Drafted genome sequences for identification of entomopathogenic fungi that act as a parasite of insects to disable them from causing harm to crops. Scientists world-wide continue to study insect pathogenic microorganisms for their potential to serve as biological control agents and reduce the need for chemical pesticide applications. Traditionally, identifications of insect pathogenic fungi have been based on visual observations of physical characteristic when grown on nutrient agar, which lead to improper identification and confusion in the reported literature. Approximately 800 entomopathogenic fungal strains were collected from around the world over many years by ARS scientists at the European Biological Control Laboratory. ARS researchers in Peoria, Illinois, have successfully completed draft genome sequencing for each of these beneficial fungi. This step has initially confirmed accurate taxonomic identification of these isolates and is expected to provide insights into their ecological evolution and their potential as biological control agents. This genomic dataset will soon be made publicly available on GenBank and is already the basis of four collaborative projects to understand how these beneficial fungi attack insect pests. Proper fungal identification is necessary for continuity of research among the many laboratories around the world. Further, this information will be used by industry and university partners to improve the development of new biopesticides for crop protection. 2. Discovered dual-purpose corn resistance genes that guard against both insect and mold damage of corn. Insect and mold damage to corn costs millions of dollars because of lower yields and added expense for pesticide applications. Ear molds often take advantage of insect damage to begin their infection. Then these fungi contaminate the grain with toxins that are harmful to people and animals.  Host plant resistance can reduce mold damage, but often focuses on controlling either the insect or the fungus, but not both. ARS researchers in Peoria, Illinois, found several corn genes that code for natural compounds and showed that these compounds help protect the plant. Specialized cell-culture techniques in the laboratory validated the resistance activity of these genes. Corn cells that express these gene simultaneously reduced the growth of both pest insects and toxin-producing fungal diseases. These new dual-purpose resistance genes differ from traditional host plant resistance genes in that the previous genes control only one pest. By breeding the new genes into food and feed varieties of corn, we will add to our integrated pest control arsenal, reducing the need for chemical pesticides. Dual-purpose host plant resistance will be most beneficial for producers and consumers of pesticide free organic corn production. Beyond corn, it is likely that future research will expand to find similar dual-purpose resistance genes for pest control in other crop plants. **Review Publications**
                            *Behle, R.W., Wu, S., Toews, M.D., Duffield, K.R., Shapiro-Ilan, D.I. 2022. Comparing production and efficacy of Cordyceps javanica with Cordyceps fumosorosea. Journal of Economic Entomology. 115(2):455-461. https://doi.org/10.1093/jee/toac002.*
                                *Dowd, P.F., Naumann, T.A., Johnson, E.T. 2022. A maize gene coding for a chimeric superlectin reduces growth of maize fungal pathogens and insect pests when expressed transgenically in maize callus. Plant Gene. 30. Article 100359. https://doi.org/10.1016/j.plgene.2022.100359.*
                                *Soni, R., Keharia, H., Dunlap, C.A., Pandit, N., Doshi, J. 2022. Functional annotation unravels probiotic properties of a poultry isolate, Bacillus velezensis CGS1.1. LWT - Food Science and Technology. 153. Article 112471. https://doi.org/10.1016/j.lwt.2021.112471.*
                                *Dowd, P.F., Johnson, E.T. 2022. Different maize (Zea mays L.) inbreds influence the efficacy of Beaveria bassiana against major maize caterpillar pests, which is potentially affected by maize pathogen resistance. Biocontrol Science and Technology. 32(7):847-862. https://doi.org/10.1080/09583157.2022.2055745.*
                                *Duffield, K.R., Hunt, J., Sadd, B.M., Sakaluk, S.K., Oppert, B.S., Rosario-Cora, K., Behle, R.W., Ramirez, J.L. 2021. Active and covert infections of cricket iridovirus and Acheta domesticus densovirus in reared Gryllodes sigillatus crickets. Frontiers in Microbiology. 12. Article 780796. https://doi.org/10.3389/fmicb.2021.780796.*


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