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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** 2021 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 collaboration with ARS scientists at Byron, Georgia, and researchers at University of Georgia, we continue to jointly study the production and use of a new strain of insect pathogenic fungus collected from infected whiteflies in a Georgia cotton field during the summer of 2017. Studies on media compositions used for liquid culture showed the benefits of low carbon to nitrogen ratios resulting in high numbers of blastospores. This level of production supports development of prototype formulations to provide for storage stability and to protect the fungus from environmental degradation after application. Insecticidal efficacy in laboratory and field experiments demonstrating some benefits of this strain have been published and support the submitted patent application for this beneficial fungus. 
We continue to support licensed technology on the production of microsclerotia by insect pathogenic fungus by participating in regular conference calls with industry scientists. A cooperative project studied fungal microsclerotia as a seed coating to limit damage caused by larva of the corn rootworm that feeds on corn roots. Unfortunately, studies showed that optimal conditions for seed storage at 25°C and 50% relative humidity resulted in rapid degradation of the fungus, which stores best under extreme dryness. Fungal degradation is sufficiently rapid to prevent commercial application of this technology unless methods progress to extend fungal survival under seed storage conditions.
We are studying impacts of repeated liquid culturing on beneficial fungi. Repeated dry cultures have been shown to select for adverse characteristics such as poor spore production or reduced insecticidal efficacy. Three selected fungal strains have completed 34 liquid culture cycles and three additional fungal strains have completed 22 cycles. Culture samples are collected every 15 cycles. Assays for direct comparisons of collected samples will be conducted at the completion of 60 culture cycles. The knowledge gained will help to direct the selection among economical methods for production of individual fungi for use as biopesticides.
In a separate project, we collaborated with scientists from EMBRAPA to identify a fungal pathogen infecting eucalyptus snout beetles, a pest insect of eucalyptus plantations. The study characterized the basic properties of the pathogen and its occurrence in field and laboratory colony insects. Understanding the natural pathogen pressures on insects is important to developing control strategies for these pests. The presence of natural pathogens may positively or negatively impact the efficacy of pest control products and need to be understood for predictable insect management. The current study enhances our knowledge by identifying an unknown natural pathogen infecting a forest pest, which can be further evaluated to determine its impact on pest control products and strategies.

Objective 2.  We successfully extracted DNA from approximately 1,000 entomopathogenic fungal strains to support genome sequencing. Genomes of 200 fungal strains have already been sequenced and assembled as draft genomes. The sequencing provides fundamental insights into fungal taxonomy and ecological evolution of the strains. 
We optimized infection bioassay conditions with non-conventional insects. This technique uses crickets as host models to study development of fungal diseases in insects. The fundamental knowledge gained from these studies is expected to help in streamlining selection of the most effective strains to use as biopesticides.  
Collaborations continue between ARS and Illinois State University scientists to identify and isolate microbes that cause diseases of crickets. These diseases reduce production of crickets grown in cultures for human food and animal feed.  Identifying common disease pathogens and their distribution among cricket production colonies will aid in developing technologies for their control and support more economical production of the insect as a commodity.
While researching host plant resistance, two genes were identified in corn that provide plants with resistance to fungal disease. We discovered that when the two genes were expressed together in transgenic corn, the plants had increased resistance to diseases, but unfortunately, also reduced efficacy of an insect biocontrol fungus. If successfully applied to commercial corn hybrids, these genes could reduce plant diseases caused by these fungi and accumulation of their toxins. Reducing disease results in better quality and safer corn for commercial users and consumers, but strategies dependent on fungal bioinsecticides for insect pest control may need to be altered.
We continue to study interactions among production technologies to identify more sustainable production practices. Research on insect biocontrol agents showed that insect pests were killed at different rates over time when they fed on different sweet corn varieties previously treated with these agents. This information suggests improvement in insect biocontrol can result when more compatible varieties of corn and strains of biocontrol agents are used together, resulting in safer and more effective control of corn insect pests benefitting both producers and consumers. **Accomplishments**
                                

1. New protein from corn inhibits pests. Insect and mold damage to corn causes millions of dollars in damage. Genes from corn that inhibit pests are a useful way of reducing damage. ARS scientists at Peoria, Illinois, isolated a gene from corn and put it in corn cells so the protein would always be produced. When insects fed on these corn cells, they grew slower and were up to 65% smaller than normal, and fungal growth on these cells was also reduced by as much as 70%. Using this gene in corn should reduce pest damage, resulting in improved yields and higher crop quality. 2. Identifying fungal genes for biopesticide production. The large-scale economical production of beneficial fungi is a key step in developing biopesticide products for crop protection. ARS researchers at Peoria, Illinois, collaborated with scientists from EMBRAPA to study the genes expressed by a biopesticide fungus during large-scale production. Understanding how these fungi respond to different culturing conditions allows us to determine the best methods of producing these fungi. The goal of this study was to identify the genes associated with the development of a specific fungal structure known as a blastospore. Blastospores are the desired product of these fungal cultures for many biopesticide products. The study also provided new insight into the structure of the cell wall of the fungus, which improves our understanding of how these cells survive stress. These findings will be used to develop new processes for producing these beneficial fungi as crop protection products. 3. Identifying a cricket disease virus infecting commercially grown crickets. Insect farming of crickets, as well as other insects, will help to provide sustainable food production for the world’s growing population. Like all livestock systems, crickets get diseases that hamper production. Diseases caused by viruses are especially troublesome because they often spread widely among crickets grown in production colonies before symptoms are seen. ARS scientists at Peoria, Illinois, and Illinois State University collaborated to identify a viral pathogen from lab-reared crickets. The whole genome of this virus was sequenced to provide much needed information to improve diagnostic methods for early identification of viral diseases in cricket production colonies. Earlier detection will allow growers in this rapidly growing industry to start control measures to limit transmission and production losses. **Review Publications**
                            *Dara, S.K., Behle, R.W., Arthurs, S.P. 2021. Editorial: Entomopathogens for sustainable food production. Frontiers in Sustainable Food Systems. 5. Article 672404. https://doi.org/10.3389/fsufs.2021.672404.*
                                *Dowd, P.F. 2021. Enhanced rates of lethality to fall armyworms (Spodoptera frugiperda) after association of Beauveria bassania strain Ant 03 with sweet corn leaves. Biocontrol Science and Technology. 31(8):877-882. https://doi.org/10.1080/09583157.2021.1895071.*
                                *Mutschlechner, M., Lackner, N., Markt, R., Salvenmoser, W., Dunlap, C.A., Wagner, A.O. 2020. Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant. Antonie Van Leeuwenhoek. 114:45-54. https://doi.org/10.1007/s10482-020-01497-0.*
                                *Mascarin, G.M., Iwanicki, N.S., Ramirez, J.L., Delalibera, Jr, I., Dunlap, C.A. 2021. Transcriptional responses of Beauveria bassiana blastospores cultured under varying glucose concentrations. Frontiers in Cellular and Infection Microbiology. 11. Article 644372. https://doi.org/10.3389/fcimb.2021.644372.*
                                *Clifton, E.H., Gardescu, S., Behle, R.W., Hajek, A.E. 2020. Optimizing application rates of Metarhizium brunneum (Hypocreales: Clavicipitaceae) microsclerotia for infecting the invasive Asian longhorned beetle (Coleoptera: Cerambycidae). Journal of Economic Entomology. 113(6):2650-2656. https://doi.org/10.1093/jee/toaa222.*
                                *Behle, R.W. 2020. Emergence of walnut husk maggot adults in Central Illinois and potential for control with Metarhizium brunneum. Journal of Insect Science. 20(6). Article 33. https://doi.org/10.1093/jisesa/ieaa134.*
                                *Wu, S., Toews, M.D., Hofman, C.O., Behle, R.W., Simmons, A.M., Shapiro Ilan, D.I. 2020. Environmental tolerance of entomopathogenic fungi: a new strain of cordyceps javanica isolated from a whitefly epizootic versus commercial fungal strains. Insects. 11(10). Article 711. https://doi.org/10.3390/insects11100711.*
                                *Johnson, E.T., Dowd, P.F., Skory, C.D., Dunlap, C.A. 2021. Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures. Antonie Van Leeuwenhoek. 114:37-44. https://doi.org/10.1007/s10482-020-01495-2.*


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