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





- Ramirez, Jose
- Crop Protection & Quarantine (304)

Research Project: **Location:
                            Crop Bioprotection Research** Project Number: 5010-22410-023-004-I
                        
                        Project Type: Interagency Reimbursable Agreement Start Date: May 1, 2024
                        
                        End Date: Sep 30, 2024 **Objective:**
                        
                        Primary goal:
Conduct microsporidian pathogen surveillance in recently introduced locations to establish pathogen spread dynamics as a base for improved microbial control of Japanese beetles.
Objectives:
1. Collect Japanese beetles at 30 recently established locations in Illinois and Colorado for pathogen detection, identification, and quantification.
2. Develop an improved methodology for absolute quantification of the microsporidian O. popilliae to determine pathogen copy numbers per beetle and establish pathogen prevalence and infection load. These are key quantitative measurements that will improve our understanding of the microsporidian pathogen success at infecting Japanese beetles in these newly established locations. 
3. Determine microsporidian specificity and pathogen host range by collecting and evaluating infection with O. popilliae in other beetles collected from recently introduced locations. 
4. Determine O. popilliae phylogenetic relatedness from recently established locations, to verify source of infection, corroborate success of pathogen introduction methodology, and determine the existence of O. popilliae strains currently in the U.S. **Approach:**
                        
                        1. State cooperators, NCAUR and APHIS Field Operations staff will collect Japanese beetles from selected study sites via the use of Japanese beetle-specific traps. Beetles will be collected in DNA/RNA Shield reagent to preserve the genetic integrity of the samples and sent to the Crop Bioprotection Research Unit (NCAUR) for DNA extraction and pathogen detection/quantification.   
2. NCAUR researchers will work on current methodology to improve detection and quantification parameters of both O. popilliae and Japanese beetle genetic material. Insect genetic material will be used to normalize O. popilliae copies and to serve as a control to evaluate efficiency of DNA extraction. The budget includes materials and supplies to generate oligo standards for absolute quantification of O. popilliae via qPCR. Results from the analysis will be used to estimate O. popilliae prevalence (fraction of the Japanese beetle population infected with O. popilliae) and intensity (level of infection, # of O. popilliae per beetle). These quantitative parameters are crucial for understanding the microsporidian pathogen success at infecting Japanese beetles in these newly established locations. 
3. State cooperators, NCAUR and APHIS Field Operations staff will collect beetles from the recently established locations and ship them to the Crop Bioprotection Research Unit laboratory (NCAUR) for DNA extraction and O. popilliae detection and quantification.  Identification to the family level of collected beetles will be performed via morphological characterization on a stereoscope prior to DNA extraction. If the beetle sample tests positive for O. popilliae, it will be further characterized molecularly via sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (COI).
4. NCAUR researchers will work to determine O. popilliae phylogenetic relatedness across well established and recently introduced sites. Here, PCR and sequence analysis of the small subunit ribosomal DNA (ssrDNA) from O. popilliae will be used to infer relatedness across all study sites. Sequence analysis will also include samples collected from well-established locations. Phylogenetic analysis will be correlated to distribution, prevalence and intensity of infection determined in steps 1-3 above to infer the potential existence of O. popilliae strains that are better at infecting Japanese beetles.


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