Using targeted sequencing and TaqMan approaches to detect acaricide (bifenthrin, bifenazate, and etoxazole) resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards.

Tetranychus urticae (Koch) is an economically important pest of many agricultural commodities world-wide. Multiple acaricides, including bifenazate, bifenthrin, and extoxazole, are currently registered to control T. urticae. However, populations of T. urticae in many different growing regions have developed acaricide resistance through multiple mechanisms. Within T. urticae, single nucleotide polymorphisms (SNPs) have been documented in different genes which are associated with acaricide resistance phenotypes. The detection of these mutations through TaqMan qPCR has been suggested as a practical, quick, and reliable tool to inform agricultural producers of acaricide resistance phenotypes present within their fields and have potential utility for making appropriate acaricide application and integrated pest management decisions. Within this investigation we examined the use of a TaqMan qPCR-based approach to determine genotypes which have been previously associated with acaricide resistance in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results suggest that a TaqMan qPCR approach accurately genotypes T. urticae populations for SNPs that have been linked to Bifenazate, Bifenthrin, and Etoxazole resistance. The results also demonstrated that different populations of mites in Washington and Idaho displayed varying frequencies of the examined SNPs. While we were able to detect the SNPs associated with the examined acaricides, the mutation G126S was not an appropriate or accurate indicator for bifenazate resistance.

Multiplex Polymerase Chain Reaction Reveals Unique Trends in Pathogen and Parasitoid Infestations of Alfalfa Leafcutting Brood Cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. M. rotundata is a solitary cavity nesting bee that forms brood nests where its larvae can develop. During the developmental stages of growth, brood can be preyed upon by multiple different fungal pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollination services provided by M. rotundata. Reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. The presence of multiple Ascosphaera species in a single brood cell was observed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species, including multiple predators and pathogenetic species not previously documented in association with M. rotundata brood cells.

Examining Historical Rates of Leafcutting Bee Brood Cell Pathogens, Parasitoids, and Predators to Establish Baseline Infectivity Rates for Alfalfa Seed Growers.

The alfalfa leafcutting bee Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae) is the primary pollinator for the alfalfa seed industry. It is a solitary cavity nesting bee that utilizes leaf lined brood cells provisioned with pollen for larval development and pupation into the adult stage. During development, multiple pathogens, parasitoids, and predators can prey upon or use the larvae as a host, resulting in the loss of the future adult bee. As such, the presence of invertebrate pests and fungal pathogens presents a major concern for commercial alfalfa seed growers. In the present study, we used historic data from the Parma Cocoon Diagnostic Laboratory to determine baseline rates of pathogens, parasitoids, and predators of Megachile rotundata brood cells and used this analysis to determine cutoffs for management practices to inform growers when the purchase of new bee stocks should be considered. Additionally, we compared the presence of chalkbrood, predators, and parasitoids in samples collected from both grower-produced stocks and newly purchased Canadian bees. The results of the investigation provide historic averages of the presence of chalkbrood, predators, and parasitoids, show a significant increase in chalkbrood and predators in 2007-2011, and find a significant difference in rates of chalkbrood and predators between samples from Canadian suppliers and grower stocks. We speculate that these differences may have resulted from economic conditions that increased the cost of Canadian Megachile rotundata cells and likely resulted in increased reliance on 2nd-year U.S. grower stocks and subsequently increased infection rates during this time period.