The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. Megachile rotundata is a solitary bee that forms brood cocoons where its larvae can develop. During the developmental stages of growth, broods can be preyed upon by multiple different fungal and bacterial 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 pollinator services provided by Megachile rotundata and 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 the most common 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. Co-infection of single brood cells by multiple Ascosphaera species was confirmed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species including multiple new predators and pathogenetic species not previously documented in associated with M. rotundata brood cells.

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.

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.

Tetranychus urticae (Koch) is an economically important pest of many agricultural commodities in the Pacific Northwest. Multiple miticides are currently registered for control including abamectin, bifenazate, bifenthrin, and extoxazole. However, populations of Tetranychus urticae have developed miticide resistance through multiple mechanisms, in many different growing regions. Producers of agricultural commodities where Tetranychus urticae infestations are problematic rely on integrated pest management tools to determine optimal control methods. Within this species multiple single nucleotide polymorphisms have been documented in different genes which are associated with miticide 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 miticide resistance phenotypes present within their fields and have potential utility for making appropriate miticide application and integrated pest management decisions. Within this investigation we examined the use of a TaqMan qPCR-based approach to determine miticide resistance genotypes in field-collected populations of Tetranychus urticae from mint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results suggest the TaqMan approach accurately genotypes Tetranychus urticae populations collected from agricultural fields. The interpretation of the results, however, provide additional challenges for integrated pest management practitioners, including making miticide application recommendations where populations of Tetranychus urticae are a mix of resistant and wildtype individuals.