Development of a real-time PCR assay for detection of hemp russet mite (Aculops cannabicola).

Abstract

Of the many arthropod species affecting hemp (Cannabis sativa L.) cultivation in the United States, one species of particular importance is the hemp russet mite (Aculops cannabicola, HRM). Hemp russet mite is a microscopic arthropod which feeds on all parts of hemp plants. Due to its minute size, HRM can proliferate undetected for a long time, complicating management efforts and causing serious economic losses. DNA sequencing and PCR assays can facilitate accurate identification and early detection of HRM in infested-plants. Therefore, a real-time SYBR Green based species-specific PCR assay (quantitative PCR, qPCR) was developed for the identification of HRM DNA by amplification of a 104 bp Internal Transcribed Spacer 1 (ITS1) sequence. The detection limit was estimated to be approximately 48 copies of the HRM marker gene sequence. The real-time-PCR assay is rapid, detects all life stages of mite under 2 hours. A 10-fold serial dilution of the plasmid DNA containing the ITS1 insert were used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay showed a strong linear relationship with HRM DNA with R2 of 0.96. The assay was tested against several commonly found hemp pests including two-spotted spider mite and western flower thrips to determine specificity of the assay and to show that no non-target species DNA was amplified. The outcomes of this research will have important applications for agricultural biosecurity through accurate identification of HRM, early detection and timely deployment of management tactics to manage and prevent pest outbreaks.