A Real-Time PCR Assay for Rapid Identification of Tuta absoluta (Lepidoptera: Gelechiidae)

Abstract

The tomato leafminer, Tuta absoluta (Meyrick), is a highly destructive pest of tomatoes, causing damage to leaves, stalks, buds, and fruits. Native to South America, T. absoluta is now found throughout Europe, South Asia, Africa, parts of Central America, and the Caribbean. Adults are small, with a wingspan of approximately one cm and lack distinctive markings, making morphological identification difficult. Larvae are also difficult to identify and resemble those of many other gelechiids. Due to the extensive time spent and expertise required for morphological identification, and the imminent threat to the North American tomato crop, we have developed a rapid molecular test for discriminating individual specimens of T. absoluta using a probe-based real-time polymerase chain reaction (PCR) assay. The assay is able to quickly distinguish T. absoluta from similar-sized moth specimens that are attracted to T. absoluta pheromone lures in the United States and is also able to identify larvae of T. absoluta. Decreased identification time for this critical pest will lead to more rapid identification at ports of entry and allow for more efficient trap screening for domestic monitoring programs.

Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae)

“…A few methods have been published using multiplexed PCR, qPCR, and digital droplet PCR to detect P. absoluta (Sint et al 2016, Zink et al 2020). We find our qPCR assay is equally sensitive as the method published in Zink et al (2020), with both being able to detect 0.01 to 0.02 ng of input P. absoluta DNA.…”

“…A few methods have been published using multiplexed PCR, qPCR, and digital droplet PCR to detect P. absoluta (Sint et al 2016, Zink et al 2020). We find our qPCR assay is equally sensitive as the method published in Zink et al (2020), with both being able to detect 0.01 to 0.02 ng of input P. absoluta DNA. Our assay has the added advantage of being able to identity an additional two moth species which are morphologically nearly identical to P. absoluta , occupy similar host crops, and are already established in the United States.…”

“…Molecular diagnostics, on the other hand, would allow for accurate and rapid identification without entomological expertise. To date, several molecular diagnostics have been developed, all using polymerase chain reaction (PCR) to amplify target DNA presence before detecting signal either by fluorescence, gel electrophoresis, or mass spectrometry (Sint et al 2016, Tabuloc et al 2019, Zink et al 2020). A potential limitation of these PCR-based diagnostics is the requirement for a thermocycler, as well as specialized equipment to visualize results.…”

CO1 barcodes resolve an asymmetric biphyletic clade for Diabrotica undecimpunctata subspecies and provide nucleotide variants for differentiation from related lineages using real-time PCR

“…After overnight incubation, specimens were removed from the lysis buffer, rinsed with absolute ethanol, and returned to pins (museum specimens) or ethanol (intercepted specimens). The remaining extraction was carried out following the manufacturer's instructions with modifications as described by Zink et al (30). After elution, DNA concentration and purity were measured for a 2mL sample using a NanoDrop 2000 v 1.6 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).…”

A review on ecology, biology, and management of a detrimental pest, Tuta absoluta (Lepidoptera: Gelechiidae)

“…A portion of the significant pests of tomatoes includes armyworms and fruitworms, cabbage loopers, leafminers, pinworms, aphids, thrips, stink bugs, and whiteflies (Trumble, 2020). However, at present, a neotropical and tenacious rural vermin is pulverizing these harvests by assaulting the plants and debilitating their development and yield limit, giving direct consequences for the market, processing units, and nurseries Mkonyi et al, 2020;Moeini-naghade et al, 2020;Zink et al, 2020;Abdel-baky et al, 2021). The Tuta absoluta is generally known as Tomato leaf miner (Lepidoptera: Gelechiidae) is an oligophagous, notorious, multivoltine, and an r-rated species that attacks countless Solanaceae plants and is viewed as a genuine danger to tomato cultivation around the world (Desneux et al, 2010;Abadi, 2014;Jac, 2017;Shiberu & Getu, 2017;Rostami et al, 2018;Coleman, 2020;Hogea, 2020;Abdel-baky et al, 2021).…”