Abstract
Maize chlorotic mottle virus (MCMV) has driven the emergence of maize lethal necrosis worldwide, where it threatens maize production in areas of East Africa, South America, and Asia. It is thought that MCMV transmission through seed may be important for introduction of the virus in new regions. Identification of infested seed lots is critical for preventing the spread of MCMV through seed. Although methods for detecting MCMV in leaf tissue are available, diagnostic methods for its detection in seed lots are lacking. In this study, ELISA, RT-PCR, and RT-qPCR were adapted for detection of MCMV in maize seed. Purified virions of MCMV isolates from Kansas, Mexico, and Kenya were then used to determine the virus detection thresholds for each diagnostic assay. No substantial differences in response were detected among the isolates in any of the three assays. The RT-PCR and a SYBR Green-based RT-qPCR assays were >3,000 times more sensitive than commercial ELISA for MCMV detection. For ELISA using seed extracts, selection of positive and negative controls was critical, most likely because of relatively high backgrounds. Use of seed soak solutions in ELISA detected MCMV with similar sensitivity to seed extracts, produced minimal background, and required substantially less labor. ELISA and RT-PCR were both effective for detecting MCMV in seed lots from Hawaii and Kenya, with ELISA providing a reliable and inexpensive diagnostic assay that could be implemented routinely in seed testing facilities.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Highlights
Identification of infested seed lots is critical for preventing the spread of Maize chlorotic mottle virus (MCMV) through seed
Purified virions of MCMV isolates from Kansas, Mexico, and Kenya were used to determine the virus detection thresholds for each diagnostic assay
MCMV isolates from around the world have a high degree of genome sequence identity (Braidwood et al 2018; Redinbaugh and Stewart 2018), differences in virus detection among laboratories (Adams et al 2013; Mahuku et al 2015), among samples collected from different regions, or using different antibodies or primer pairs have led to speculation that ELISA and RT-PCR might not be useful for routine detection of MCMV (Adams et al 2013; Fentahun et al 2017)
Summary
Identification of infested seed lots is critical for preventing the spread of MCMV through seed. ELISA, RT-PCR, and RT-qPCR were adapted for detection of MCMV in maize seed. Purified virions of MCMV isolates from Kansas, Mexico, and Kenya were used to determine the virus detection thresholds for each diagnostic assay. The RT-PCR and a SYBR Green-based RT-qPCR assays were >3,000 times more sensitive than commercial ELISA for MCMV detection. MCMV with similar sensitivity to seed extracts, produced minimal background, and required substantially less labor. ELISA and RT-PCR were both effective for detecting MCMV in seed lots from Hawaii and Kenya, with. MCMV alone can cause production losses in maize, coinfection with any of several maize-infecting potyvirids results in the synergistic disease maize lethal necrosis (MLN), which has much greater economic impact (Redinbaugh and Stewart 2018).
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