Detection of Diverse Maize Chlorotic Mottle Virus Isolates in Maize Seed

Abstract

Maize chlorotic mottle virus (MCMV) has driven the emergence of maize lethal necrosis (MLN) 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 SYBRTM 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.

Transmission, localization, and infectivity of seedborne maize chlorotic mottle virus

“…All seed were stored at 4˚C in sealed bags, except for those collected at R2 and R4, which were sealed and stored at -20˚C. MCMV infested and un-infested seed lots used for localization and transmission experiments were described previously [27].…”

“…Pools of poorly growing or deceased plant samples were weighed and extracted in 2 volumes (w/v) GEB. For each sample, two 100 μl aliquots were tested for the presence of MCMV by DAS-ELISA (Agdia, Elkhart, IN, USA) as previously described [27]. Within each plate, MCMV positive controls were separated from other samples by a row of buffer to prevent and monitor possible cross-contamination.…”

“…To ensure positive pools resulted from seed transmission of MCMV and not from cross-contamination, the individual plants from which discs in positive pools were collected were resampled as described above. RNA was extracted from each individual and used for RT-PCR as previously described [27,41]. To minimize the presence of false negative tests, samples from pools with mean absorbances greater than the mean of the healthy controls plus three times the standard deviation of the mean absorbance of the healthy controls (>HC + 3SD) were resampled and retested by ELISA or RT-PCR.…”

“…However, these processes are currently poorly understood. While MCMV is present and detectable on seed from infected plants [27], little is known about the viability and localization of the virus in seed. The localization of viruses within plant seed tissues is an important indicator of transmissibility and can inform disease management.…”

Maize Lethal Necrosis disease: review of molecular and genetic resistance mechanisms, socio-economic impacts, and mitigation strategies in sub-Saharan Africa

“…The disease may also be transmitted through infected maize seeds [ 40 ]. Diverse MCMV isolates have been detected in maize seeds [ 41 ]. Therefore, usage of virus-free seeds is important in controlling MLN in the African continent.…”