Transmission and Detection of Maize Chlorotic Mottle Virus in Maize Seed

Abstract

Maize lethal necrosis (MLN) is an important disease in Kenya and eastern Africa countries causing up to 100% yield losses in farmers field when not controlled. Decrease of production of maize is a threat to food security. The disease is caused by a synergetic infection of two viruses: maize chlorotic mottle virus (MCMV) and a cereal-infecting potyvirus. The frequently identified potyvirus in eastern Africa is sugarcane mosaic virus (SCMV). Maize chlorotic mottle virus is transmitted via insect vectors, mechanically, through plant residues and soil and also by seed. The main objective of this study was to determine the various aspects of seed as a source of transmission of MCMV- Kenya isolate. Serological and nucleic-acid based methods were evaluated for their effectiveness and sensitivity in detecting MCMV using purified virus and seed and leaf tissue. The methods were available and some in progress of development in the country at the time of study. The limit of detection of one infected seed in different sample size and the current sample size in use for seed certification (400-seed) was evaluated for effectiveness in detection of MCMV using ungerminated seed and 10-day grow out seedlings, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The seed transmission rate was determined using MCMV-infected commercial seed lots while seeds from mechanically inoculated plants were used to determine the effect of genotypes on seed transmission of MCMV. The effect of crop stage at time of infection on seed transmission was determined using three genotypes, inoculated at four crop stages. Study of infectivity of the virus was carried out using contaminated seed which were soaked in inoculation buffer and the soak solution as well as seed extract used to inoculate seedlings of a maize genotype PH30G19. Data was analysed using descriptive statistics, analysis of variance (means separated by least significant differences), proportional odds logistic regression, pooled prevalence tests, Kruskal-Wallis test and binomial probabilities using R- software packages and Epitools calculator. The sensitivity levels of detection for MCMV using purified virus were 0.1-0.01 ng/μl, 0.1 pg/μl and 0.001 fg/μl for DAS-ELISA, IC-RT-PCR and lateral flow strips (AGDIA); and up to 0.1 fg/μl, 1 pg/μl and 0.001 fg/μl for RT-PCR, RT-LAMP and real-time RT-PCR, respectively. However, the sensitivity of detection of DAS-ELISA reduced when crude leaf sap and seed extract were used to dilute the purified virus unlike for IC-RT-PCR. The probability of detecting MCMV in a 400-seed sample was higher ungerminated ground seed samples unlike when grow outs from xviii contaminated seed of similar size were used. Seed transmission rates observed for four seed lots (45,939 seedlings) evaluated ranged from 0-0.57% while that of seed from different genotypes with varying severity scores of MCMV infection ranging from 0 to 1.04%. Higher transmission was recorded from seeds obtained from plants infected at four-leaf stage (0.19%). Infection of 1.64% PH30G19 seedlings was recorded when inoculated with soak solution and seed extract from MCMV contaminated seeds. The methods available for detection of MCMV determine the reliability of the results. While DAS-ELISA is a commonly used method in virus diagnosis, the method is prone to false results when detecting MCMV in seedling grow outs. However, it has been found to be reliable when the samples are ground seeds. Nucleic-acid methods are more reliable as the samples are further purified from the crude state. The use of one 400 seed-sample may lead to a false analysis of seed lot when the seedling grow outs are tested. The study suggests the use of more than one replicate of the sample from a seed lot to determine the presence of transmitted MCMV, or an increase in the number of seeds in a drawn composite sample from a seed lot. Seed transmission of MCMV in all the samples tested was below 1.05%, showing low transmission. The low transmission was despite the level of severity of MCMV in the plant where the seeds were obtained. The results emphasize the need for continuous seed health testing of all the genotypes. The transmission of MCMV was higher at early crop stage, though proper management of MCMV infection should be implemented at all stages, as symptoms were observed on all inoculated plants. Maize chlorotic mottle virus from contaminated seed led to infection to seedlings when used as inoculum. This information is important in ensuring proper detection of MCMV in seed health.