DEVELOPMENT OF METHODS IN SCREENING FOR TOLERANCE AGAINST MAIZE LETHAL NECROSIS DISEASE (MNLD) AND DETECTION OF MLND CAUSAL VIRUSES

Abstract

Maize lethal necrosis disease (MLND), a viral disease resulting from double infection by Maize Chlorotic Mottle Virus (MCMV) and Sugarcane Mosaic Virus (SCMV) is the most devastating viral disease of maize which has impacted negatively to maize production in Kenya. This study aimed to test the prospect of using small RNA transcriptome profiling in detection of MLND causal viruses and subsequent development of markers for future use in detecting the viruses. Next generation sequencing of small RNAs (sRNASeq) from leaf samples of symptomatic plants collected from Bomet, Nyamira and Kericho counties was done using BGIseq500 sequencing platform and mapping and differential expression analysis of the resulting reads done using available pipelines with reference to viral and host genomes. Five viruses including maize chlorotic mottle virus, sugarcane mosaic virus, maize streak virus, maize associated totivirus and maize yellow mosaic virus were identified through sRNAseq. Capsid protein and P7b movement protein domains of MCMV and P3 domain in SCMV were identified as the highly expressed domains and were used subsequently in design of primers for development of a diagnostic kit and in identification of the alternate host. These markers were used to identify non-maize hosts for the viruses from plants collected in fields infested with the disease. Here, sugar cane (Saccharum officinarum), Napier grass (Pennisetum purpureum) Proso millet (Panicum miliaceum), Sorghum (Sorghum versicolor), Finger millet (Eleusine coracana) and Wandering Jew (Commelina benghalensis) all tested positive for SCMV and MCMV. Further, the study aimed to test the applicability of detached leaf assay (DLA) as a pre-screening technique for tolerance against MLND. Three MLND-tolerant maize genotypes (CHMLND0093, CKIR11027, CKIR12032) from International Maize and Wheat Improvement Center (CIMMYT) and susceptible genotypes Namba nane and Hybrid 144 from Kenya Agricultural and Livestock Research Organization (KALRO) were used for the study. Agar media supplemented with 3% sucrose, 20mg/l gibberellic acid and 10mg/l kinetin was determined as the best media regime in retaining the green color and was subsequently used in assaying for the development of MLND symptoms on detached and artificially infected maize leaves. Detached leaf discs at 28, 42 and 63-day after planting xviii were infected with an inocula consisting of MCMV and SCMV and maintained on this media and scored for disease severity for 19 consecutive days using CIMMYT MLND scoring scale. Detached leaf assay from plants at 28th day of growth was able to distinct different genotypes as either tolerant or susceptible with tolerance increasing with plant growth age. Overall, the results of this study showed the potential of small RNAs for use in MLND diagnosis as well as in development of markers for future detection and further revealed that MLND causal viruses are co-hosted by majority of grass family members. The potential of DLA as a pre-screening technique was demonstrated and could accelerate the identification of promising germplasm for use as candidates in development and screening for tolerant germplasm in MLND breeding programs prior to extensive field testing.