Characterization of Exserohilum turcicum isolates from South African maize production areas

Abstract

Northern corn leaf blight (NCLB) is a yield-limiting foliar disease that results from infection with the fungal pathogen, Exserohilum turcicum (Pass.). Maize, sorghum and related grass species such as Johnson grass and Sudan grass serve as hosts for E. turcicum. NCLB is characterized by cigar-shaped lesions that are tan to grey in colour. In severe cases, lesions coalesce to blight the entire leaf surface, thereby reducing the photosynthetic potential of affected leaves and resulting in yield losses. The biology, life cycle and infection strategy of E. turcicum has been studied. The population structure and mode of reproduction in countries such as Kenya, Uganda, Mexico, China, and various locations in the United States of America and Europe have been investigated with random amplification of polymorphic DNA, amplified fragment length polymorphism or isozymes. The majority of previous studies performed crosses with tester strains to determine the mating type of isolates. In this dissertation, robust and reproducible simple sequence repeat (SSR) markers were developed to study the population genetic structure of E. turcicum in South Africa. In addition, a mating type multiplex PCR assay was designed to easily and rapidly determine the mating types of isolates. Of the 32 SSR primers identified from the available E. turcicum genome, thirteen markers amplified regions that were polymorphic between E. turcicum isolates from maize collected from regions within the Free State, Mpumalanga, North West, Gauteng, KwaZulu-Natal and the Northern Cape during 2005 – 2011. Screening of the markers on two isolates of sorghum collected from the Free State in 2011 revealed ten private alleles and two haplotypes unique to sorghum-infecting isolates. Three mating type PCR primers were designed from the available mating type idiomorph sequences and were successfully used in a multiplex PCR assay to determine the mating type of E. turcicum isolates. One SSR marker amplified in two isolates of E. rostratum. No amplification of E. rostratum isolates was observed with the mating type multiplex PCR assay, indicating that it may be useful to differentiate isolates of E. turcicum and E. rostratum. To study the population genetic structure of E. turcicum in South Africa, thirteen polymorphic microsatellite markers were screened on 268 E. turcicum isolates collected from maize during 2012 – 2013 from Machadodorp in Mpumalanga and four locations in the KwaZulu-Natal. The distribution of mating types was investigated among three E. turcicum populations from KwaZulu-Natal. Isolates collected from South Africa during 2012 – 2013 were grouped into three clusters based on STRUCTURE analyses, with isolates from different locations clustering together. The three groups may have resulted from multiple introductions of the E. turcicum into South Africa from other countries where it occurs. Gene and genotypic diversity was high in all populations analysed, suggesting sexual recombination events may occur among isolates from South Africa. Shared haplotypes were detected between isolates from different years and locations, although private alleles were also detected in isolates from each location. Mating type distributions revealed evidence for sexual recombination in one population from KwaZulu-Natal. The markers developed in this study will be useful in future global population genetic studies.