Characterization of Alternaria alternata isolates causing brown spot of potatoes in South Africa

Abstract

Alternaria alternata (Fr.) Keissler is now widely recognized as the causal agent of brown spot and black pit of potatoes. Brown spot is a foliar disease with potential to cause 30% yield loss and postharvest losses of up to 10%. Losses are mainly due to premature defoliation. In this study, morphological and epidemiological characteristics of A. alternata were studied in order to understand the extent to which different light regimes influence radial growth, sporulation and pathogenicity of this pathogen. The role of low temperatures on initial sporulation was also investigated. Exposure of isolates to low temperatures (4°C) in the dark increased number of spores produced by isolates under all light conditions. Light did not have any effect on pathogenicity and isolate genetic capability had no influence on radial growth of isolates. The combined isolate and light effect (gene-environment interaction), had significant influence on both radial growth and disease severity. The taxonomy of the genus Alternaria has been highly debateable over the years, especially in small spored Alternaria species where identification is based entirely on morphological characteristics. This is mainly due to presence of closely related taxa such as Ulocladium, Macrosporium and Stemphylium that complicate correct identification of this genus. An investigation was carried out to determine the phylogenetic relationship as well to determine the relationship between molecular characterization and morphological identification. All isolates were identified as A. alternata based on morphology. The identity was further confirmed by molecular phylogeny using the GAPDH, EF1α and a combined phylogeny of these gene regions. All isolates formed one section with A. alternata. The isolates also grouped together with A. arborescens, A. tenuissima, A. longipes and A. gaisen, which were all recently characterized into the Alternata section. Comparison of RFLP digests of the ITS1 and ITS4 region revealed no genetic variability. The GAPDH and EF gene regions can therefore be used to delineate among Alternaria isolates and was in congruence with morphological identification. PCR-RFLP can be a useful tool in detecting genetic variability among isolates. Control of brown spot has mainly been through the use of strobilurins; however, recent disease epidemics on potatoes in South Africa led to an investigation into the failure of strobilurins to control brown spot. Samples were collected during the 2012-2013 growing season and eight Alternaria isolates were recovered from five growing regions. In vitro sensitivity tests showed that six of the eight isolates had reduced sensitivity to azoxystrobin. Sequence analysis of the cytochrome b gene revealed a mutation that led to an amino acid substitution which consequently led to reduced sensitivity. This study will lead to a better understanding of this new disease of potatoes that has proven to be of economic importance. Correct identification is paramount in disease management and this study has shown some reliable molecular technics that can be used to identify species in this genus correctly. This study was also able to link failure to control brown spot to fungicide resistance, and alternative control strategies can now be recommended to control this pathogen.