Characterisation of phytoalexin accumulation in maize inoculated with Cercospora zeina, the causal organism of grey leaf spot disease

Grey Leaf Spot (GLS) is a fungal disease of Zea mays (maize) that is caused by Cercospora zeina. It thrives in sub-tropical climates and causes devastating crop losses of up to 60% in southern Africa where maize is grown as a staple food source. Phytoalexins are low molecular weight anti-microbial bio-chemicals that are synthesised in planta in response to biotic stress. Related studies have characterised many phytoalexins produced in various plants against several diseases. In maize, phytoalexins fall into to two terpenoid groups: kauralexins and zealexins. To date no studies have been carried out that examine the accumulation in maize of phytoalexins in response to C. zeina. This research project found that in maize samples inoculated with C. zeina, kauralexin accumulation significantly increased with disease development stages (T0 - 0 days post inoculation, T1 - 17 dpi, T2 - 18 dpi and T3 - 24 dpi) while zealexins did not change. Gene expression of the phytoalexin biosynthesis genes TPS6 and TPS11 (both encoding the protein terpene synthase 6/11, specific for zealexins) and CPPS2 (encoding ent-copalyl diphosphate synthase 2, specific for kauralexins) increased significantly at each time point, reaching a maximum level at T2. Infiltration of maize leaves with a chitosan elicitor to mimic fungal pathogen associated molecular pattern (PAMP), and a subsequent callose assay showed positive induction of a callose defence response. However, gene expression and phytoalexin accumulation did not change following chitosan treatment, although zealexin accumulation was higher than kauralexins. Previous studies have shown that phytoalexins accumulate transiently in seedlings. Six diverse Southern African maize lines were compared for phytoalexin accumulation at seedling stage. Zealexin accumulation was generally higher than kauralexins and there were significant differences in both zealexin and kauralexin accumulation in different lines. Gene expression analysis using Genevestigator looked at microarray files and found that expression of TPS6/11 (zealexin biosynthesis) and CPPS2 (kauralexin biosynthesis) genes to be largely co-regulated and highly expressed in response to fungal pathogens, nematodes, insect pests and abiotic stresses; Ustilago maydis, Phytophthora cinnamomi, Fusarium moniliforme, Colletotrichum graminicola, Sporisorium reilianum, Meloidogyne incognita, Ostrinia nubilalis, waterlogging and drought stress. Finally promoter region analysis showed similar cis-acting regulatory elements in the 1kb region upstream of the promoter of both genes and defence specific elements. Thus kauralexin phytoalexins are produced in response to C. zeina inoculation, chitin is not likely to be the key PAMP leading to phytoalexin accumulation, phytoalexin accumulation in seedlings is genotype-dependent and phytoalexin biosynthesis genes are expressed under different conditions suggesting a wider range of action beyond repelling fungal pathogens