A molecular study of the causal agent of bacterial leaf streak disease

Abstract

Bacterial leaf streak disease (BLSD) of maize is caused by the pathogen currently classified as Xanthomonas campestris pv. zeae. The disease has only been reported in South Africa and is a potential threat to commercial maize practices. Limited information is available regarding the taxonomy of the causal agent. A specific and reliable diagnostic tool has not yet been developed for effective detection of the pathogen. The aim of this study was to use a molecular and bioinformatics approach in order to gain more insight into the taxonomy and for detection of the pathogen. The molecular study involved whole genome sequencing and analysis of the genomic composition of the causal agent. Genome sequences of the BLSD causal agent were assembled and compared to reference genomes of Xanthomonas relatives. Six Xanthomonas housekeeping genes (atpD, dnaK, fyuA, recA, rpoB and rpfB) were selected from the whole genome sequence data. These complete gene sequences were compared to gene sequences of other Xanthomonas species to determine nucleotide variation between the BLSD causal agent and relatives. Housekeeping gene sequences were used to construct phylogenetic relationships between the causal agent and close relatives. Phylogenetic trees for individual genes revealed different clustering of the BLSD pathogen within the genus. Based on phylogenetic analysis, closest relatives of the causal agent were X. vasicola, X. oryzae, X. axonopodis and X. hortorum pv. hederae. The BLSD causal agent never clustered with the X. campestris species, regardless of the gene that was analysed. Results from this study supported the conclusion that X. campestris pv. zeae is not part of the X. campestris species. As the pathogen was not placed consistently with the same related species in phylogenetic analysis of different genes, the pathogen is likely a novel Xanthomonas species. Regions of the dnaK, recA and rpoB genes were mined for DNA markers that were potentially unique to the pathogen. Six primer pairs, targeting variable regions within the genes, were designed to amplify these sequences and specificity was assessed via PCR. Five primer pairs amplified DNA of all BLSD isolates but also one or both of the reference strains and were thus deemed not sufficiently discriminatory. One primer set (XanZ_rpoB) allowed for completely specific amplification of DNA from the BLSD isolates. This primer pair targets a sufficiently variable region within the rpoB gene and was a potential molecular marker of the BLSD organism for use in detection of the pathogen. This collection of new molecular data regarding the BLSD causal agent will help to establish the pathogen's taxonomy within the Xanthomonas genus as well as enable the development of reliable detection techniques for identification of the pathogen.