Location: Sugarcane Field StationTitle: PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographic distribution in P. kuehnii Author
Submitted to: Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2010
Publication Date: 5/9/2010
Publication URL: http://hdl.handle.net/10113/43853
Citation: Glynn, N.C., Dixon, L.J., Castlebury, L.A., Szabo, L.J., Comstock, J.C. 2010. PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographic distribution in P. kuehnii. Plant Pathology. 59:703-711. Interpretive Summary: Two rust diseases are recognized in sugarcane, orange rust caused by the pathogen Puccinia kuehnii and brown rust caused by P. melanocephala. Brown rust has been prevalent in the sugarcane industries of the Americas since the early 1980’s. However, the distribution of orange rust is still emerging having increased rapidly in the Caribbean basin since its introduction to the region in 2007. The only confirmed reports of orange rust prior to 2007 were in Asia and Australia and since there are no confirmed reports in South America and the continent of Africa these areas are under considerable threat from the pathogen. Early detection and spore morphology can be used to distinguish P. kuehnii and P. melanocephala but has limited applications, relies on mature spores on well preserved leaf material and is not a quantitative method. In this study, molecular methods were developed that allowed the sensitive detection and quantification of P. kuehnii and P. melanocephala DNA. The assays proved useful for detecting inoculum of the two pathogens in leaf material prior to the development of disease symptoms. An additional assay was developed to detect a variation in P. kuehnii DNA isolates obntained from around the world. One of the variations was observed only in samples from Asia and not in P. kuehnii samples from the Western Hemisphere. The assays reported in this study will be useful for monitoring the spread of P. kuehnii through sugarcane industries threatened by orange rust and in studies aimed at improving the understanding of epidemiology of the brown and ornage rust pathogens.
Technical Abstract: Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane respectively. P. kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5.8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labeled hydrolysis probes were designed for each pathogen for use in real-time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimised real-time PCR conditions allowed the detection of 0.19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer-introduced restriction analysis-PCR (PIRA-PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.