Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing
Article first published online: 20 AUG 2012
© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 113, Issue 5, pages 1145–1153, November 2012
How to Cite
Van de Wouw, A.P. and Howlett, B.J. (2012), Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing. Journal of Applied Microbiology, 113: 1145–1153. doi: 10.1111/j.1365-2672.2012.05413.x
- Issue published online: 17 OCT 2012
- Article first published online: 20 AUG 2012
- Accepted manuscript online: 26 JUL 2012 12:15AM EST
- Manuscript Accepted: 19 JUL 2012
- Manuscript Revised: 18 JUL 2012
- Manuscript Received: 21 JUN 2012
- avirulence genes;
- Brassica napus ;
- Leptosphaeria maculans ;
To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores of Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus).
Methods and Results
The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90-bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble.
This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops.
Significance and Impact of the Study
Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white-nose syndrome has been detected.