Genetic diversity and differentiation of Leptosphaeria biglobosa on oilseed rape in China

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans and L. biglobosa, is the most devastating disease in oilseed rape (canola) production worldwide. In this study, 84 Leptosphaeria isolates were collected in China from rapeseed (Brassica napus) plants with blackleg symptoms and identified using multiplex PCR and rDNA-ITS sequence analysis. Results showed that all these isolates are L. biglobosa and no L. maculans was detected. A total of 99 isolates was analyzed, including 15 other L. biglobosa isolates from the UK, Canada and Poland using rDNA-ITS sequences, and all isolates were found to belong to the L. biglobosa 'brassicae' subclade. The genetic variation of these 99 isolates was tested using ISSR (inter simple sequence repeats) markers using 24 polymorphic primers pairs that generated 241 bands by ISSR-PCR amplification. A dendrogram based on weighted pair group mean analysis of these bands revealed that L. biglobosa isolates from China clustered into different groups from those obtained from Canada and Europe. Furthermore, Chinese isolates from seven provinces, except those from Jiangsu, could be placed within the same cluster, based on geographical location. Nei’s gene diversity ranged from 0.25 in Jiangsu to 0.11 in Sichuan, and Shannon’s information index from 0.16 to 0.37. Genetic identity ranged from 0.784 to 0.834 when isolates from China were compared with those from Canada and the UK, respectively. Analysis of gene flow among populations (Nm) indicated that almost no gene exchange has occurred between L. biglobosa in China and the populations of the fungus from the UK or Canada.