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The biotrophic basidiomycete Phakopsora pachyrhizi is the causal agent of Asian soybean rust (ASR), which has become a serious soybean disease in South America. Control of this disease is mainly based on fungicide applications, with demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) representing a large market. DMIs have been the key component for ASR-control for many years. This ongoing selection pressure has led to an adaptation of P. pachyrhizi resulting in a continuous shift of the population towards reduced sensitivity. The objective of the present study was to characterize the resistance of P. pachyrhizi to DMIs and the underlying mutations in the target gene (CYP51), using single spore isolates for the first time. The fungal populations from which these isolates were obtained, originated from infected soybean leaves from South America. Sequence analyses of the CYP51-gene confirmed three newly identified mutations (V130A, I145V, F154Y), occurring in one allele in combination with previously described mutations. A total of nine mutations were observed in CYP51 of P. pachyrhizi (F120L, V130A, Y131F/H, K142R, I145V/F, F154Y, I475T), present in different combinations. Their locations in the enzyme were shown by CYP51-protein modeling. The newly identified mutation combinations F120L + V130A + Y131F, F120L + Y131H + I145V and F120L + Y131H + F154Y are associated with different sensitivities to DMIs. The sensitivity studies also demonstrated incomplete cross-resistance of P. pachyrhizi to DMIs caused by different mutation combinations. Furthermore, using an isolate mixture (defined population), it could be shown that different DMIs select different mutation combinations. P. pachyrhizi produces dikaryotic uredospores, and moreover, this study confirmed that each spore contains a total of 6 CYP51-copies. [ABSTRACT FROM AUTHOR]

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