LAMP Detection and Identification of the Blackleg pathogen Leptosphaeria biglobosa ‘brassicae’

Abstract

Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/L. biglobosa (Lb), which are composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, however, under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb ‘brassicae’ (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb ‘canadensis’ (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. In order to monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp-long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China anddeveloping strategies for management of the blackleg disease.