Whole-genome alignment-based development of molecular markers for detecting Leptosphaeria maculans and Leptosphaeria biglobosa, the causal agents of blackleg disease in Brassicas

Accurate diagnosis of the differentially aggressive fungi Leptosphaeria maculans 'brassicae' and L. biglobosa 'brassicae' causing blackleg disease in Brassica crops is crucial. Prior to the availability of fungal whole-genome sequences, gene-specific markers were designed on specific genes. But, the availability of the whole-genome sequences of several isolates now offers a greater opportunity to develop more precise and novel markers. The draft genomes of L. maculans and L. biglobosa that are available in the repository were aligned, and species-specific primers were designed from the variable sequences that encode for effector proteins. Three primer sets namely, Lm1, Lm2 and Lm5 and two primer sets namely Lb1' and Lb3 specifically detected the isolates of target species in PCR assay. The primers Lm5 and Lb1' were multiplexed for detection of L. maculans 'brassicae' and L. biglobosa 'brassicae', respectively, from a single PCR reaction. The markers were highly sensitive in amplifying target species from crude 'pseudothecia and ascospore suspension' without requiring DNA extraction. These markers, solitarily or in combination, could be used for precise, sensitive and rapid detection of L. maculans 'brassicae' and L. biglobosa 'brassicae' species and will be helpful for surveillance, management and transboundary quarantine of the devastating blackleg disease.

Automated summary

By analyzing the whole-genome sequences of fungi, specific primers were designed for rapid detection of the two aggressive fungi causing blackleg disease, providing a new approach and method for monitoring and managing blackleg disease.