Isothermal molecular detection of Alternaria alternata by RPA-LFD

Abstract

Alternaria species are the most important fungal pathogens with a wide range of host including apple, pear, potato, tobacco, strawberry and tomato. Tobacco brown spot disease (TBS) is a disease caused by A. alternata that mainly occurs in maturation period of tobacco, and often leads to enormous economic losses. For the rapid detection of A. alternata in tobacco, recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection method was established in this work. Specific primers were designed based on the nucleotide sequence of the conidial pigment polyketide synthase gene ALM1. All investigated A. alternata isolates showed positive result using the specific primers, while other fungal species demonstrate negative result. The optimal temperature and reaction time were 39 °C for 10 min. Under this optimum conditions, the detection limitation of the RPA-LFD assay was 1 pg of genomic DNA of A. alternata. Furthermore, the practicality of RPA-LFD method was verified in vivo successfully by detection of A. alternata from infected leaves. In summary, we established a practical and reliable method for rapid diagnosis of TBS caused by A. alternata in tobacco in early stage of disease development.