LAMP Assay for Distinguishing Fusarium oxysporum and Fusarium commune in Lotus (Nelumbo nucifera) Rhizomes

Abstract

Yields of edible rhizome from cultivation of the perennial hydrophyte lotus (Nelumbo nucifera) can be severely reduced by rhizome rot disease caused by Fusarium species. There is a lack of rapid field-applicable methods for detection of these pathogens on lotus plants displaying symptoms of rhizome rot. Fusarium commune (91%) and Fusarium oxysporum (9%) were identified at different frequencies from lotus samples showing symptoms of rhizome rot. Because these two species can cause different severity of disease and their morphology is similar, molecular diagnostic-based methods to detect these two species were developed. Based on the comparison of the mitochondrial genome of the two species, three specific DNA loci targets were found. The designed primer sets for conventional PCR, quantitative PCR, and loop-mediated isothermal amplification (LAMP) precisely distinguished the above two species when isolated from lotus and other plants. The LAMP detection limits were 10 pg/μl and 1 pg/μl of total DNA for F. commune and F. oxysporum, respectively. We also carried out field-mimicked experiments on lotus seedlings and rhizomes (including inoculated samples and field-diseased samples), and the results indicated that the LAMP primer sets and the supporting portable methods are suitable for rapid diagnosis of the lotus disease in the field. The LAMP-based detection method will aid in the rapid identification of whether F. oxysporum or F. commune is infecting lotus plants with symptoms of rhizome rot and can facilitate efficient pesticide use and prevent disease spread through vegetative propagation of Fusarium-infected lotus rhizomes.