LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR THE RAPID DETECTION OF CURTOBACTERIUM FLACCUMFACIENS pv. FLACCUMFACIENS FOR QUARANTINE AND FIELD APPLICATIONS

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is a Gram-positive bacterium, and the causal agent of a systemic vascular disease known as bacterial wilt of beans (Phaseolus vulgaris).Bean seedlings rapidly die after infection, while older plants can survive the attack and produce mature seeds, infected by Cff via the vascular system. These seeds are generally asymptomatic, with rare yellow, orange, or purple pigmentation on white bean seeds, depending on the Cff strains/variants. Within or on infected seeds Cff remains viable for decades, and seeds are the main means of dissemination of Cff over short and long distances. This bacterium is present since the early 20th century in North America, where it still causes high economic losses, and is endemic in many bean producing countries. Although Cff is a quarantine pathogen for Europe (EPPO A2 list), so far the control of imported seeds for Cff is not mandatory, and inspections often consist just on visual examination. Recently, the isolation of Cff from diseased soybean in Germany dramatically focused on the threat posed by its further spread. Therefore, it is urgently needed to implement rapid, easy and specific tests for Cff, having the potential to be routinely used at the ports of entry. A loop-mediated isothermal amplification (LAMP) assay for Cff was developed, targeting a nucleotide sequence exclusive of Cff strains pathogenic on bean. This LAMP test has a sensitivity of 10 pg/reaction, and the entire procedure takes only about 1.5 h.