Xylella fastidiosa field detection using loop-mediated isothermal amplification (LAMP) and MinION Mk1C

Xylella fastidiosa (XF) is a xylem phytopathogenic bacterium that halts plants from feeding, leading to losses and damage to large agricultural and forestry areas causing great economic and environmental impacts. Approximately 563 plant species can be infected by XF and due to the destructive potential, the European and Mediterranean Plant Protection Organization (EPPO) described XF as a quarantine organism (A2 List).

The XF impact will be determined by the subspecies, the host, the vectors transmission, and environmental factors, but an early detection will help farmers to take preventive measures by reducing impact, costs, and damage.

In this line, Loop-mediated isothermal amplification (LAMP) and Oxford Nanopore Technology are two methods that are becoming important due to their rapidity, sensitivity and their portable format applied to various environments and for plant pathogen detection. This present work proposes to develop a simple, accurate and sensitive real time detection tool to be used in the field. This consists in coupling LAMP with a 3rd generation sequencing technology, the MinION MK1c device.

LAMP allows, without prior DNA extraction (Yaseen et al., 2015), a portable, simple, fast, and inexpensive sensitive and specific detection and ONT MinION MK1C assures the diagnostic accuracy. For Xylella fastidiosa LAMP detection, we used previously designed primers for rimM gene (Harper et al., 2010) and newly designed primers for at least Multi-Locus Sequence Typing (MLST) of three housekeeping genes: cysG, malF and rpoD (Minsavage et al., 1994;EPPO 2019).

LAMP positive samples are confirmed (eliminating false positives) by real-time sequencing for Xylella fastidiosa presence. Foremost, MLST gene primers will allow the subspecies identification and sequence type (ST) by finding possible new outbreaks or hosts. Hence, this method allows to reduce diagnosis from weeks to hours. This study is the first step towards applying fast, accurate and sensitive methods to prevent plant diseases in an early stage, avoiding pathogen field dissemination.