A Powerful LAMP Weapon against the Threat of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens

Abstract

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is a Gram-positive phytopathogenic bacterium attacking leguminous crops and causing systemic diseases such as the bacterial wilt of beans and bacterial spot of soybeans. Since the early 20th century, Cff is reported to be present in North America, where it still causes high economic losses. Currently, Cff is an emerging plant pathogen, rapidly spreading worldwide and occurring in many bean-producing countries. Infected seeds are the main dissemination pathway for Cff, both over short and long distances. Cff remains viable in the seeds for long times, even in field conditions. According to the most recent EU legislation, Cff is included among the quarantine pests not known to occur in the Union territory, and for which the phytosanitary inspection consists mainly of the visual examination of imported bean seeds. The seedborne nature of Cff combined with the globalization of trades urgently call for the implementation of a highly specific diagnostic test for Cff, to be routinely and easily used at the official ports of entry and into the fields. This paper reports the development of a LAMP (Loop-Mediated Isothermal Amplification) specific for Cff, that allows the detection of Cff in infected seeds, both by fluorescence and visual monitoring, after 30 min of reaction and with a detection limit at around 4 fg/μL of pure Cff genomic DNA.

Keywords: Curtobacterium flaccumfaciens pv. flaccumfaciens; LAMP; Loop-Mediated Isothermal Amplification; bacterial wilt of bean; molecular diagnostics; quarantine plant pathogen; tan spot of soybean.

Figure 1

Optimization of LAMP reaction for Cff DNA detection. Isothermal amplification was carried out on Cff ICMP 2584^T DNA (40 ng/reaction) used as a template, and at temperatures ranging from 62 °C to 67 °C. Data assessment was based on 2% agarose gel electrophoresis analysis (A) and real-time fluorescence monitoring (B). (A) M - Gene Ruler 100 pb Plus DNA Ladder; C = negative control; lanes 1 to 6: amplicons in the temperature range 62 °C–67 °C. (B) Optimization of reaction time, according to real-time fluorescence amplification plots; NC = negative control. 1 cycle = 1 min reaction.

Figure 2

Determination of the analytical sensitivity of the LAMP assay for Cff detection. Isothermal amplifications were carried out on tenfold diluted test samples of Cff ICMP 2584^T DNA (from 100 ng to 100 fg per 25 µL reaction). Data assessment was based on (A) real-time fluorescence monitoring and (B) 2% agarose gel electrophoresis analysis of the LAMP products. (A) NC = negative control. 1 cycle = 1 min reaction. (B) Lanes 1–7: 100 ng, 10 ng, 1 ng, 0.1 ng, 10 pg, 1 pg, and 0.1 pg of genomic DNA/reaction, respectively; M - Gene Ruler 100 pb Plus DNA Ladder; C = negative control.

Figure 3

LAMP assay results from Cff-artificially infected bean plants cv. Cannellino. The Cff type strain ICMP 2584 was used for artificial inoculation. (A) Cff infection was confirmed by conventional PCR carried out using the primer pair CffFOR2/CffREV4. Lanes: 1, 2, 3, 4: Cff-inoculated bean plants; 5: uninoculated bean plant; 6: Cff ICMP 2584^T pure DNA; 7: negative control, sterilized molecular grade water as a template; M - Gene Ruler 100 pb Plus DNA Ladder. (B) Real-time monitoring of LAMP reaction for Cff detection on bean plants artificially inoculated with Cff ICMP 2584^T or with SPS. Sterilized molecular grade water was used as a negative control (NC).

Figure 4

Visual detection of LAMP products obtained with bean seed samples cv. “Pak” for Cff detection. Visual detection of LAMP products from bean seed samples cv. “Pak” for Cff detection. Positive samples show a color change, from pink (negative samples) to yellow. Uninfected (samples 1 and 2) and naturally Cff-infected (samples 3, 4, 5, 6, 7). Molecular grade water was used as a template in negative controls (sample 8).