A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

Abstract

Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop‐mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on‐site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high‐throughput and user‐friendly method for detection and screening of plant material in support of quarantine regulations.

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description

“…Access to a strain is crucial to understand how the pathogen is transferred between countries. To prevent further spread of the disease, highly specific and effective methods have been designed for detection of X. fragariae [ 44, 71, 72 ].…”

“…Strains were selected in order to obtain a collection with various years of isolation and geographical origins. Strain identity was confirmed using the X. fragariae -specific loop-mediated isothermal amplification (LAMP) assay [ 44 ]. The draft genomes of LMG 25863 (accession number: AJRZ01000000 ) [ 45 ] and of two American isolates, Fa P21 (accession numbers: CP016830–CP016832) and Fa P29 (accession numbers: CP016833–CP016835) [ 46 ] were obtained from GenBank and were added to the in silico analysis.…”

“…Strains were grown on agar plates with Wilbrinks-N medium [ 56 ] for 72 h. A full loop of bacteria was transferred to a 2 ml tube with 500 µl distilled water, boiled for 15 min at 95 °C and diluted tenfold in distilled water. Prior to PCR, verification with the LAMP detection system specific to X. fragariae [ 44 ] was applied to all samples to confirm them as being X. fragariae samples. This control is crucial in the eventuality of negative amplifications of a sample with all three primer sets, which could be caused by the occurrence of a deletion affecting one of more spacers targeted by primers.…”

Comparative genomics of Xanthomonas fragariae and Xanthomonas arboricola pv. fragariae reveals intra- and interspecies variations

“…fragariae and X. fragariae could be co-isolated from symptomatic strawberry leaves (Scortichini and Rossi 2003;Vandroemme et al 2013b). In order to avoid enrichment and biased growth between both bacterial species, the recently designed X. fragariae loopmediated isothermal amplification (LAMP) could be used to directly detect plant tissue (Gétaz et al 2017a). For detection of X. arboricola pv.…”

“…fragariae is complicating diagnostics from symptomatic plants, and wrong conclusions may be drawn when picking a single colony from plate. Tools for diagnostics directly from infected plant material like LAMP are already available for the specific detection of X. fragariae (Wang and Turechek 2016;Gétaz et al 2017a). A similar test would need to be designed as well for detection of X. arboricola pv.…”