First report of Curtobacterium flaccumfaciens pv. flaccumfaciens the causal agent of bacterial wilt disease in bean plants (Phaseolus vulgaris) in The Netherlands.

Abstract

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), a European quarantine organism, is the causal agent of bacterial wilt in several members of the Fabaceae family (Osdaghi et al., 2020). Recently, several Cff findings have been reported on the European continent (Tarakanov et al., 2022; EPPO 2022; EPPO 2024). In August 2024, following the Cff finding in an imported common bean seed lot, asymptomatic common bean plants were sampled from three fields in the Netherlands, ranging from approximately 0.5 to 4.8 ha in size, where the crop was cultivated for human consumption. Each sample consisted of 200 pieces of stem of 2-4 cm long. After short surface sterilization in 70% EtOH the pieces were crushed in a Stomacher bag and incubated in 50 mL 0.05 M phosphate buffer with 0.02% Tween 20 at 100 rpm at room temperature for 30 minutes. For bacteria isolation, 20 µL of the plant extract was plated onto YPG medium (5 g/L yeast extract, 10 g/L peptone, 5.5 g/L glucose, 15 g/L agar) and modified King's B medium (38 g/L Pseudomonas agar F, 10 g/L sucrose, 100 mg/L cycloheximide) by dilution plating. After incubation at 28 °C for 4 days, one typical colony (shiny, slimy, cream-colored) per sample was selected from the modified King's B medium for purification. For preliminary identification MALDI-TOF MS analysis was performed (Bruker, Germany) comparing the spectra of the three purified isolates with in-house made reference spectra of several C. flaccumfaciens (Cf) strains. The DNA of isolates that were identified as Cf was isolated by thermal lysis and used for identification at the pathovar level using the conventional PCR by Tegli et al. (2002). The genomes of these isolates, generated using Illumina sequencing (accession numbers JBKYKM000000000, JBKYKN000000000 and JBKYKO000000000) share between 96.0-97.3% average nucleotide identity (ANI) with the Cf type strain CFBP 3418 confirming they fall within the species boundary. Recently, yellow-pigmented Cf strains showed higher than 94 % ANI among themselves and with CFBP 3418 but below 94% ANI with members of the pink-/orange-/red-pigmented strains (Osdaghi et al., 2024). A pathogenicity test on Phaseolus vulgaris var. Ferrari plants, performed according to EPPO (2011), confirmed the virulence of these isolates. Per isolate, 7-8 out of 10 plants showed typical Cff symptoms at 7 dpi, including wilting and interveinal chlorotic areas in the leaves, similar to the positive control plants. Negative control plants remained asymptomatic. The isolates were successfully re-isolated from the inoculated plants and identified as Cff using the above mentioned methods. The diagnostic procedure followed justifies the classification of the three Cf isolates as Cff. This is the first confirmed report of Cff on common bean in the Netherlands. Eradication measures have been imposed on the fields from which plants tested positive, including destruction of the crop and crop remains, hygiene measures, and prohibition to cultivate Fabaceae for the coming two years. Following this, samples were taken from composite weed samples growing in the near vicinity of the infected fields. In one of these samples, the presence of Cff has been confirmed as well (accession nr JBKYKL000000000), following the same diagnostic procedure as for the common bean samples. No bean plants with Cff symptoms were observed in these three fields.