First Report of <i>Curtobacterium flaccumfaciens</i> pv. <i>flaccumfaciens,</i> the Causal Agent of Bacterial Wilt, in Bean Seeds Produced in Greece

In December 2024, four samples of bean (Phaseolus vulgaris L.) seeds (500g /sample) were collected from four 0.6- to 1.2-ha field crops, in the Regional Unit of Florina (Western Macedonia, Greece) during the harvesting season by the local Phytosanitary Inspectors in the frame of the annual National Survey Program, and sent to the Laboratory of Bacteriology of the Benaki Phytopathological Institute for examination for the quarantine bacterium Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff). No symptoms of Cff, the cause of bacterial wilt in several species of the Fabaceae family (Osdaghi et al., 2020), had been observed on these crops or seeds. Each seed sample (500g or 1000 seeds) was soaked overnight (4 oC) in 2.5 x Thousand Seed Weight (in mL) sterile buffer (10 mM PB with 0.02 % v/v Tween 20), i.e. 1250 mL. An aliquot of 50 ml of each sample seed extract was used for DNA extraction, using the DNeasy mericon Food kit (Qiagen, Germany). Cff was detected in the DNA extract of one of the four tested seed samples by two qPCR assays, using: a) CffF3/CffB3 primers (Tegli et al. 2022), and b) Cff1-F/Cff1-R primers and Cff1-P probe (Venneman and Van Vaerenbergh 2024). Yellowish colonies were isolated on modified King's B medium (Volkers et al., 2025) from this positive sample, and one of these isolates was identified as Cff by qPCR (Tegli et al. 2022) and indirect immunofluorescence (IF) assay with a Cff-specific polyclonal antiserum (PRI, Netherlands). Cff was not isolated from the other three seed samples. The pathogenicity of the Cff isolate was confirmed via inoculation of five bean (cv. Borlotto) seedlings at the stage of two trifoliate leaves. A 10 μl drop of the isolate’s suspension (108 cfu/ml) was inserted by needle prick on the node of the first leaves to introduce the inoculum in the vascular strand of the stem. After 7 days of incubation at 25-28 oC/14 h daylight, the plants exhibited leaf flaccidity and wilt, typical symptoms of Cff infection. The pathogen was re-isolated from the main vein of a symptomatic leaf of an inoculated plant, and its identification was confirmed by IF and qPCR (Tegli et al. 2022), as before, fulfilling Koch’s postulates. The Cff strain NCPPB 559 and sterile distilled water were used as positive and negative controls respectively. Furthermore, partial sequencing (Eurofins, Austria) of the 16S rDNA, gyrB and atpD genes of the same isolate was performed using the primer pairs: pA/pH (EPPO 2021), 2F/6R (Richert et al. 2005) and aptD2F/aptD2R (Jacques et al. 2012), respectively. The obtained sequences for the 16S rDNA (1455 bp, PV770169), gyrB (897 bp, PV779358) and atpD (1016 bp, PV779359) genes were 100%, 98.77% and 99,70%, respectively, identical to those of the Cff type strain CFBP 3418 (NCPPB 1446; CP045852.1) and were deposited in the GenBank. This is the first confirmed report of the presence of Cff in Greece. This finding is added to three other recent Cff outbreaks reported in European countries: the Netherlands, Belgium and Switzerland (Volkers et al. 2025), and underlines the importance of seeds as a means of Cff introduction in an area. Currently official phytosanitary measures have been imposed in the affected area to eradicate Cff, whereas a wider monitoring of bean crops is ongoing to assess any possible Cff spread in Florina, which is one of the main bean-producing Regional Units in Greece, and the produced beans are registered as a ‘Protected Geographical Indication’ product.