First report of Pseudomonas syringae pv. alisalensis causing bacterial blight of arugula (Eruca vesicaria subsp. sativa) in Greece

In March of 2007, leaf symptoms were observed on two commercial fields of arugula (Eruca vesicaria subsp. sativa) grown in Heraklion, Crete. Approximately 20% of plants were affected. Likewise, similar symptoms were observed on samples which were collected from freshly packeted arugula leaves in the market, and plants originating from the areas of West Peloponnese (Ilia) and Attica (Marathonas and Megara) in Greece. In the early infection stage, small angular water-soaked spots (2-5mm) were observed on leaves, often surrounded by chlorotic haloes visible on both leaf surfaces. At later stages these water-soaked lesions became necrotic with a brown to tan centre, remaining angular in shape, expanding progressively and resulting in leaf collapse (Fig. 1 A, C). Pure cultures of blue-green fluorescent bacteria (aerobic Gram negative rods) were consistently isolated on King’s medium B from leaf lesions of infected plants. In LOPAT assays, arugula isolates exhibited the phenotype [+ - - - +] of Lelliot’s Ia group, which includes Pseudomonas syringae pathovars. Biochemical tests were performed as described by Goumas et al.(1999). All the arugula isolates tested were positive for D(-) tartrate utilisation while negative for erythritol. Trigonelline and ascorbic acid tests were able to differentiate those isolates from Pseudomonas syringae pv. maculicola. Based on these results, we hypothesized that the isolates correspond to Pseudomonas syringae pv. alisalensis, recently renamed as P. cannabina pv. alisalensis (Cintas et al., 2002; Bull et al., 2004; Bull et al., 2010).

To confirm our hypothesis, we performed both BOX and ERIC-PCR, as described by Weingart & Völksch (1997) and Marques et al., (2008), on two out of ten isolates (R1-3 and R6) using P. s. pv. maculicola (CFBP 1657), P. s. pv. tomato (DC3000) and P. s. pv. alisalensis (CFBP 6866 and CFBP 6870) as controls. The BOX and ERIC-PCR patterns obtained with  P. s. pv. alisalensis CFBP 6866 and CFBP 6870 and R1-3 and R6  isolates were identical, while those obtained with P. s. pv. maculicola and P. s. pv. tomato were clearly different (Fig. 2). Cultures were grown on nutrient agar for 24h at 27°C and adjusted in sterile water to 1 x 10⁶ cfu/ml.  Arugula leaves of two to three-week-old plants were inoculated by spraying until runoff with a hand mister. Inoculated plants were incubated in plastic polyethylene bags with high humidity for 48h at 22-2°C. Sterile distilled water was used as control. Symptoms were visible one week after inoculation and similar to those observed under natural infection (Fig.1B-C, Fig 3). To fulfil Koch's postulates we re-isolated P. s. pv. alisalensis from symptomatic tissue of arugula plants. Control plants remained symptomless. Using similar inoculation and incubation methods (except for spot inoculating detached Brussels sprouts heads with 10 µl of bacterial suspension at 1x10⁸ cfu/ml in sterile water), these isolates were found to be pathogenic on broccoli (Brassica oleracea var. italica), brussel sprouts (B. oleracea var. gemmifera), turnip (B. napus) and broccoli raab (B. rapa var. rapa), radish (Raphanus sativus), oats (Avena sativa) and tomato (Solanum lycopersicum). Based on morphological, biochemical, physiological, molecular and pathogenicity tests, we conclude that the arugula isolates identified here belong to P. s. pv. alisalensis. To our knowledge this is the first report of P. s. pv. alisalensis in the European area (Bull et al., 2010). Two isolates (R1-3 and R6) isolates have been deposited in the Benaki Phytopathological Institute Collection, Greece with the reference numbers BPIC 2127 and BPIC 2128 respectively.