First report of black rot disease in Eruca vesicaria subsp. sativa caused by Xanthomonas campestris pv. campestris in Belgium.

Abstract

Eruca vesicaria subsp. sativa (Mill.) Thell. (arugula or rocket) is a leafy vegetable originating from the Mediterranean region primarily being sold in bagged salads. From 2014 to 2017, plants (cv. Montana) exhibiting blackened leaf veins and irregular V-shaped chlorotic to necroic lesions at the leaf margins were observed in commercial greenhouses in Flanders, Belgium (Figure S1A). Symptoms started after harvest of the first cut, indicating that leaf injury favours disease development. By the last cut, infections had spread uniformly across the plots, with symptoms advanced to the point where harvesting was no longer profitable. Excised surface-sterilized necrotic leaf tissue and seeds were homogenized in phosphate buffer (PB), followed by dilution plating on Pseudomonas Agar F containing sucrose. After four days at 28°C, bright yellow round, mucoid, convex Xanthomonas-like colonies were obtained, both from leaves and seeds. For confirmation, DNA was extracted from pure cultures after which a partial fragment of gyrB was amplified and sequenced (Holtappels et al. 2022). Amplicons were trimmed to 530 nucleotides (Genbank ON815895-ON815900) according to Parkinson et al. (2007) and compared with the NCBI database. Strain GBBC 3139 shares 100% sequence identity with Xanthomonas campestris pv. campestris (Xcc) type strain LMG 568 and with RKFB 1361-1364, isolated from arugula in Serbia (Prokić et al. 2022). The other isolates from Belgian rocket - GBBC 3036, 3058, 3077, 3217 and 3236 - all have a gyrB sequence 100% identical to that of Xcc strain ICMP 4013, among others. To determine the genetic relatedness to other pathogenic Xc strains, the genomes of GBBC 3077, 3217, 3236 and 3139 were sequenced using a MinION (Nanopore) and non-clonal sequences were submitted to NCBI (BioProject PRJNA967242). Genomes were compared by calculating Average Nucleotide Identity (ANI). This revealed that the Belgian strains cluster together with Xc isolates originating from Brassica crops and separate from strains identified as Xc pv. barbareae, pv. incanae and pv. raphani (Figure S2A). Their designation as pv. campestris is supported by maximum likelihood clustering of concatenated gyrB-avrBs2 sequences (EPPO, 2021; Figure S2B,C). Finally, pathogenicity was verified on five-week-old rocket 'Pronto' plants grown in a commercial potting mix by cutting the leaves along the midrib with scissors dipped into a suspension of 108 cfu/ml of each strain or PB as control (4 plants/strain). Plants were kept in closed polypropylene boxes for 48 hr to support high humidity and facilitate infection. They were then maintained at 25 ± 2 °C. Lesions like those observed on commercial plants developed on the inoculated leaves within one week (Figure S1B). Bacterial colonies reisolated from symptomatic tissue were identified based on gyrB as the strains used for inoculation, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of black rot disease in arugula caused by Xcc in Belgium. Previously, Xcc on arugula has been reported in Argentina, California and Serbia as well (Romero et al. 2008; Rosenthal et al. 2017; Prokić et al. 2022). Arugula being a minor crop in Belgium, challenged by Xcc infections and strong import competition, many growers have abandoned the sector in recent years. Therefore, this study makes a strong case for early detection of disease symptoms and timely application of relevant management strategies in vulnerable crop settings.