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

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 (Prokic 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; Prokic 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.

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens.

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

Chitin Mixed in Potting Soil Alters Lettuce Growth, the Survival of Zoonotic Bacteria on the Leaves and Associated Rhizosphere Microbiology.

Chitin is a promising soil amendment for improving soil quality, plant growth, and plant resilience. The objectives of this study were twofold. First, to study the effect of chitin mixed in potting soil on lettuce growth and on the survival of two zoonotic bacterial pathogens, Escherichia coli O157:H7 and Salmonella enterica on the lettuce leaves. Second, to assess the related changes in the microbial lettuce rhizosphere, using phospholipid fatty acid (PLFA) analysis and amplicon sequencing of a bacterial 16S rRNA gene fragment and the fungal ITS2. As a result of chitin addition, lettuce fresh yield weight was significantly increased. S. enterica survival in the lettuce phyllosphere was significantly reduced. The E. coli O157:H7 survival was also lowered, but not significantly. Moreover, significant changes were observed in the bacterial and fungal community of the lettuce rhizosphere. PLFA analysis showed a significant increase in fungal and bacterial biomass. Amplicon sequencing showed no increase in fungal and bacterial biodiversity, but relative abundances of the bacterial phyla Acidobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Proteobacteria and the fungal phyla Ascomycota, Basidiomycota, and Zygomycota were significantly changed. More specifically, a more than 10-fold increase was observed for operational taxonomic units belonging to the bacterial genera Cellvibrio, Pedobacter, Dyadobacter, and Streptomyces and to the fungal genera Lecanicillium and Mortierella. These genera include several species previously reported to be involved in biocontrol, plant growth promotion, the nitrogen cycle and chitin degradation. These results enhance the understanding of the response of the rhizosphere microbiome to chitin amendment. Moreover, this is the first study to investigate the use of soil amendments to control the survival of S. enterica on plant leaves.

Characterization of Novel Bacteriophages for Biocontrol of Bacterial Blight in Leek Caused by Pseudomonas syringae pv. porri.

Pseudomonas syringae pv. porri, the causative agent of bacterial blight in leek (Allium porrum), is increasingly frequent causing problems in leek cultivation. Because of the current lack of control measures, novel bacteriophages were isolated to control this pathogen using phage therapy. Five novel phages were isolated from infected fields in Flanders (vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, vB_PsyM_KIL4, and vB_PsyM_KIL5), and were complemented with one selected host range mutant phage (vB_PsyM_KIL3b). Genome analysis of the phages revealed genome sizes between 90 and 94 kb and an average GC-content of 44.8%. Phylogenomic networking classified them into a novel clade, named the "KIL-like viruses," related to the Felixounalikevirus genus, together with phage phiPsa374 from P. syringae pv. actinidiae. In vitro characterization demonstrated the stability and lytic potential of these phages. Host range analysis confirmed heterogeneity within P. syringae pv. porri, leading to the development of a phage cocktail with a range that covers the entire set of 41 strains tested. Specific bio-assays demonstrated the in planta efficacy of phages vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, and vB_PsyM_KIL3b. In addition, two parallel field trial experiments on three locations using a phage cocktail of the six phages showed variable results. In one trial, symptom development was attenuated. These data suggest some potential for phage therapy in controlling bacterial blight of leek, pending optimization of formulation and application methods.

Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.

Four bacterial strains, designated M89, M92, M97(T), and M106, were isolated in a previous study from surface-sterilized leaves of rice (Oryza sativa) or murainagrass (Ischaemum rugosum) at three sites in Mali, Africa. Here they were examined by a polyphasic taxonomic approach and analysis of a whole-genome sequence. Phylogenetic analyses based on 16S rRNA sequence and multilocus sequence analysis of seven genes showed that these four strains formed a distinct lineage representing a novel species within the genus Xanthomonas. This was supported by whole-genome average nucleotide identity values calculated from comparisons of strain M97(T) with established Xanthomonas species. The strains can be differentiated from the known Xanthomonas species on the basis of their fatty acid and carbohydrate utilization profiles. Population growth studies on rice confirmed that these bacteria multiply in rice leaves without causing symptoms. Identification of this novel species can be accomplished by using diagnostic primer sets or by gyrB gene sequence analysis. We propose to classify these rice- and grass-associated bacteria as Xanthomonas maliensis sp. nov. with strain M97(T) = CFBP7942(T) = LMG27592(T) as the type strain.