We describe a novel species isolated from walnut (Juglans regia) which comprises non-pathogenic and pathogenic strains on walnut. The isolates, obtained from a single ornamental walnut tree showing disease symptoms, grew on yeast extract–dextrose–carbonate agar as mucoid yellow colonies characteristic of Xanthomonas species. Pathogenicity assays showed that while strain CPBF 424T causes disease in walnut, strain CPBF 367 was non-pathogenic on walnut leaves. Biolog GEN III metabolic profiles disclosed some differences between strains CPBF 367 and CPBF 424T and other xanthomonads. Multilocus sequence analysis with seven housekeeping genes (fyuA, gyrB, rpoD, atpD, dnaK, efp, glnA) grouped these strains in a distinct cluster from Xanthomonas arboricola pv. juglandis and closer to Xanthomonas prunicola and Xanthomonas arboricola pv. populi. Average nucleotide identity (ANI) analysis results displayed similarity values below 93 % to X. arboricola strains. Meanwhile ANI and digital DNA–DNA hybridization similarity values were below 89 and 50 % to non-arboricola Xanthomonas strains, respectively, revealing that they do not belong to any previously described Xanthomonas species. Furthermore, the two strains show over 98 % similarity to each other. Genomic analysis shows that strain CPBF 424T harbours a complete type III secretion system and several type III effector proteins, in contrast with strain CPBF 367, shown to be non-pathogenic in plant bioassays. Taking these data altogether, we propose that strains CPBF 367 and CPBF 424T belong to a new species herein named Xanthomonas euroxanthea sp. nov., with CPBF 424T (=LMG 31037T=CCOS 1891T=NCPPB 4675T) as the type strain.

We present the complete genome sequences of two Xanthomonas euroxanthea strains isolated from buds of a walnut tree. The whole-genome sequences of strains CPBF 367 and CPBF 426 consist of two circular chromosomes of 4,923,218 bp and 4,883,254 bp and two putative plasmids of 45,241 bp and 17,394 bp, respectively. These data may contribute to the understanding of Xanthomonas species-specific adaptations to walnut.

Xanthomonas arboricola pv. juglandis is the etiologic agent of important walnut (Juglans regia L.) diseases, causing severe fruit drop and high economic losses in walnut production regions. Rapid diagnostics and knowledge of bacterial virulence fitness are key to hinder disease progression and apply timely phytosanitary measures. This work describes an X. arboricola pv. juglandis-specific real-time quantitative PCR (qPCR) using X. arboricola pv. juglandis-specific DNA markers to quantify the bacterial load in infected walnut plant tissues. Method validation was achieved using calibration curves obtained with serial dilutions of X. arboricola pv. juglandis chromosomal DNA and standard curves obtained from walnut samples spiked with X. arboricola pv. juglandis cells. High correlations (R2 > 0.990 and > 0.995) and low limits of detection (35 chromosomes/qPCR reaction and 2.7 CFU/qPCR reaction) were obtained for both markers considering the calibration and standard curves, respectively. Assessment of qPCR repeatability, reproducibility, and specificity allowed us to demonstrate the reliability and consistency of the method. Furthermore, in planta quantification of X. arboricola pv. juglandis bacterial load using infected walnut fruit samples showed a higher detection resolution compared with standard PCR detection. By allowing quantification of virulence fitness of distinct X. arboricola pv. juglandis strains in planta, the proposed qPCR method may contribute to assertive risk assessment of walnut diseases caused by X. arboricola pv. juglandis and ultimately help to improve phytosanitary practices.

The recent report of distinct Xanthomonas lineages of Xanthomonas arboricola pv. juglandis and Xanthomonas euroxanthea within the same walnut tree revealed that this consortium of walnut-associated Xanthomonas includes both pathogenic and nonpathogenic strains. As the implications of this co-colonization are still poorly understood, in order to unveil niche-specific adaptations, the genomes of three X. euroxanthea strains (CPBF 367, CPBF 424T, and CPBF 426) and of an X. arboricola pv. juglandis strain (CPBF 427) isolated from a single walnut tree in Loures (Portugal) were sequenced with two different technologies, Illumina and Nanopore, to provide consistent single scaffold chromosomal sequences. General genomic features showed that CPBF 427 has a genome similar to other X. arboricola pv. juglandis strains, regarding its size, number, and content of CDSs, while X. euroxanthea strains show a reduction regarding these features comparatively to X. arboricola pv. juglandis strains. Whole genome comparisons revealed remarkable genomic differences between X. arboricola pv. juglandis and X. euroxanthea strains, which translates into different pathogenicity and virulence features, namely regarding type 3 secretion system and its effectors and other secretory systems, chemotaxis-related proteins, and extracellular enzymes. Altogether, the distinct genomic repertoire of X. euroxanthea may be particularly useful to address pathogenicity emergence and evolution in walnut-associated Xanthomonas.

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