Xanthomonas citri pv. eucalyptorum strain 4866-2_S43 (formerly X. axonopodis pv. eucalyptorum): the causal agent of bacterial leaf blight on eucalypts recovered in Argentina

Abstract

We report the draft genome assembly of strain 4866-2_S43 isolated from a eucalyptus lesion in Argentina and what until recently was caused by Xanthomonas citri pv. eucalyptorum (Xce). The genome size is 5 188 607 bp, with a G+C content of 64.66%. Comparative analysis reveals that the closest relative of strain 4866-2_S43 is Xce LPF 602, isolated in Brazil. Comparison of the whole genome sequences revealed an average nucleotide identity (ANI) of 99.96% between the two strains. ANIs were determined between the whole genome sequence of strain 4866-2_S43 and the genomes of all currently validated Xanthomonas spp. These results revealed that strain 4866-2_S43 shared >95% similarity with X. citri pv. citri and X. citri pv. phaseoli, and <95% with X. euvesicatoria pv. alfalfae, X. euvesicatoria pv. perforans, and X. euvesicatoria pathovars euvesicatoria and eucalyptii.

Data Summary

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession no. JAZGJM000000000. The annotated assembly has been deposited at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) under accession no. Gp0621574.

Announcement

Bacterial leaf blight (BLB) constitutes a significant disease affecting eucalyptus trees, particularly in regions characterized by abundant rainfall, both in the open field and nursery settings [1]. Typical symptoms of BLB include leaf distortion and water-soaking associated with lesions, progressing to necrosis that may lead to the formation of shotholes in lesions, ultimately resulting in pronounced defoliation [2]. A comprehensive investigation conducted in 2008, utilizing strains collected from diverse geographical locations, identified Xanthomonas axonopodis pv. eucalyptorum (Xae) [later X. citri pv. eucalyptorum (Xce)] as the causal agent of BLB in Eucalyptus spp. within Brazil [2]. More recently, the emergence of a novel pathovar, X. euvesicatoria pv. eucalyptii (Xee), was documented as the causal agent of BLB in Eucalyptus spp. [3].

Eighteen Xce strains were isolated on nutrient agar (NA) from Eucalyptus grandis hybrids in the Experimental Station of INTA Bella Vista (Corrientes province, Argentina) in 2017. All the Xce strains elicited a hypersensitive reaction when infiltrated into grapefruit leaves (Citrus paradisi). Initially, species identification was performed using standard biochemical methods. The strains were stored at −80 °C in 30% glycerol containing 0.8% nutrient broth (NB, Difco, Becton Dickinson). One of the strains, Xce 4866-2_S43, was grown on an NA plate for 24 h and processed for sequencing, and further bioinformatics analyses were done using the methodology as outlined by Subedi et al. [4]. The whole genome of Xae 4866-2 was sequenced on the Illumina MiSeq, generating a total of 725948 paired ends with a read length of 2×250 bp, resulting in a mean coverage of 8×. Raw sequencing reads were subjected to quality control and adapter trimming using FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic v0.38, respectively [5]. Clean reads were then de novo assembled using SPAdes v3.15.4 [6], removing contigs smaller than 500 bp to ensure sequencing quality, leaving a total of 209 contigs containing 5  188  607 bp. The maximum and minimum scaffold lengths were 146  011 and 519 bp, respectively. The scaffold N50 and N90 metrics were 51 103 and 14  158 bp, respectively. Furthermore, calculated G+C content was 64.66%. Genome annotation was accomplished by prokka software [7]. A total of 4416 coding sequences and 48 tRNAs were identified in the Xce 4866-2_S43 strain genome.

Average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) analyses of the whole genome of strain Xce 4866-2_S43 were performed with the reference genomes of Xanthomonas available in the NCBI genome database to determine the correct species identification and estimate the relationship of Xce 4866-2_S43 with other Xanthomonas spp. ANI and DDH values were determined using ANIb [8,9] and GGDC [10]. The ANI value of Xce 4866-2_S43 compared with 30 NCBI reference genomes of the genus Xanthomonas and 16 X. axonopodis genomes was >99% to Xce LPF 602 (RWJW00000000.1). Also, DDH comparison with same dataset of genomes showed a DDH value >99% to Xce LPF 602 (Fig. 1). Based on our result of multiple genome comparisons through several in silico analyses, the strain isolated in Argentina is placed as Xanthomonas citri pv. eucalyptorum and had an ANI >95% with reference strains of X. citri pv. citri and X. citri pv. phaseoli.

Fig. 1. ANI values of strain 4866-2 with the type strains of Xanthomonas spp.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession no. JAZGJM000000000. Annotation of the assembled genomes was done using the IMG Annotation Pipeline (v5.0.23) at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) (https://img.jgi.doe.gov) under accession no. Gp0621574.

Abbreviations

ANI

average nucleotide identity

DDH

DNA–DNA hybridization

NA

nutrient agar

Xae

Xanthomonas axonopodis pv. eucalyptorum

Xce

Xanthomonas citri pv. eucalyptorum