High-Quality Complete Genome Sequence of Xanthomonas phaseoli pv. dieffenbachiae Outbreak Strain D182: The Causative Agent of Anthurium Bacterial Blight in Hawaii

Xanthomonas phaseoli pv. dieffenbachiae (Xpd) formerly known as X. axonopodis pv. dieffenbachiae (Constantin et al. 2016), the causal agent of bacterial blight of anthurium (Anthurium andraeanum Lind. ex André), is regarded as one of the most devastating pathogens, causing significant crop losses in commercial foliage plant production and cut flowers (Alvarez et al. 2006). Due to the economic importance of anthurium cut flowers in the European Union, Xpd is listed as an A2 quarantine pathogen on the European and Mediterranean Plant Protection Organization (EPPO) list (Cottyn et al. 2018; EPPO 2023).

The epidemic of the disease in Hawaii reached its peak between 1985 and 1989, resulting in the widespread devastation of approximately 200 small farms and a drop in production from 30 million stems in 1980 to 15.6 million stems in 1990 (Alvarez et al. 2006; Shehata 1992). The disease symptoms begin with water-soaked spots at the margins of the foliage, which later cause the surrounding tissue to turn chlorotic. Lesions eventually coalesce, forming large areas of necrotic tissue at the leaf margins. The pathogen can invade the vascular tissue of petioles and stems, leading to water stress symptoms. In cases of systemic infection, the main stem turns dark brown, leading to deterioration of the growing point and, ultimately, death of the plant. When infection occurs through stomata, there is limited colonization of mesophyll tissue, which does not necessarily result in systemic infection (Fukui et al. 1998).

In this study, the complete genome of Xpd strain D182, representative of the Hawaiian anthurium blight outbreak (1981 to 1986) was sequenced. Strain D182 was isolated in 1986 from an anthurium plant infected during the outbreak in Hawaii and stored in the Pacific Culture Collection, Honolulu, HI. The bacterium was revived from the lyophilized vial of strain D182 and streaked on dextrose peptone agar (DPA: peptone 10 g/liter, dextrose 5 g/liter, and agar 17 g/liter) (modified from Norman and Alvarez 1989) and incubated at 28°C for 24 h. A single colony was streaked again onto DPA and incubated at 28°C for 24 h. Genomic DNA was extracted from this culture using the QIAGEN Genomic-tip 100/G following the manufacturer's instructions (Qiagen, Germantown, MD). The quantity and quality of the isolated DNA were assessed using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA) and agarose (1%) gel electrophoresis (Boluk et al. 2021). Strain D182 underwent whole-genome sequencing at the University of Washington Genome Sciences PacBio Facility using the PacBio Sequel II System (Pacific Biosciences of California, Menlo Park, CA) employing a single-molecule real-time (SMRT) technology, was de novo assembled with the Hierarchical Genome Assembly Process (HGAP v4) using default parameters (500-bp minimum subread length; 6-kb minimum seed read length), and was polished with the Quiver algorithm (Chin et al. 2013). The assembled and polished genome was submitted to online automated annotation systems, including Prokaryotic Genomes Automatic Annotation Pipeline (PGAP) at the National Center for Biotechnology Information (NCBI) (Tatusova et al. 2016), Rapid Annotation using Subsystem Technology (RAST) (Brettin et al. 2015), and Integrated Microbial Genomes pipeline version 4.10.5 from the Joint Genome Institute (IMG-JGI; Huntemann et al. 2015) for downstream analyses. The general genomic features of strain D182 are presented in Table 1, and graphical circular maps of the chromosome and plasmid are illustrated in Figure 1. The average nucleotide identity (ANI) was determined using the Nucleotide MUMmer algorithm (ANIm) through the JSpecies Web Server (Richter et al. 2016), digital DNA–DNA hybridization (dDDH) was computed utilizing version 2.1 of the genome-to-genome distance calculator (GGDC version 2.1) with the recommended formula 2, and BLAST+ alignment criteria was found to be 99.86 and 98.9% with Xpd strain LMG 695^PT, respectively.

TABLE 1 General features of the complete genome of Xanthomonas phaseoli pv. dieffenbachiae strain D182

Parameter	D182 genome
Genome size (bp)	5,217,888
GC content (%)	64.89
Number of chromosomes	1
Chromosome size (bp)	5,144,406
Number of plasmids	1
Plasmid size (bp)	73,482
Number of coding genes	4,461
Total coding number of bases	4,483,246
Number of tRNA genes	55
Number of rRNA genes	6
Number of 5S rRNA, 16S rRNA, and 23S rRNA	2, 2, and 2
CRISPRs	2
Coarse consistency (%)	99.7
Completeness (%)	100
CheckM2 contamination	0.23
NCBI GenBank accession numbers	CP041380; CP041381 (plasmid)

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The annotated genome was searched for pathogenicity determinants including type secretion systems (TSSs) and xanthan gum. Genome analysis indicated that the D182 strain harbors Type II, III, IV, and VI secretion systems similar to those reported by Constantin et al. (2017). The Type IV secretion system (T4SS) is described as an important factor aiding bacterial adaptation to new hosts (Saenz et al. 2007); 12 genes known to contribute to the secretion of virulence factors, namely virB1 to virB11 and virD4, were identified in strain D182. The T2SS plays a crucial role in secreting toxins and cell wall-degrading enzymes in various Xanthomonas species (Ryan et al. 2011). Whole-genome analysis revealed the presence of two clusters, xps and xcs, containing 11 (xpsD to xpsN) and 12 (xcsC to xcsN) genes, respectively. The T3SS in Xanthomonas is crucial for delivering effector proteins directly into host cells, thereby facilitating bacterial pathogenicity and manipulation of the host (Cornelis 2006; Galán and Wolf-Watz 2006). The T3SS encoded by the hrp gene cluster in strain D182 contains all 27 genes, with identical organization to those reported by Jalan et al. (2011) and Constantin et al. (2017). The T6SS in Xanthomonas acts as a versatile mechanism for both bacterial competition and host infection (Shyntum et al. 2015). The genome of strain D182 contains two clusters of T6SS, with genes from ecfK to tssA in cluster I and from tssB to clpV in cluster II.

Xanthan gum is an exopolysaccharide that plays a crucial role in biofilm formation and virulence of pathogens listed in the Xanthomonadaceae family (Katzen et al. 1998). This cluster harbors genes from gumB to gumN, which is consistent with the findings reported by Constantin et al. (2017). The genome was also analyzed for the flagella synthesis clusters, which consisted of all four gene clusters needed for flagellar biosynthesis (Moreira et al. 2010). Cluster I encompassed a 18,134-bp region that comprised 18 genes from fliS to flgN, with a presence of two hypothetical genes in strain D182 when compared with LMG695^PT; cluster III consisted of motA and motB genes coding for flagellar motor protein (2,023-bp region), which is similar to the flagellar cluster described by Constantin et al. (2017) and Moreira et al. (2010). Flagellar gene cluster II of strain D182 has the similar gene organization as X. fuscans subsp. aurantifolii reported by Moreira et al. (2010). Cluster II consisted of 27 genes from the cheA to fliE genes, spanning a 31,339-bp region with three hypothetical genes inserted in this cluster and the presence of a par gene encoding a partitioning protein. These genes are responsible for bacterial membrane interaction and code for flagellar motor components. This cluster differs in the arrangement of the genes compared to the previously reported LMG25940 strain of Xpd reported by Constantin et al. (2017) but is similar to LMG695^PT. Cluster IV, consisting of seven genes from cheA to motA, was located close to cluster I. The gene clusters involved in flagellum biosynthesis and regulation are shown in Figure 2. The images were generated using clinker and clustermap.js (Gilchrist and Chooi 2021). Furthermore, multiple copies of methyl-accepting chemotaxis protein (mcs), which is reported to play a role in chemotaxis, were also found in the genome of D182 (Constantin et al. 2017; da Silva et al. 2002).

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The genome was analyzed for genomic islands (GIs) using the IslandViewer 4 webserver, which integrates four different and accurate prediction tools: IslandPath-DIMOB, SIGI-HMM, IslandPick, and Islander (Bertelli and Brinkman 2018; Bertelli et al. 2017; Hudson et al. 2015; Langille et al. 2008). The GIs, indicative of horizontal gene transfer events, are reported to contain genes associated with adaptive traits, virulence, and resistance to new environments (Constantin et al. 2017; Li et al. 2022). IslandViewer 4 predicted 45 GIs, ranging in size from 4.02 to 108.55 kb. The largest GIs (108.55 kb) contain genes for various functions, including oxidoreductase, dioxygenase, endonuclease, methylase, Type I secretion system permease, HlyD family type I secretion periplasmic adaptor subunit, Type IV conjugative transfer system, pilus assembly protein, caspase family protein, ParA protein, and many hypothetical proteins. The genome was also searched for regions containing prophage-like elements using the PHASTER webserver (Phage Search Tool Enhanced Release), which predicted the presence of putatively defective (questionable) prophage-like elements within a size range of 12.3 kb (4,517,572 to 4,529,966) (Arndt et al. 2016; Zhou et al. 2011). The assembled genome of Xpd D182 provides valuable insights into its genomic characteristics, which will aid in studies focusing on its interactions with anthurium, as well as in phylogenetic and evolutionary genomics research.