Three new species, Xanthomonas hawaiiensis sp. nov., Stenotrophomonas aracearum sp. nov., and Stenotrophomonas oahuensis sp. nov., isolated from the Araceae family

Xanthomonas and Stenotrophomonas are closely related genera in the family Lysobacteraceae. In our previous study of aroid-associated bacterial strains, most strains isolated from anthurium and other aroids were reclassified as X. phaseoli and other Xanthomonas species. However, two strains isolated from Spathiphyllum and Colocasia were phylogenetically distant from other strains in the Xanthomonas clade and two strains isolated from Anthurium clustered within the Stenotrophomonas clade. Phylogenetic trees based on 16S rRNA and nine housekeeping genes placed the former strains with the type strain of X. sacchari from sugarcane and the latter strains with the type strain of S. bentonitica from bentonite. In pairwise comparisons with type strains, the overall genomic relatedness indices required delineation of new species; digital DNA–DNA hybridization and average nucleotide identity values were lower than 70 and 95%, respectively. Hence, three new species are proposed: S. aracearum sp. nov. and S. oahuensis sp. nov. for two strains from anthurium and X. hawaiiensis sp. nov. for the strains from spathiphyllum and colocasia, respectively. The genome size of X. hawaiiensis sp. nov. is ~4.88 Mbp and higher than S. aracearum sp. nov. (4.33 Mbp) and S. oahuensis sp. nov. (4.68 Mbp). Gene content analysis revealed 425 and 576 core genes present in 40 xanthomonads and 25 stenotrophomonads, respectively. The average number of unique genes in Stenotrophomonas spp. was higher than in Xanthomonas spp., implying higher genetic diversity in Stenotrophomonas.


Introduction
Genera Xanthomonas (Dowson, 1939) and Stenotrophomonas (Palleroni and Bradbury, 1993) are the groups of gram-negative and aerobic bacteria belonging to the family Lysobacteraceae (syn.Xanthomonadaceae) of Lysobacterales (syn.Xanthomonadales) order of Gammaproteobacteria class in the phylum Proteobacteria (Kumar et al., 2019;Parte et al., 2020;Bansal et al., 2021a,b;Bansal et al., 2023).Stenotrophomonas and Xanthomonas are phylogenetically closely related genera along with Xylella and Pseudoxanthomonas in Lysobacteraceae (Bansal et al., 2021b;Bansal et al., 2023).Before the current generic name, Xanthomonas was described as Bacterium in 1921 and later reclassified into the genus Phytomonas in 1923 (Doidge, 1921;Bergey et al., 1923;Dowson, 1939).The taxonomy of the first reported xanthomonad from pepper and tomato was changed several times, as described below.The pathogen was originally classified as B. vesicatorium and then X. vesicatoria; subsequently, it was given the trinomial pathovar vesicatoria first under X. campestris (Young et al., 1978) and later under X. axonopodis (Vauterin et al., 1995) and finally reclassified as a separate species, X. euvesicatoria (Jones et al., 2004;Constantin et al., 2016).Interestingly, before designating the genus Stenotrophomonas, the first stenotrophomonad isolated from pleural fluid of a hospitalized patient had been referred to as X. maltophilia; initially identified within the Bacterium genus, it was subsequently reclassified under Pseudomonas for a decade (Hugh and Ryschenkow, 1961;Swings et al., 1983;Palleroni and Bradbury, 1993).In 1993, due to distinct phylogenetic lineage from the other phytopathogens in Xanthomonas, X. maltophilia was replaced by S. maltophilia (Palleroni and Bradbury, 1993).At the time of writing this manuscript, there are 36 and 17 validly published species of Xanthomonas and Stenotrophomonas, respectively, as well as some other invalid species shown in quotation marks ("") throughout the rest of this article in the List of Prokaryotic names with Standing in Nomenclature (LPSN, last accessed on December 2022) (Parte et al., 2020).
Among monocot plants, the Araceae family includes the most economically important ornamental plants in Hawaii, especially the genus Anthurium.During the 1980s to 1990s, the anthurium industry was seriously damaged due to X. phaseoli pv.dieffenbachiae outbreaks (formerly called X. axonopodis pv.dieffenbachiae) (Alvarez et al., 2006;Constantin et al., 2016).Hundreds of bacterial strains were isolated from various plant genera in Araceae worldwide, including the strains collected during the outbreaks in Hawaii, and stored in the Pacific Bacterial Collection at the University of Hawaii at Manoa. 1 In our previous five-gene multilocus sequence analysis (MLSA) of Lysobacteraceae strains isolated from the Araceae family, a strain from Spathiphyllum and another strain from Colocasia clustered within the Xanthomonas clade but formed a distinct monophyletic lineage, while two strains from Anthurium grouped with the Stenotrophomonas clade instead of the Xanthomonas clade (Chuang, 2023).Moreover, these two stenotrophomonads were distinct from the former two xanthomonads based on the utilizations of N-acetyl-D-galactosamine (GalNAc) and D-serine, and the inability to oxidize D-galactose, glycerol, pectin, and sucrose based on Biolog GEN III microplate assays (Chuang, 2023).
Hence, we sequenced the whole genomes of the former strains isolated from Araceae, which are potential novel species, comparing them with the genomes of Xanthomonas spp.and Stenotrophomonas spp.type strains.Based on the nine-gene MLSA, overall genomic relatedness index (OGRI) values, and pan-core genomic analyses, strains A6251 T from Spathiphyllum and A2111 from Colocasia are described as new species X. hawaiiensis sp.nov., strain A5588 T from Anthurium is described as S. aracearum sp.nov., and strain A5586 T from Anthurium is described as S. oahuensis sp.nov.
For the phylogenetic analysis of 16S rRNA gene, the full-length sequences of 65 Stenotrophomonas and Xanthomonas species type strains 2 https://www.bv-brc.org/were retrieved from their whole genome sequences and downloaded from GenBank on NCBI (Supplementary Table 1).The multiple alignment was performed using Geneious Prime 2021.2.2. 3 The module of finding the best DNA/Protein model for the multiple alignment data was conducted, and the maximum likelihood (ML) phylogenetic tree was built using MEGA X (Kumar et al., 2018).The consistency of the phylogenetic tree was assessed by computing 1,000 bootstrapping analyses.
Additionally, the precise MLSA was performed to reveal the phylogenetic relations between the novel species and other Stenotrophomonas and Xanthomonas species.Nine housekeeping genes (atpD, dnaA, dnaK, gltA, gyrB, nuoD, ppsA, rpoH, and uvrB) used from the previous studies (Ramos et al., 2011b;Vasileuskaya-Schulz et al., 2011;Chuang, 2023) were retrieved from downloaded genomes.The sequences of the nine housekeeping genes were aligned with free end gaps algorithm separately.After trimming the both sequence ends of each gene, nine gene sequences were concatenated in alphabetic order using Geneious Prime for further analyzing.The ML phylogenetic tree was formed using MEGA X following the process as detailed above.The phylogenetic trees with bootstrapping analyses were created using web-based tool Interactive Tree Of Life (iTOL v6)4 (Letunic and Bork, 2021).

Genome similarity
To define new species, the pairwise comparisons of overall genomic relatedness indices (OGRIs) among the genomes of new species strains and other type strains of Stenotrophomonas and Xanthomonas species retrieved from NCBI database were calculated.The pairwise ANI and AP (alignment percentage) values were calculated using CLC Genomics Workbench 22.0.2(CLC Bio-QIAGEN, Arahus, Denmark).Due to the inclusion of some incomplete genomes, OrthoANI (Average Nucleotide Identity by Orthology), which only considered the orthologous fragment pairs, was additionally calculated by performing Orthologous Average Nucleotide Identity tool (OAT) (Lee et al., 2016).Moreover, the pairwise dDDH values and the differences in G + C content (mol%) were inferred by estimating precise distance from whole genome sequences using the Genome-Genome Distance Calculator (GGDC) v3.0 on Type Strain Genome Server (TYGS) web server 5 (Meier-Kolthoff et al., 2013, 2022).

Pan-genome analysis
Whole genome sequences of the new species and closely phylogenetically related species in each genus were used for pan-genome and core-genome analyses.Prokka v1.14.6 (Seemann, 2014) was used to re-annotate representative genomes, and the output gff files were used as input files for the Roary v3.13.0 pipeline (Page et al., 2015).For Roary, core and accessory genes were assessed with 80% minimum BLASTp identity, and multi-FASTA alignment of the core genome was generated using highly accurate PRANK, which is a 10. 3389/fmicb.2024.1356025Frontiers in Microbiology 04 frontiersin.orgprobabilistic multiple alignment program (Löytynoja, 2014;Page et al., 2015).The number of core and unique genes among species of each genus was assessed from the Roary output and was used for the flower plots by computing R script in RStudio (R Core Team, 2022).A core gene phylogenetic tree was established using an ML tree inference tool Randomized Axelerated Maximum Likelihood -Next Generation (RAxML -NG) v0.8.0 (Kozlov et al., 2019), which combine the strengths of RAxML (Stamatakis, 2014) and Exascale Maximum Likelihood (ExaML) (Kozlov et al., 2015).The DNA substitution model, General Time Reversible (GTR) + GAMMA (G), was performed and ran separately with core genomes of type species of Xanthomonas spp.and Stenotrophomonas spp., with 1,000 bootstrap replicates.The core genome phylogenetic tree was displayed using a web-based tool Interactive Tree Of Life (iTOL v6, see text footnote 4) (Letunic and Bork, 2021).The Roary matrix with the presence and absence of core and accessory genes was combined with the core genome ML tree, and the results were visualized by conducting roary_ plots.py(Page et al., 2015).

Antibiotic sensitivity assay
Antibiotic sensitivity assays were performed using disc diffusion methods described by Klair et al. (2022).Single colonies were picked from the pure culture plates of four new species strings and incubated in 10 mL of Luria-Bertani (LB) broth at 28°C with shaking at 200 rpm for 16 h.Light absorbance at 600 nm (OD600) of bacterial inoculum was adjusted to the value ~1.0, and 100 μL of inoculum was spread evenly on nutrient agar (NA, CRITERION™, Hardy Diagnostics).Seven antibiotics with different concentrations of bacitracin (50 mg/ mL), chloramphenicol (50 mg/mL), gentamicin (50 mg/mL), kanamycin (50 mg/mL), penicillin (50 mg/mL), tetracycline (40 mg/ mL), and polymyxin B sulfate (50 mg/mL) were tested.One Petri dish was divided into four zones, and three discs impregnated with each antibiotic solution and one disc soaked with sterile distilled water as control were placed in the center of each zone.Inhibition zones were observed and measured after incubating the plates at 28°C for 24 h.

Genome assembly and annotation
The high-quality genomes of the strains A6251 T , A5588 T , and A5586 T were assembled using Unicycler v0.4.8, whereas strain A2111 had a better de novo assembly using another hybrid genome assembler, Flye v2.9.1 (Table 1).The genome sizes of new species strains from anthurium, A5588 T and A5586 T , are 4.33 Mbp and 4.68 Mbp with 66.44 mol% and 65.3 mol% of GC content, respectively.In comparison, the GC content was higher in the other two strains, i.e., A6251 T (4.88 Mbp) from spathiphyllum and A2111 (4.87 Mbp) from colocasia, with 68.93 mol% and 68.88 mol% GC content, respectively (Table 1).Based on the annotation of NCBI-PGAPservice, the average CDS number of the four strains was 4,016.The strain A5588 T has the lowest CDS number, whereas the strain A5586 T has the highest number (Table 1).The CheckM completeness estimates were 99.9% in A6251 T and A2111 and 100% in A5588 T and A5586 T (Table 1).Although the CDS numbers estimated by RAST web server were slightly different from PGAP annotation, the strain A5588 T had the lowest CDS number which correlated with its genome size (data not shown).By contrast, the coverage of subsystem features presented in A5588 T was the highest and in A5586 T was the lowest (Figure 1A).Four strains comprised 23 out of the total 27 subsystem feature categories including virulence, stress response, membrane transport, DNA, and protein metabolism (Figure 1B).Notably, only strains A6251 T and A2111 contained proteins in the iron acquisition and metabolism subsystem but not strains A5588 T and A5586 T (Figure 1B).Stenotrophomonas maltophilia was reported to use two putative iron acquisition systems for the mediation of siderophores and heme as iron starvation (Kalidasan et al., 2018), implying that strains A5588 T and A5586 T from anthurium were different from the opportunistic human pathogen.

Phylogenetic analyses
The partial sequences of 16S rRNA gene were amplified using primer set P16S-F1 and P16S-R1 and deposited in the NCBI GenBank database under accession numbers OP962219 (A6251 T ), OP962220 (A2111), OP964727 (A5586 T ), and OP964728 (A5588 T ).The 16S rRNA gene sequences were retrieved from the whole genomes of new species strains, and 38 type strains of Xanthomonas species and 23 type strains of Stenotrophomonas species published in the NCBI database (Supplementary Table 1).The sequences of the nearly entire 16S rRNA gene ranging from 1,415 bp (S. bentonitica LMG 29893 T ) to 1,421 bp (S. chelatiphaga DSM 21508 T ) were analyzed for phylogenetic relationships.The 16S rRNA gene sequences of A6251 T and A2111 were identical with X. sacchari CFBP 4641 T and only one base was different from "X. sontii" PPL1 T .A5588 T and A5586 T were closely related to each other and S. bentonitica LMG 29893 T and showed higher similarity values of 16S rRNA ranging from 99.6 to 99.8%.In the maximum likelihood (ML) phylogenetic tree, 16S rRNA gene sequences depicted better resolution within Stenotrophomonas species than Xanthomonas species because of very poor species discrimination, which was higher than the 98.7% cutoff of 16S similarity (Figure 2).
For more detailed phylogenetic analysis, nine housekeeping genes (atpD, dnaA, dnaK, gltA, gyrB, nuoD, ppsA, rpoH, and uvrB) were selected and retrieved from whole genomes of formerly mentioned type strains of Xanthomonas and Stenotrophomonas species.Total length of concatenated sequence with nine genes in alphabetic order was approximately 14.3 Kb, which contained the maximum ~2,443 bp of gyrB gene and the minimum ~879 bp of uvrB gene sequences.The similarity of the concatenated gene sequences of two strains A6251 T and A2111 was 99.3%; strain A5588 T and strain A5586 T showed 89.8% similarity.Based on nine housekeeping genes, the ML tree indicated that two major phylogenetic clades, Clade I and Clade II, were present within the Xanthomonas clade with high bootstrapping value support (Figure 3).Similar Clade I and II phylogenetic groupings were reported in the previous studies (Koebnik et al., 2021;Mafakheri et al., 2022;Rana et al., 2022).The strains A6251 T and A2111 formed a monoclade clustering with X. sacchari, X. indica, X. sontii, and X. albilineans in Clade I, which also include X. surreyensis, X. bonasiae, X. traslucens, X. hyacinthi, X. theicola, and X. youngii (Figure 3).Stenotrophomonas bentonitica consistently clustered with strains A5588 T and A5586 closely related to the A5588 T -A5586 T -S.bentonitica clade (Figure 3); however, no grouping was formed in the 16S rRNA phylogenetic tree (Figure 2).

Overall genomic relatedness indices
To examine the accurate taxonomic classification, the overall genomic relatedness indices (OGRIs) including the values of ANI and dDDH of A6251 T from spathiphyllum and A2111 from colocasia were analyzed with other type strains of Xanthomonas species.Meanwhile, A5588 T and A5586 T strains were compared with other type strains in Stenotrophomonas.The general cutoff values of ANI and dDDH for species delineation are lower than 95-96 and 70%, respectively (Goris et al., 2007;Richter and Rosselló-Móra, 2009;Meier-Kolthoff et al., 2013).Strains A6251 T and A2111 shared 98.4% ANI and 85.2% dDDH with each other, which indicated that two strains belong to the same species.Based on the pairwise comparisons of the other Xanthomonas spp.reference genomes with either A6251 T or A2111, the ANI and dDDH values were 83.4-94.9% and 22.3-59.3%,respectively, which strongly signified that A6251 T and A2111 are distinguished from the others and should be considered a novel lineage (Table 2).Despite that X. sacchari CFBP 4641 T shared slightly higher OrthoANI values (95.04, 95.1) with A6251 T and A2111, other OGRIs supported the assignment as a new species (Table 2).The estimations of ANI and dDDH of anthurium strains, A5588 T and A5586 T , were 86.4 and 28.2%, respectively.Both strains shared ANI and dDDH values lower than 90% (83.4-86.8%)and 30% (20.7-29.9%)with other type strains of Stenotrophomonas spp., respectively, except for that A5588 T and S. bentonitica LMG 29893 T shared 94.7% of ANI and 56.4% of dDDH sequence identities (Table 3).In addition to ANI and dDDH values, other OGRIs including AP, OrthoANI, and G + C differences supported that A5588 T and A5586 T are two novel species (Table 3).To combine the phylogenetic analyses and evidence of OGRIs, three novel species were proposed, i.e., X. hawaiiensis sp.nov.strains A6251 T and A2111; S. aracearum sp.nov.strain A5588 T ; and, S. oahuensis sp.nov.strain A5586 T .

Pan-and core-genomic analyses
Among 40 reference genomes of Xanthomonas spp.including X. hawaiiensis sp.nov.strains A6251 T and A2111, 425 core orthologous genes (99% ≤ strains ≤100%) and 28,285 cloud genes (0% ≤ strains <15%) were found (Figure 4A).The lowest two numbers of unique genes present in A6251 T and A2111 were 87 and 103, respectively, and follow X. sacchari CFBP 4641 T which had 171 unique genes, as shown in the Figure 4B.The number of exclusive hypothetical protein encoded genes was comparatively lower in strains A6251 T and A2111, whereas 50 common hypothetical proteins existed in all type strains of Xanthomonas spp.(Figure 4C).Based on the phylogenetic tree constituted with 425 core genes, the closest relative of X. hawaiiensis sp.nov.was X. sacchari CFBP 4641 T , which successively clustered with "X.sontii" PPL1 T and "X.indica" CFBP 9039 T in Xanthomonas clade I species (Figure 5).The groupings were concordant with the previously described MLSA tree (Figure 3).The 34,713 gene clusters estimated in the Roary matrix revealed that the genomes of xanthomonads were highly diversified (Figure 5).On the other hand, the pan genome size of 20 Stenotrophomonas spp.type strains, including S. aracearum sp.nov.(A5588 T ) and S. oahuensis sp.nov.(A5586 T ), was 31,069 with 576 core genes (Figures 6A, 7).The genome of the strain A5588 T contained 396 unique genes, 317 of which were hypothetical protein encoding genes; whereas, a high number of hypothetical protein encoding genes (1,242 genes) were harbored in the genome of the strain A5586 T , possessing total 1,526 unique genes (Figures 6B-C).As presented in the 9-gene ML tree (Figure 3), A5588 T and S. bentonitica DSM 103927 T were closely clustered together and grouped with A5586 T , which was a sister group of the clade formed with S. rhizophila DSM 14405 T and "S.nematodicola" CPCC 101271 T (Figure 7).The average number of unique genes with unknown functions was higher in 25 Stenotrophomonas spp.than 40 Xanthomonas spp.(806 > 538), implying higher genetic diversity within stenotrophomonads, which warrants further investigations on Stenotrophomonas species.
The type strain A5586 T = D-31 T = ICMP 25024 T = LMG 33201 T was isolated from Anthurium (Araceae family) in 1981 in Hawaii, USA.

Discussion
The genera Xanthomonas and Stenotrophomonas are phylogenetically and evolutionarily linked and are also found frequently together in several niches, including environmental reservoirs (plants and soil) and biofilters used for waste gas treatment of animal-rendering plants (Lipski and Altendorf, 1997;Finkmann et al., 2000;Ryan et al., 2009).Although more studies are focused on phyto-and human-pathogenic species, the versatility of Xanthomonas and Stenotrophomonas spp.has the potential to be applied to many different fields and needs to be explored further.
The well-known industrial biopolymer, which is also a food additive, is xanthan gum produced by X. campestris and other Xanthomonas species (Margaritis and Zajic, 1978;Kennedy and Bradshaw, 1984;Gumus et al., 2010).Production of other bioactive secondary metabolites from xanthomonads include the siderophore xanthoferrin, which acts as a bioproduction agent under low iron   The species name between two ditto marks (") indicates the invalidly published species.Alignment Percentage (AP) and Average Nucleotide Identity (ANI) values were calculated using CLC Genomics Workbench 22.0.2;Average Nucleotide Identity by Orthology (OrthoANI) values were estimated using Orthologous Average Nucleotide Identity tool (OAT); digital DNA-DNA Hybridization (dDDH) and the differences of G + C content (mol%) were inferred on Type Strain Genome Server (TYGS) web server.conditions (Pandey et al., 2017), and the pigment xanthomonadin, analogs of which have antioxidant potential (Madden et al., 2019).The subsystem features of iron acquisition and metabolism based on RAST annotation webserver (Figure 1B) suggest that X. hawaiiensis sp.nov.strains, A6251 T and A2111, are capable of surviving inside the hosts (Expert et al., 1996).The xss gene cluster encodes proteins including XssABCDE (Xanthomonas siderophore synthesis) and XsuA (Xanthomonas siderophore utilization), which are homologous to PvsABCDE (Vibrioferrin biosynthesis) and PsuA (Vibrioferrin receptor) (Pandey and Sonti, 2010;Pandey et al., 2017).The xss gene loci involved in biosynthesis, uptake, and export of xanthoferrin is found in both X. hawaiiensis sp.nov.strains A6251 T and A2111.In addition, the xanC gene, which encodes an acyl carrier protein and is essential for yellow xanthomonadin pigment biosystem (Cao et al., 2018), is harbored in the genomes of A6251 T and A2111.
The increasing number of studies on non-pathogenic xanthomonads isolated from rice, banana, citrus, walnut, and so on suggests that they have the potential for biocontrol and bioprotection against the causal agent of their host plants (Fernandes et al., 2021;Bansal et al., 2021a,b;Rana et al., 2022).For example, X. sontii strain R1 (formerly misclassified as X. sacchari) isolated from rice seed was reported to have an antagonistic ability against Burkholderia glumae, which caused rice panicle blight disease (Xie et al., 2003;Ham et al., 2011;Fang et al., 2015).In addition, Xanthomonas sp. from ryegrass, which was phylogenetically closely related to X. translucens, showed bioprotection activities against broad tested fungal pathogens (Li et al., 2020).In the previous studies (Leite et al., 1994;Lee et al., 2020), the gene cluster involved in type III secretion system (T3SS) formation was amplified to identify the non-pathogenicity and pathogenicity strains of X. campestris.While deciphering the genomes of X. hawaiiensis sp.nov.strains, the T3SS gene cluster was missing in both genomes of A6251 T and A2111 (Chuang, 2023).The absence of T3SS was also observed in non-pathogenic strains of X. campestris (Lee et al., 2020), X. sacchari NCPPB 4393 and R1 strains (Studholme et al., 2011;Fang 10.3389/fmicb.2024.1356025Frontiers in Microbiology 13 frontiersin.orget al., 2015), and considerably commensal X. arboricola CFBP 6771 (Cesbron et al., 2015;Merda et al., 2017).Furthermore, based on 16S rRNA and nine housekeeping genes, the ML trees (Figures 2, 3) revealed that X. hawaiiensis sp.nov.strains A6251 T and A2111 were placed in Clade I along with X. sacchari and X. traslucens, which have potential biocontrol and bioprotective agents, as previously described.
Hence, the genomic constituents of A6251 T and A2111 strains not only suggest that X. hawaiiensis sp.nov.strains isolated from Araceae should   be commensal but also provide insight into the potential biocontrol capabilities of X. hawaiiensis sp.nov.
Recent research has begun to unravel the potential for biotechnological applications and biological control of stenotrophomonads.In agriculture, for example, Stenotrophomonas strains are known for promoting plant growth, protecting plants against biotic and abiotic stresses, and serving as biocontrol agents for plant diseases (Zhang and Yuen, 1999;Wolf, 2002;Messiha et al., 2007;Alavi et al., 2013;Berg and Martinez, 2015).As bioremediators and phytoremediators, Stenotrophomonas strains are capable of metabolizing and degrading a broad range of organic compounds, such as benzene and toluene, and tolerating antibiotics and heavy metals, such as mercury and silver (Binks et al., 1995;Alonso et al., 2000;Lee et al., 2002;Pages et al., 2008).Although S. aracearum sp.nov.A5588 T strain and S. oahuensis sp.nov.A5586 T strain showed no subsystem features of iron acquisition and metabolism, the higher number of RNA metabolism in A5586 T strain and protein metabolism in A5588 T strain (Figure 1B) might shed light on some unique metabolic activities in these new Stenotrophomonas species.Interestingly, the high number of unique genes and hypothetical protein encoding genes unraveled from detailed genomic contents of the novel species, especially in S. oahuensis sp.nov., imply that novel or useful enzymatic properties and metabolic capabilities of Xanthomonas and Stenotrophomonas spp.from different environmental sources are worth exploring for biocontrol and bioprotection purposes.Preliminary data from pathogenicity tests on anthurium indicated that strains A5588 T and A5586 T from anthurium are non-pathogenic stenotrophomonads due to lack of symptom development on their original host.In this study, we proposes three new species, namely, X. hawaiiensis sp.nov., S. aracearum sp.nov., and S. oahuensis sp.nov., isolated from Araceae and provides high quality whole genome sequences for further studies relative to their pathogenicity on Araceae host plants and other possible bioactivities.

FIGURE 1
FIGURE 1Subsystem annotation summary of new species strains, A6251 T , A2111, A5588 T , and A5586 T by conducting RAST web server.(A) The percentages of protein-coding genes present (orange portions) or absent (blue portions) in the RAST subsystem.(B) The pie chart and the number of subsystem features in total 27 categories found in four genomes of aroid strains.

FIGURE 2 Maximum
FIGURE 2 Maximum Likelihood phylogenetic tree based on almost full-length 16S rRNA gene sequences among three new species strains and type strains of Xanthomonas and Stenotrophomonas species.The tree scale bar indicates the number of nucleotide substitutions per sequence position.The range of gray triangles represents the degree of bootstrapping values.

FIGURE 3 Maximum
FIGURE 3Maximum Likelihood phylogenetic tree based on concatenated sequence set of nine housekeeping genes, atpD, dnaA, dnaK, gltA, gyrB, nuoD, ppsA, rpoH, and uvrB of Xanthomonas and Stenotrophomonas species type strains.The scale bar represents the nucleotide substitutions per site.The range of purple triangles indicates the degree of bootstrapping support.

FIGURE 4
FIGURE 4Pan-genome analyses of Xanthomonas hawaiienses sp.nov.(A6251 T and A2111) with other type strains of Xanthomonas species.(A) Numbers of core, soft-core, shell, and cloud genes within 40 genomes of type strains of Xanthomonas species.(B) Floral plot showing the number of core orthologous genes in the center and the number of unique genes on each petal.(C) The number of common hypothetical protein encoding genes in the center of floral plot and the number of unique hypothetical protein encoding genes of each Xanthomonas strain on each petal.

FIGURE 5
FIGURE 5 Core-and pan-genome analyses of 40 Xanthomonas species including new species strains.(A) Core genome-based ML phylogenetic tree of Xanthomonas hawaiienses sp.nov.(A6251 T and A2111) with other type strains of Xanthomonas species.Tree scale bar represents the nucleotide substitutions per site.The range of purple circles indicates the percentage of bootstrapping confidence.(B) Pan genome-based Roary matrix of the presence and absence of genes among 40 coordinated Xanthomonas species.Dark blue blocks represent genes and pale blue blocks are missing genes in the genomes.

FIGURE 6
FIGURE 6 Pan-genome analyses of Stenotrophomonas aracearum sp.nov.(A5588 T ) and S. oahuensis sp.nov.(A5586 T ) with other type strains of Stenotrophomonas species.(A) Numbers of core, soft-core, shell, and cloud genes within 25 genomes of type strains of Stenotrophomonas species.(B) Floral plot showing the number of core orthologous genes in the center and the number of unique genes on each petal.(C) The number of common hypothetical protein encoding genes in the center of floral plot and the number of unique hypothetical protein encoding genes of each Stenotrophomonas species on each petal.

FIGURE 7
FIGURE 7 Core-and pan-genome analyses of 25 Stenotrophomonas species including two new species type strains.(A) Core genome-based ML phylogenetic tree of S. aracearum sp.nov.(A5588 T ) and S. oahuensis sp.nov.(A5586 T ) along with other type strains of Stenotrophomonas species.Tree scale bar represents the substitutions per nucleotide position.The purple circles represent 100% bootstrapping support.(B) Pan genome-based Roary matrix of the presence and absence of genes among all coordinated Stenotrophomonas species.Dark blue blocks indicate genes present and pale blue blocks indicate genes absent in the genomes.

TABLE 2
Overall genomic relatedness indices (OGRIs) comparison of new species, Xanthomonas hawaiienesis sp.nov., strains with other type strains of Xanthomonas species.

TABLE 3
Overall genomic relatedness indices (OGRIs) comparison of new species, Stenotrophomonas oahuensis sp.nov.and S. aracearum sp.nov.within other species in the genus.