Phylogenomic Analysis Supports the Transfer of 20 Pathovars from Xanthomonas campestris into Xanthomonas euvesicatoria

Abstract

The Gram-negative bacterial genus Xanthomonas includes numerous infra-specific taxa known as pathovars, which are defined primarily on host range and disease symptoms. With the advent of molecular sequence data, many pathovars have been transferred from X. campestris into other Xanthomonas species to better harmonise taxonomy and phylogeny. We performed whole-genome shotgun sequencing on pathotype strains of the following X. campestris pathovars: blepharidis, carissae, clerodendri, convolvuli, coriandri, daturae, euphorbiae, fici, heliotropii, ionidii, lawsoniae, mirabilis, obscurae, paulliniae, pennamericanum, spermacoces, uppalii, vernoniae, viegasii and zingibericola. These genomes showed more than 98% average nucleotide identity with the type-strain of X. euvesicatoria and less than 88% with the type-strain of X. campestris. We propose the transfer of these pathovars into X. euvesicatoria and present an emended species description for X. euvesicatoria.

1. Introduction

The genus Xanthomonas contains Gram-negative plant-associated bacteria, many of which are pathogens of crops, ornamentals, or other plants [1,2]. An important feature of Xanthomonas pathogen classification and nomenclature is the inclusion of infra-specific taxa known as pathovars, defined primarily on their host range. The taxonomy of pathovars is governed by the International Standards for Naming Pathovars of Phytopathogenic Bacteria [3]. These standards are devised by the Committee on the Taxonomy of Plant Pathogenic Bacteria under the auspices of the International Society for Plant Pathology.

Recently, there have been significant efforts to reconcile the taxonomy of Xanthomonas with phylogenetic relationships inferred from molecular sequence data as well as biochemical traits. Nevertheless, the taxonomic positions of some taxa are incongruent with their apparent evolutionary relationships. This is the case for more than 20 infra-specific taxa (i.e., pathovars) that are classified within the species X. campestris, yet appear to be only distantly related to the typestrain of this species, according to preliminary phylogenetic analysis based on the gyrB genetic locus [4]. In the present study, we perform a more robust phylogenetic analysis based on whole-genome sequencing rather than just that single locus. Thereby, we clarify the phylogeny and propose taxonomic revisions to reflect this.

The current inconsistencies between phylogeny and taxonomy can be explained by the historical context. Collectively, Xanthomonas bacteria cause disease in hundreds of plant species. However, most of these bacterial strains each causes disease on a narrow range of hosts, often a single plant genus or species. Before the advent of molecular methods, Xanthomonas pathogens were difficult to distinguish phenotypically other than by their host ranges and disease symptoms. Historically, the commonly accepted approach to taxonomy was to propose a new Xanthomonas species for each new host. This led to a proliferation of Xanthomonas species, with over 100 being listed in the 1951 edition of Elliott’s Manual of Bacterial Plant Pathogens [5]. Many of these species were subsequently lumped together as pathovars within a single species, X. campestris, in the 1974 edition of Bergey’s Manual of Determinative Bacteriology [6] and a 1978 proposal on nomenclature and classification for plant pathogenic bacteria [7].

After the lumping together of diverse pathogens into X. campestris, there followed a period of splitting. Based on DNA—DNA hybridisation studies, a major revision of Xanthomonas taxonomy created several new Xanthomonas species. Some pathovars were transferred out of X. campestris into these new species [8]. In that study, 66 pathovars were also identified as being ‘formerly’ within X. campestris but not yet transferred to any of the new species. The authors recommended referring to these as “Xanthomonas species”, without using a species name [8]. Although these authors generated fatty acid methyl ester (FAME) profiles for most of those 66 pathovars [9], they did not include these pathovars in their DNA—DNA hybridisation experiments nor their phenotypic analysis using the Biolog GN microplate system [8]. Therefore, this 1995 major update of Xanthomonas taxonomy did not assign these 66 pathovars to species [8].

Parkinson and colleagues furthered the case for additional Xanthomonas species in 2009. They performed an extensive survey of partial gyrB gene sequences across the genus [4]. This identified a number of X. campestris pathovars whose gyrB sequences were highly divergent from the X. campestris type strain. Many of these appeared to belong to what Parkinson called the “X. euvesicatoria species complex”. However, phylogenetic analysis based on a single genetic locus can be unrepresentative of the wider genome. Therefore, Constantin and colleagues subjected pathotype strains to multi-locus sequence analysis (MLSA), whole-genome sequencing, DNA—DNA hybridisation and phenotypic profiling [10]. This polyphasic study proposed the transfer of several X. campestris pathovars into X. euvesicatoria, X. citri, X. axonopodis, and X. phaseoli [10]. Recent studies formally proposed transfer of a further 20 X. campestris pathovars to X. citri [11] and two to X. vasicola [12].

Despite previous progress in resolving taxonomy of X. campestris pathovars, there remain many whose position is yet to be addressed, perhaps because of the limited genetic data available for these. In the present study, we perform whole-genome sequencing and phylogenetic analysis to argue that pathovars uppalii, lawsoniae, clerodendri, zingibericola, viegasii, pennamericanum, mirabilis, obscurae, paulliniae, daturae, convolvuli, carissae, fici, heliotropii, euphorbiae, vernoniae, spermacoces, coriandri, blepharidis, ionidii should be transferred from X. campestris into X. euvesicatoria. Furthermore, we confirm that pathovars aberrans, armoraciae, barbaraeae, incanae, papavericola, plantaginis, and raphani are phylogenetically close to the type-strain of X. campestris and so do not require transfer.

2. Materials and Methods

The bacterial strains used in this study are listed in

Table 1. Bacterial pathotype strains used in this study. Fatty acid methyl ester (FAME) clusters are shown according to their designation in the study by Yang and colleagues [9]. Those that belong to Yang’s FAME clusters 1 and 14 are indicated with those respective numbers. (1) Strains are related (>0.4 similarity index) to FAME cluster 1. Strains are remotely related (0.2–0.4 similarity index) to FAME cluster 1 [1].

Pathovar	Strain NCPPB Number	Host	FAME Cluster	References
Controls: X. campestris
aberrans	2986	Brassica oleracea var. capitata	2	[14]
armoraciae	347	Iberis sp.	1	[15,16,17]
barbareae	983	Barbarea vulgaris	7	[18]
incanae	937	Matthiola sp.	2	[19]
papavericola	2970	Papaver rhoeas	2	[20]
plantaginis	1061	Plantago lanceolata	3	[21]
raphani	1946	Raphanus sativus	2	[22]
Controls: X. euvesicatoria
alangii	1336	Alangium lamarckii	1	[10,23]
physalidis	1756	Physalis minima	1	[10,24]
Controls: X. citri
merremiae	3114	Merremia gangetica	1	[11,25]
trichodesmae	585	Trichodesma zeylanicum	1	[11,26]
Control: X. phaseoli
passiflorae	2346	Passiflora edulis	1	[27]
Transfer to Xanthomonas euvesicatoria
blepharidis	1757	Blepharis boarhaavifolia	1	[28]
carissae	2373	Carissa carandas	1	[29]
clerodendri	575	Clerodendron sp.	1	[30]
convolvuli	2498	Convolvulus arvensis	(1)	[31]
coriandri	1758	Coriandrum sativum	1	[32]
daturae	2932	Datura metel	N.d.	[33]
euphorbiae	1828	Euphorbia acalyphoides	1	[34]
fici	2372	Ficus religiosa	(1)	[35]
heliotropii	2057	Heliotropium sudanicum	[1]	[34]
ionidii	1334	Ionidium heterophyllum	1	[36]
lawsoniae	579	Lawsonia inermis	[1]	[37]
mirabilis	4348	Mirabilis jalapa	N.d.	[38]
obscurae	3759	Ipomea obscura	N.d.	[39]
paulliniae	3079	Paullinia cupana	(1)	[40]
pennamericanum	4349	Pennisetum americanum	N.d.	[41]
spermacoces	1760	Spermacoce hispida	14	[28]
uppalii	586	Ipomoea muricata	(1)	[42]
vernoniae	1787	Vernonia cinerea	(1)	[43]
viegasii	4351	Pachystachys lutea	N.d.	[44]
zingibericola	4352	Zingiber officinale	(1)	[45]

and were purchased from the National Collection of Plant Pathogens (NCPPB), York, United Kingdom. For the preparation of genomic DNA, bacteria were grown in plates with King’s B agar medium [13] at 28 °C for 48 h. Single colonies were picked and transferred to Universals with 10 mL King’s B broth medium and incubated overnight at 28 °C at 220 rpm. Next, 1.8 mL of culture was transferred to a 2 mL Eppendorf tube and centrifuged at 5000× g for 10 min. The supernatant was discarded, and another 1.8 mL of culture was added to this pellet, and tubes were centrifuged for 2 min at 5000× g. The pellets were used for DNA extraction immediately or flash-frozen in liquid nitrogen and stored at −20 °C until extraction. This procedure for bacterial growth is documented in protocols.io at https://dx.doi.org/10.17504/protocols.io.ewov1nr92gr2/v1 (accessed on 19 November 2022).

Genomic DNA was extracted using the Qiagen MagAttract HMW DNA kit following the manufacturer’s instructions with some modifications. This modified procedure for DNA extraction is documented in protocols.io at https://dx.doi.org/10.17504/protocols.io.5jyl89428v2w/v1 (accessed on 19 November 2022).

Sequencing libraries were prepared using the NEBNext^® Ultra™ II FS DNA Library Prep kit (New England Biolabs) according to the manufacturer’s instructions. Libraries were sequenced at the University of Exeter’s DNA sequencing facility on the Illumina NovaSeq 6000 platform to generate paired 150-bp reads.

Sequence reads were assembled de novo using SPAdes version 3.15.1 [46] as described in protocols.io at https://dx.doi.org/10.17504/protocols.io.kxygxzrqzv8j/v1 (accessed on 19 November 2022). The resulting genome assemblies were annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [47]. Assembly quality was assessed using CheckM version 1.2.2 [48], selecting its markers for genus Xanthomonas. Pairwise average nucleotide identities (ANI) between genome assemblies were calculated using FastANI [49]. Phylogenomic analysis used PhaME [50] and FastTree [51] as described in the protocol at https://dx.doi.org/10.17504/protocols.io.261geny57g47/v1 (accessed on19 November 2022). The resulting phylogenetic tree was visualised using the Interactive Tree of Life [52].

3. Results

3.1. Genome Sequencing and Assembly

We sequenced the genomes of the pathotype strains for each of 32 pathovars of X. campestris, which are listed in Table 1. These sequenced strains included 20 pathovars whose gyrB sequences suggested a closer relationship to X. euvesicatoria rather than to X. campestris [4]. It also includes, as controls, strains that have recently been transferred from X. campestris into X. euvesicatoria [10] or X. citri [11]. A further set of controls comprised seven pathovars that are closely related to the type strain of X. campestris [4]. Finally, we include the pathotype strain of X. axonopodis pv. passiflorae [53], synonymous with X. campestris pv. passiflorae, which has been mentioned in the literature as “X. phaseoli pv. passiflorae” [54].

The raw sequencing reads and draft-quality genome assemblies are available in the Sequence Read Archive [55,56] via BioProject accessions PRJNA742925 and PRJNA774128. Summary statistics for the genome assemblies are provided in

Table 2. Summary statistics for genome sequence assemblies. All data can be accessed via BioProjects PRJNA742925 and PRJNA774128. All sequences strains are the pathotype strains for their respective pathovars.

Assembly Accession	Strain (NCPPB Number)	Length (b.p.)	Number of Contigs	N50 Length (b.p.)
GCA_020813115.1	aberrans 2986	5,136,506	75	145,536
GCA_020731405.1	armoraciae 347	5,063,577	72	149,552
GCA_020813315.1	barbareae 983	4,982,365	62	208,499
GCA_020813295.1	incanae 937	4,916,318	50	191,313
GCA_020813015.1	papavericola 2970	5,509,044	46	220,536
GCA_020813005.1	plantaginis 1061	5,214,263	76	153,285
GCA_020813075.1	raphani 1946	4,898,270	53	196,893
GCA_019193005.1	alangii 1336	4,984,130	20	399,322
GCA_019192985.1	physalidis 1756	5,125,394	37	354,850
GCA_019201545.1	merremiae 3114	5,092,415	53	310,143
GCA_019201525.1	trichodesmae 585	5,572,310	82	194,166
GCA_019201075.1	passiflorae 2346	5,022,320	68	164,613
GCA_019201485.1	blepharidis 1757	4,969,587	58	167,765
GCA_019201365.1	carissae 2373	5,089,141	136	93,135
GCA_019201225.1	clerodendri 575	5,097,109	39	312,137
GCA_019201245.1	convolvuli 2498	4,982,289	54	201,220
GCA_019201305.1	coriandri 1758	5,049,250	34	522,126
GCA_019201325.1	daturae 2932	5,173,277	46	333,897
GCA_019201425.1	euphorbiae 1828	4,926,792	26	995,918
GCA_019201375.1	fici 2372	4,880,201	87	109,976
GCA_019201405.1	heliotropii 2057	5,161,499	120	107,075
GCA_019201465.1	ionidii 1334	4,974,037	50	264,595
GCA_019201185.1	lawsoniae 579	5,050,397	112	114,657
GCA_019201145.1	mirabilis 4348	5,091,739	25	745,972
GCA_019201105.1	obscurae 3759	5,007,609	61	242,019
GCA_019201285.1	paulliniae 3079	4,806,543	18	540,544
GCA_019201165.1	pennamericanum 4349	5,014,174	87	133,002
GCA_019201445.1	spermacoces 1760	5,070,626	45	344,807
GCA_019201115.1	uppalii 586	4,954,104	37	305,235
GCA_019201345.1	vernoniae 1787	4,961,493	98	150,860
GCA_019201265.1	viegasii 4351 PT	5,007,865	38	232,093
GCA_019201205.1	zingibericola 4352	4,926,474	31	267,819

. Assemblies consisted of between 18 and 136 contigs with N₅₀ lengths ranging between 93 and 996 kb. CheckM reported each assembly as more than 99.6% complete and no more than 1.71% contamination (see

Table A2. Assessment of completeness and contamination using CheckM.

GenBank Accession	Pathovar	Completeness (%)	Contamination (%)
GCA_020813115.1	aberrans	99.89	0.03
GCA_019193005.1	alangii	99.94	0.61
GCA_020731405.1	armoraciae	99.89	0.03
GCA_020813315.1	barbareae	99.85	0.03
GCA_019201485.1	blepharidis	99.89	0.03
GCA_020813135.1	campestris	99.77	0
GCA_019201365.1	carissae	99.94	0.41
GCA_019201225.1	clerodendri	99.94	0.36
GCA_019201245.1	convolvuli	99.94	0.03
GCA_019201305.1	coriandri	99.89	0.3
GCA_019201325.1	daturae	99.87	0.53
GCA_019201425.1	euphorbiae	99.89	0.03
GCA_019201375.1	fici	99.94	0.68
GCA_019201405.1	heliotropii	99.82	0.32
GCA_020813295.1	incanae	99.64	0.53
GCA_019201465.1	ionidii	99.89	0.15
GCA_019201185.1	lawsoniae	99.64	0.03
GCA_019201545.1	merremiae	99.81	0.34
GCA_019201145.1	mirabilis	99.94	0.22
GCA_019201105.1	obscurae	99.94	0.07
GCA_020813015.1	papavericola	99.89	1.71
GCA_019201075.1	passiflorae	99.89	0.42
GCA_019201285.1	paulliniae	99.89	0.03
GCA_019201165.1	pennamericanum	99.89	0
GCA_019192985.1	physalidis	99.94	0.03
GCA_020813005.1	plantaginis	99.89	0.53
GCA_020813075.1	raphani	99.89	0.11
GCA_019201445.1	spermacoces	99.89	0.03
GCA_019201525.1	trichodesmae	99.76	0.93
GCA_019201115.1	uppalii	99.62	0.25
GCA_019201345.1	veroniae	99.89	0.37
GCA_019201265.1	viegasii	99.94	0.03
GCA_019201205.1	zingibericola	99.89	0.18

in Appendix B).

3.2. Phylogenomic Reconstruction

The phylogenetic positions of many of these pathovars had previously been inferred on the basis of partial sequences of the gyrB locus. Therefore, we used our newly generated genome assemblies to explore the levels of genomic similarity, going beyond that single locus. We used PhaME [50] to infer the phylogeny based on whole-genome sequence data (Figure 1). The resulting tree included a clade that includes the type-strain of X. euvesicatoria together with the pathotype strains of X. euvesicatoria pathovars perforans, alangii, allii, raphani and physalidis (Figure 1). Into this clade also fell pathovars blepharidis, carissae, clerodendri, convolvuli, coriandri, daturae, euphorbiae, fici, heliotropii, ionidii, lawsoniae, mirabilis, obscurae, paulliniae, pennamericanum, spermacoces, uppalii, vernoniae, viegasii and zingibericola, suggesting that these 20 pathovars phylogenetically fall within the species X. euvesicatoria. Consistent with previous studies [4,11], pathovars merremiae and trichodesmae showed phylogenetic proximity to X. citri. Pathovars aberrans, armoraciae, barbaraeae, incanae, papavericola, plantaginis and raphani all fall close to the type-strain of X. campestris, as expected. Pathovar passiflorae falls closer to the type-strain of X. phaseoli than to X. axonopodis.

3.3. Species Delineation Based on Average Nucleotide Identity

The results of the genome-based phylogenetic analysis supported the proposition that 20 pathovars are evolutionarily much closer to X. euvesicatoria than to X. campestris (Figure 1). However, to delineate the bounds of bacterial species, the standard approach is to use genome-wide average nucleotide identity (ANI). Therefore, we calculated pairwise ANI between each of the pathotype strains and type strains of relevant Xanthomonas species. These ANI values are summarised in Table 3.

Pathovars blepharidis, carissae, clerodendri, convolvuli, coriandri, daturae, euphorbiae, fici, heliotropii, ionidii, lawsoniae, mirabilis, obscurae, paulliniae, pennamericanum, spermacoces, uppalii, vernoniae, viegasii and zingibericola each share more than 97% ANI with the type-strain of X. euvesicatoria. This is above widely accepted thresholds for delineating species boundaries at 94–96% [57,58,59,60,61]. On the other hand, these pathovars showed less than 86% ANI with the X. campestris type strain, well below the threshold for inclusion. This supports the transfer of these 20 pathovars from X. campestris into X. euvesicatoria.

Consistent with phylogeny, pathovars aberrans, armoraciae, barbaraeae, incanae, papavericola, plantaginis and raphani show high levels of ANI with the X. campestris type strain. These ANI values are all above 96%, supporting their current taxonomic position with this species. As expected, X. citri pv. merremiae, X. citri, pv. trichodesmae, X. euvesicatoria pv. alangii and X. euvesicatoria pv. physalidis each had >96% ANI to their respective species type-strains. Pathovar passiflorae showed 98.16% ANI with X. phaseoli but only 93.42% with X. axonopodis LMG 982^T. This supports the transfer of this pathovar from X. axonopodis to X. phaseoli.

Table 3. Percentage average nucleotide identities (ANI) between genomes of the newly sequenced X. campestris pathotype strains versus genomes of type strains of X. campestris, X. citri and X. euvesicatoria [10,62,63]. Values of ANI exceeding 96% are highlighted in bold.

Pathovar and NCPPB Number	X. euvesicatoria LMG 27970 T	X. campestris ATCC 33913	X. citri LMG 9322	X. phaseoli 49119
X. campestris pv. aberrans 2986	85.80	98.66	85.65	85.88
X. campestris pv. armoraciae 347	85.74	98.16	85.79	85.88
X. campestris pv. barbareae 983	85.79	97.31	85.84	85.95
X. campestris pv. incanae 937	85.78	97.29	85.79	85.89
X. campestris pv. papavericola 2970	85.66	96.49	85.60	85.78
X. campestris pv. plantaginis 1061	85.88	96.45	85.78	85.79
X. campestris pv. raphani 1946	85.83	97.23	85.72	85.80
X. phaseoli pv. passiflorae 2346	93.86	85.96	93.65	98.16
X. citri pv. merremiae 3114	93.85	85.81	96.20	93.91
X. citri pv. trichodesmae 585	93.77	85.83	96.16	93.82
X. euvesicatoria pv. alangii 1336	98.01	85.86	94.33	94.18
[X. campestris] pv. blepharidis 1757	97.90	85.86	94.45	94.11
[X. campestris] pv. carissae 2373	97.54	85.80	94.73	94.12
[X. campestris] pv. clerodendri 575	97.98	85.85	94.31	94.08
[X. campestris] pv. convolvuli 2498	98.13	85.93	94.50	94.14
[X. campestris] pv. coriandri 1758	97.98	85.89	94.47	94.10
[X. campestris] pv. daturae 2932	98.73	85.85	94.27	94.01
[X. campestris] pv. euphorbiae 1828	97.57	85.91	94.41	94.10
[X. campestris] pv. fici 2372	98.00	85.91	94.40	94.19
[X. campestris] pv. heliotropii 2057	97.53	85.97	94.31	94.06
[X. campestris] pv. ionidii 1334	97.92	85.88	94.38	94.08
[X. campestris] pv. lawsoniae 579	98.04	85.90	94.45	94.12
[X. campestris] pv. mirabilis 4348	98.00	85.84	94.36	94.17
[X. campestris] pv. obscurae 3759	98.42	85.93	94.37	94.11
[X. campestris] pv. paulliniae 3079	97.69	85.85	94.36	94.37
[X. campestris] pv. pennamericanum 4349	97.45	85.80	94.27	94.04
X. euvesicatoria pv. physalidis 1756	98.74	85.92	94.31	94.10
[X. campestris] pv. spermacoces 1760	97.83	85.86	94.50	94.10
[X. campestris] pv. uppalii 586	98.38	85.95	94.37	94.09
[X. campestris] pv. vernoniae 1787	98.10	85.95	94.41	94.18
[X. campestris] pv. viegasii 4351	98.05	85.94	94.31	94.18
[X. campestris] pv. zingibericola 4352	98.10	85.88	94.47	94.23

Table 3. Percentage average nucleotide identities (ANI) between genomes of the newly sequenced X. campestris pathotype strains versus genomes of type strains of X. campestris, X. citri and X. euvesicatoria [10,62,63]. Values of ANI exceeding 96% are highlighted in bold.

Pathovar and NCPPB Number	X. euvesicatoria LMG 27970 T	X. campestris ATCC 33913	X. citri LMG 9322	X. phaseoli 49119
X. campestris pv. aberrans 2986	85.80	98.66	85.65	85.88
X. campestris pv. armoraciae 347	85.74	98.16	85.79	85.88
X. campestris pv. barbareae 983	85.79	97.31	85.84	85.95
X. campestris pv. incanae 937	85.78	97.29	85.79	85.89
X. campestris pv. papavericola 2970	85.66	96.49	85.60	85.78
X. campestris pv. plantaginis 1061	85.88	96.45	85.78	85.79
X. campestris pv. raphani 1946	85.83	97.23	85.72	85.80
X. phaseoli pv. passiflorae 2346	93.86	85.96	93.65	98.16
X. citri pv. merremiae 3114	93.85	85.81	96.20	93.91
X. citri pv. trichodesmae 585	93.77	85.83	96.16	93.82
X. euvesicatoria pv. alangii 1336	98.01	85.86	94.33	94.18
[X. campestris] pv. blepharidis 1757	97.90	85.86	94.45	94.11
[X. campestris] pv. carissae 2373	97.54	85.80	94.73	94.12
[X. campestris] pv. clerodendri 575	97.98	85.85	94.31	94.08
[X. campestris] pv. convolvuli 2498	98.13	85.93	94.50	94.14
[X. campestris] pv. coriandri 1758	97.98	85.89	94.47	94.10
[X. campestris] pv. daturae 2932	98.73	85.85	94.27	94.01
[X. campestris] pv. euphorbiae 1828	97.57	85.91	94.41	94.10
[X. campestris] pv. fici 2372	98.00	85.91	94.40	94.19
[X. campestris] pv. heliotropii 2057	97.53	85.97	94.31	94.06
[X. campestris] pv. ionidii 1334	97.92	85.88	94.38	94.08
[X. campestris] pv. lawsoniae 579	98.04	85.90	94.45	94.12
[X. campestris] pv. mirabilis 4348	98.00	85.84	94.36	94.17
[X. campestris] pv. obscurae 3759	98.42	85.93	94.37	94.11
[X. campestris] pv. paulliniae 3079	97.69	85.85	94.36	94.37
[X. campestris] pv. pennamericanum 4349	97.45	85.80	94.27	94.04
X. euvesicatoria pv. physalidis 1756	98.74	85.92	94.31	94.10
[X. campestris] pv. spermacoces 1760	97.83	85.86	94.50	94.10
[X. campestris] pv. uppalii 586	98.38	85.95	94.37	94.09
[X. campestris] pv. vernoniae 1787	98.10	85.95	94.41	94.18
[X. campestris] pv. viegasii 4351	98.05	85.94	94.31	94.18
[X. campestris] pv. zingibericola 4352	98.10	85.88	94.47	94.23

4. Discussion

The species Xanthomonas campestris has encompassed numerous pathovars, which are assemblages of strains sharing similar host ranges and pathology. With the advent of cheap and easy molecular sequencing, it has become apparent that there is great genetic heterogeneity among strains classified as X. campestris and at least 38 species have been described for this genus [64]. Previous studies [4,8,10,12,65] have highlighted that many pathovars classified within X. campestris are more closely related to other Xanthomonas species than to the type strain of X. campestris. Consequently, recent taxonomic revisions have transferred many X. campestris pathovars into different species [10,11,12,66]. Nevertheless, there remain X. campestris pathovars whose taxonomy remains to be resolved in the light of genetic and genomic evidence.

Here, the results of our genome sequencing and phylogenomic analysis are consistent with recently published taxonomic revisions that place pathovars merremiae and trichodesmae within X. citri and pathovars alangii and physalidis in X. euvesicatoria [10]. These results further support the transfer of pathovar passiflorae within X. phaseoli and a further 20 pathovars into X. euvesicatoria.

We also sequenced type strains of pathovars aberrans, armoraciae, barbaraeae, campestris, incanae, papavericola, plantaginis, and raphani and investigated their evolutionary relationships. Consistent with previous studies that were limited to sequencing a single genetic locus [4], we find that these are closely related to the type strain of X. campestris and fall within the boundaries of this species as delineated by ANI [67]. Therefore, we do not here propose any changes to the taxonomy of these pathovars. However, we previously noted [68] confusion and redundancy in the nomenclature for X. campestris isolates that cause nonvascular leaf spot disease on Brassica spp. We reiterate the previous proposal that such isolates should be classified as X. campestris pv. raphani rather than armoraceae [68].

In summary, 20 X. campestris pathovars examined in the current study have not been previously transferred from X. campestris, though analysis of limited available DNA sequence suggested that phylogenetically there would be a case for doing so [4]. Here, we present draft genome assemblies for the pathotype strains of these 20 pathovars for the purpose of assigning them to species. All of these could thereby be unambiguously assigned to either X. euvesicatoria on the basis of a 96% threshold for genome-wide ANI. Below, we present emended taxonomic descriptions to implement the proposed taxonomic transfers.

Emended description of X. euvesicatoria Jones et al., 2006 [69,70] emend. Constantin et al., 2016 [10].

The characteristics of the genus and species of X. euvesicatoria are as previously described (Ah-You et al. [71], Constantin et al. [10], Vauterin et al. [8]). In addition to the pathovars described in Constantin et al. [10], we provide phylogenomic evidence for additional pathovars to constitute X. euvesicatoria.

Emended description of X. euvesicatoria pv. blepharidis (Srinivasan and Patel 1956 [28], Dye 1978 [7]) comb. nov.

=X. campestris pv. blepharidis (Srinivasan and Patel 1956 [28]) Dye 1978 [7]

Description as provided by Srinivasan and Patel [28], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1757, ATCC 17995, ICMP 5722, LMG 557.

Emended description of X. euvesicatoria pv. carissae (Moniz et al., 1964 [29], Dye 1978 [7]) comb. nov.

=X. campestris pv. carissae (Moniz et al., 1964 [29]) Dye 1978 [7]

Description as provided by Moniz et al. [29], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 2373, ICMP 3034, LMG 669.

Emended description of X. euvesicatoria pv. clerodendri (Patel et al., 1952 [30], Dye 1978 [7]) comb. nov.

=X. campestris pv. clerodendri (Patel et al., 1952 [30]) Dye 1978 [7]

Description as provided by Patel et al. [30], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 575, ATCC 11676, ICMP 445, CIP 106789, LMG 684, DSMZ 13067, CCUG 43811.

Emended description of X. euvesicatoria pv. convolvuli (Nagarkoti et al., 1973, Dye 1978) comb. nov.

=X. campestris pv. convolvuli (Nagarkoti et al., 1973 [31]) Dye 1978 [7]

Description as provided by Nagarkoti et al. [31], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 2498, ICMP 5380, LMG 685.

Emended description of X. euvesicatoria pv. coriandri (Srinivasan et al., 1961 [32], Dye 1978 [7]) comb. nov.

=X. campestris pv. coriandri (Srinivasan et al., 1961 [32]) Dye 1978 [7]

Description as provided by Srinivasan et al., 1961 [32], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1758, ATCC 17996, ICMP 5725, LMG 867.

Emended description of X. euvesicatoria pv. daturae (Jain et al., (1975) [33], Bradbury 1986) comb. nov.

=X. campestris pv. daturae (Jain et al., 1975) [33] Bradbury 1986 [72]

Description as provided by X. campestris f.sp. daturi Jain et al. [33], Bradbury [72] and phylogenomic comparisons in the present study. The original description of X. campestris pv. Daturae [72] used the epiphet “daturi”. However, the correct genitive singular form is daturae since Datura is feminine [73]. We note that daturae (rather than “daturi”) is the name adopted by other authors [4,8,74,75] and by the NCBI Taxonomy database [73] txid487861. Pathotype strain: NCPPB 2932.

Emended description of X. euvesicatoria pv. Euphorbiae (Sabet et al., 1969 [34], Dye [7] 1978) comb. nov.

=X. campestris pv. Euphorbiae (Sabet et al., 1969 [34]) Dye 1978 [7]

Description as provided by Sabet et al. [34], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1828, ICMP 5730, LMG 863.

Emended description of X. euvesicatoria pv. Fici (Cavara 1905 [35], Dye 1978 [7]) comb. nov.

=X. campestris pv. fici (Cavara 1905 [35]) Dye 1978 [7]

Description as provided by Cavara [35], Duff [76], Jindal and Patel [77], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 2372, ICMP 3036, LMG 701.

Emended description of X. euvesicatoria pv. heliotropii (Sabet et al., 1969 [34], Dye 1978 [7]) comb. nov.

=X. campestris pv. heliotropii (Sabet et al., 1969 [34]) Dye 1978 [7]

Description as provided by Sabet et al. [34], Bradbury [72] and phylogenomic comparisons in the present study. Bradbury [72] notes that gelatin is hydrolysed, but not starch. Pathotype strain: NCPPB 2057, ICMP 5778, LMG 73.

Emended description of X. euvesicatoria pv. ionidii (Padhya and Patel 1963 [36] Dye 1978 [7]) comb. nov.

=X. campestris pv. ionidii (Padhya and Patel 1963 [36]) Dye 1978 [7]

Description as provided by Padhya and Patel 1963 [36], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1755, ATCC 17993.

Emended description of X. euvesicatoria pv. Lawsoniae (Patel et al., 1951 [37], Dye 1978 [7]) comb. nov.

=X. campestris pv. lawsoniae (Patel et al., 1951 [37]) Dye 1978 [7]

Description as provided by Patel et al. [37], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 579, ATCC 11674, ICMP 319, LMG 756.

Emended description of X. euvesicatoria pv. mirabilis (ex Durgapal and Trivedi 1976 [38], Dye et al., 1991 [38]) comb. nov.

=X. campestris pv. mirabilis (ex Durgapal and Trivedi 1976 [38]) Dye et al., 1991 [78]

Description as provided by Durgapal and Trivedi [38]) Dye et al. [78], and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 4348, ICMP 8949.

Emended description of X. euvesicatoria pv. obscurae (Chand and Singh 1994 [39]) comb. nov.

=X. campestris pv. obscurae Chand and Singh 1994 [39]

Description as provided by Chand and Singh [39] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 3759 (incorrectly reported as NCPPB 3359 in Chand and Singh [39]), ICMP 12547.

Emended description of X. euvesicatoria pv. paulliniae (Robbs et al., 1982 [40]) comb. nov.

=X. campestris pv. Paulliniae Robbs et al. 1982 [40]

Description as provided by Robbs et al. [40] and phylogenomic comparisons in the present study. Pathotype strain (holotype): NCPPB 3079, ICMP 8919, LMG 9053.

Emended description of X. euvesicatoria pv. Pennamericanum (Qhobela and Claflin 1988 [41]) comb. nov.

=X. campestris pv. Pennamericanum Qhobela and Claflin 1988 [41]

Description as provided by Qhobela and Claflin [41] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 4349, ICMP 9627, ATCC 49152.

Emended description of X. euvesicatoria pv. Spermacoces (Srinivasan and Patel 1956 [28], Dye 1978 [7]) comb. nov.

=X. campestris pv. Spermacoces (Srinivasan & Patel 1956 [28]) Dye 1978 [7]

Description as provided by Srinivasan & Patel [28], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1760, ATCC 17998, ICMP 5751, LMG 868.

Emended description of X. euvesicatoria pv. Uppalii (Patel 1948 [42], Dye 1978 [7]) comb. nov.

=X. campestris pv. uppalii (Patel 1948 [42]) Dye 1978 [7]

Description as provided by Patel [42], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 586, ATCC 11641, ICMP 5756, LMG 893.

Emended description of X. euvesicatoria pv. vernoniae (Patel et al., 1968 [43], Dye 1978 [7]) comb. nov.

=X. campestris pv. vernoniae (Patel et al., 1968 [43]) Dye 1978 [7]

Description as provided by Patel et al. [43], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 1787, ICMP 5758, LMG 9058.

Emended description of X. euvesicatoria pv. viegasii (Robbs et al., 1989 [44]) comb. nov.

=X. campestris pv. viegasii Robbs et al., 1989 [44]

Description as provided by Robbs et al. [44], and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 4351, ICMP 9261, IBSF 575.

Emended description of X. euvesicatoria pv. zingibericola (Ren and Fang 1981 [45], Bradbury 1986 [72]) comb. nov.

=X. campestris pv. zingibericola (Ren and Fang 1981 [45]) Bradbury 1986 [72]

Description as provided by Ren and Fang 1981 [45], Bradbury [72] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 4352, ICMP 8787, LMG 9060.

Emended description of X. phaseoli (ex Smith 1897 [79]) Gabriel et al., 1989 [80] emend. Constantin et al., 2016 [10]

The characteristics of the genus and species of X. phaseoli are as previously described (Ah-You et al. [71], Constantin et al. [10], Vauterin et al. [8], Smith 1897 [79]) Gabriel et al., 1989 [80]). In addition to the pathovars described in Constantin et al. [10], we provide phylogenomic evidence for an additional pathovar to constitute X. phaseoli.

Emended description of X. phaseoli pv. passiflorae (Pereira 1969 [27], Dye 1978 [7], Goncalves and Rosato 2000 [53]) comb. nov.

=X. axonopodis pv. Passiflorae (Pereira 1969 [27], Dye 1978 [7]) Goncalves and Rosato 2000 [53]

Description as provided by Pereira [27], Bradbury [72], Goncalves and Rosato [53] and phylogenomic comparisons in the present study. Pathotype strain: NCPPB 2346, ICMP 3151, LMG 810.