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. 2023 Apr 17;89(5):e02101-22. doi: 10.1128/aem.02101-22

Core-Genome Multilocus Sequence Typing for Epidemiological and Evolutionary Analyses of Phytopathogenic Xanthomonas citri

R Ragupathy a, K A Jolley b, C Zamuner c, J B Jones d, J Redfern a, F Behlau e, H Ferreira c, M C Enright a,✉
Editor: Charles M Dozoisf
PMCID: PMC10231234  PMID: 37067413

ABSTRACT

Xanthomonas citri subsp. citri is the cause of bacterial citrus canker, responsible for major economic losses to the citrus industry. X. citri subspecies and pathovars are responsible for diseases in soybean, common bean, mango, pomegranate, and cashew. X. citri disease has been tracked using several typing methods, but recent studies using genomic sequencing have been key to understanding the evolutionary relationships within the species, including fundamental differences among X. citri subsp. citri pathotypes. Here, we describe a core-genome multilocus sequence typing (cgMLST) scheme for X. citri based on 250 genomes comprising multiple examples of X. citri subsp. citri pathotypes A, A*, and Aw; X. citri subsp. malvacearum; X. citri pv. aurantifolii, pv. fuscans, pv. glycines, pv. mangiferaeindicae, pv. viticola, and pv. vignicola; and single isolates of X. citri pv. dieffenbachiae and pv. punicae. This data set included genomic sequencing of 100 novel X. citri subsp. citri isolates. cgMLST, based on 1,618 core genes across 250 genomes, is implemented at PubMLST (https://pubmlst.org/organisms/xanthomonas-citri/). GrapeTree minimum-spanning tree and Interactive Tree of Life (iTOL) neighbor-joining phylogenies generated from the cgMLST data resolved almost identical groupings of isolates to a core-genome single nucleotide polymorphism (SNP)-based neighbor-joining phylogeny. These resolved identical groupings of X. citri subsp. citri pathotypes and X. citri subspecies and pathovars. X. citri cgMLST should prove to be an increasingly valuable resource for the study of this key species of plant-pathogenic bacteria. Users can submit genomic data and associated metadata for comparison with previously characterized isolates at PubMLST to allow the rapid characterization of the local, national, and global epidemiology of these pathogens and examine evolutionary relationships.

IMPORTANCE Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others. Recent genomic studies have shown that host-adapted X. citri subspecies and pathovars and X. citri subsp. citri pathotypes form distinct clades. In this study, we describe a core-genome multilocus sequence typing (cgMLST) scheme for this species that can rapidly and robustly discriminate among these ecologically distinct, host-adapted clades. We have established this scheme and associated databases containing genomic sequences and metadata at PubMLST, which users can interrogate with their own genome sequences to determine X. citri subspecies, pathovars, and pathotypes. X. citri cgMLST should prove to be an invaluable tool for the study of the epidemiology and evolution of this major plant pathogen.

KEYWORDS: citrus canker, MLST, Xanthomonas citri, cgMLST

INTRODUCTION

Bacterial citrus canker has a major economic impact on the production of all commercial citrus crops, including oranges, limes, tangerines, lemons, and grapefruit. Three pathotypes of canker are recognized: A, B, and C. Type A, caused by Xanthomonas citri subsp. citri, is the most widespread and economically damaging, whereas types B and C, caused by X. citri pv. aurantifolii, have much-reduced virulence on sweet orange and have very limited geographical spread (1). The type A (2, 3) pathotype has the broadest host range and infects most economically important citrus plants worldwide, particularly causing a major economic burden on the South American and Californian orange industries (2, 4). Two variants of pathotype A have evolved: A*, which can cause canker on all citrus but with some isolates that can infect only key lime (Citrus aurantifolia), and Aw, which infects only key lime and alemow (Citrus macrophylla) (1).

Xanthomonas citri subspecies and pathovars other than X. citri subsp. citri infect other important crop species, including common bean (X. citri pv. fuscans), Mexican lime (X. citri pv. aurantifolii), mango (X. citri pv. mangiferaeindicae), grape (X. citri pv. viticola), cotton (X. citri subsp. malvacearum), soybean (X. citri pv. glycines), Araceae (X. citri pv. dieffenbachiae), cashew (X. citri pv. anacardii), and pomegranate (X. citri pv. punicae). Previous genome sequencing studies have examined the evolution of X. citri pathovars and subspecies (5) and X. citri subsp. citri pathotypes (4), and these studies have produced robust phylogenies that clearly resolve clades corresponding to individual X. citri pathovars and X. citri subsp. citri pathotypes. Genomic sequencing has also proven useful in investigations of host-pathogen interactions through the identification of host-specific virulence factors (6).

Whole-genome sequencing has greatly advanced the study of the epidemiology and evolution of pathogenic bacteria, greatly improving the discriminatory power and portability of other approaches such as ribotyping or pulsed-field gel electrophoresis (7). Genomic sequencing and analysis tools, developed primarily for the study of human bacterial pathogens to track and investigate outbreaks of disease caused by particularly virulent or antimicrobial-resistant clones, can also be usefully employed for the study of bacterial plant disease epidemiology and evolution.

Whole-genome sequences from isolates of pathogenic bacteria are usually compared using SNP (single nucleotide polymorphism)-based approaches that involve whole-genome alignments. Such SNP-based approaches have been used in recent studies of Xanthomonas citri biology (8, 9); however, they involve identifying genomes of isolates from the literature and downloading their sequences, followed by the computationally intensive alignment of multiple genomes to generate SNP profiles, which are then used to produce phylogenetic trees using methods such as neighbor joining (NJ), maximum parsimony, or maximum likelihood. Core-genome multilocus sequence typing (cgMLST) uses whole-genome sequence data to examine genetic similarities between isolates. It is based on allelic variations at a large number of core-genome loci that are present in all, or nearly all, members of a species (10). It differs from other whole-genome sequencing approaches in that it does not include noncore, accessory genes in comparisons of genomes, and it examines variation in allelic profiles rather than core-genome SNPs. In addition, cgMLST is computationally efficient, scalable, and suited for the representation of very large numbers of genomic comparisons. cgMLST schemes have been established for a diverse range of human pathogens, and some schemes contain many thousands of genomes. For example, the curated, open-source database PubMLST (https://pubmlst.org/) contains genomic data and metadata for 655,340 genomes of >100 bacterial species, and the EnteroBase database (https://enterobase.warwick.ac.uk) contains 379,370 Salmonella and 237,066 Escherichia coli/Shigella genomes and corresponding metadata alone (as of 23 November 2022).

In this study, we describe a cgMLST scheme and website resource that can be used to rapidly and easily identify X. citri subsp. citri variants from genome sequences without the need for computationally intensive and time-consuming core-genome SNP extraction, genome alignment, and phylogenetic comparisons. The X. citri cgMLST database at https://pubmlst.org/organisms/xanthomonas-citri represents an invaluable resource for tracking the spread of pathovars of this devastating pathogen, which should also prove to be a useful, scalable tool in future national and international efforts to control citrus canker and other crop diseases.

RESULTS

Genome sequencing.

Assemblies from each X. citri isolate consisted of between 61 and 161 contigs, with N50 values of between 96,324 and 1,044,915 nucleotides (nt) and an average depth of coverage of 102× (range = 31× to 900×). For all isolates, more than 99% of reads were mapped to the family Xanthomonadaceae using Kraken (11).

rMLST.

Ribosomal MLST (rMLST) confirmed the species designations of the 250 X. citri isolates listed in Table 1 as well as 20 other Xanthomonas spp. and the 4 other species examined and listed in Table S1 in the supplemental material. Figure 1 shows a neighbor-joining tree of all 274 genomes in this study based on the 53 concatenated rRNA gene loci used in the rMLST scheme. It can be clearly seen that all X. citri isolates form a separate and distinct clade whose closest neighbors are genomes of other X. citri pathotypes and subspecies. From this analysis, the Xanthomonas species X. vasicola and X. perforans appear to be the most closely related to X. citri, with the genomes of other xanthomonads such as X. euvesicatoria being separated by greater genetic distances. Example genomes of E. coli, Pseudomonas aeruginosa, Xylella fastidiosa, and Stenotrophomonas maltophilia are separated by even larger genetic distances from the genomes of Xanthomonas spp., including X. citri.

TABLE 1.

Details of isolates and genomes used in this studya

MLST ID Isolate Alias Country Region Yr of isolation Source Plant host species X. citri pathovar or subspecies Pathotype BioProject accession no. BioSample accession no. Reference cgMLST group (≤200 mismatches)
1 306 IBSBF 1594 Brazil Paraná Leaf Sweet orange citri A PRJNA779375 NA 14 1
2 FDC102 Brazil Leaf Sweet orange citri A PRJNA779375 SAMN23028269 This study 1
3 FDC103 Argentina Corrientes Leaf Sweet orange citri A PRJNA779375 SAMN23028270 This study 1
4 FDC104 Paraguay Leaf Sweet orange citri A PRJNA779375 SAMN23028271 This study 1
5 FDC1053 Brazil Ilha Solteira, São Paulo 2004 Leaf Sweet orange citri A PRJNA779375 SAMN23028272 This study 1
6 FDC107 Uruguay Salto Leaf Sweet orange citri A PRJNA779375 SAMN23028273 This study 1
7 FDC1083 IBSBF 256 Brazil Assis, São Paulo 1980 Leaf Sweet orange citri A PRJNA779375 SAMN23028274 This study 1
8 FDC1085 IBSBF 314 Brazil Araçatuba, São Paulo 1979 Leaf Sweet orange citri A PRJNA779375 SAMN23028275 This study 1
9 FDC1087 IBSBF 338 Brazil Cândido Mota, São Paulo 1981 Leaf Sweet orange citri A PRJNA779375 SAMN23028276 This study 1
10 FDC1088 IBSBF 340 Brazil Lins, São Paulo 1981 Leaf Sweet orange citri A PRJNA779375 SAMN23028277 This study 1
11 FDC1091 IBSBF 353 Brazil São Pedro do Turvo, São Paulo 1981 Leaf Sweet orange citri A PRJNA779375 SAMN23028278 This study 1
12 FDC1094 IBSBF 438 Brazil Cajobi, São Paulo 1982 Leaf Sweet orange citri A PRJNA779375 SAMN23028279 This study 1
13 FDC1095 IBSBF 491 Brazil Santa Mercedes, São Paulo 1983 Leaf Sweet orange citri A PRJNA779375 SAMN23028280 This study 1
14 FDC1098 IBSBF 947 Brazil Presidente Prudente, São Paulo 1992 Leaf Sweet orange citri A PRJNA779375 SAMN23028281 This study 1
15 FDC1101 IBSBF 1287 Brazil Moji-Mirim, São Paulo 1996 Leaf Sweet orange citri A PRJNA779375 SAMN23028282 This study 1
16 FDC1102 IBSBF 1403 Brazil São João da Boa Vista, São Paulo 1998 Leaf Sweet orange citri A PRJNA779375 SAMN23028283 This study 1
17 FDC1104 IBSBF 1415 Brazil Engenheiro Coelho, São Paulo 1998 Leaf Sweet orange citri A PRJNA779375 SAMN23028284 This study 1
18 FDC1107 IBSBF 1428 Brazil Itirapina, São Paulo 1999 Leaf Sweet orange citri A PRJNA779375 SAMN23028285 This study 1
19 FDC1115 IBSBF 1440 Brazil General Salgado, São Paulo 1999 Leaf Sweet orange citri A PRJNA779375 SAMN23028286 This study 1
20 FDC1116 IBSBF 1449 Brazil Presidente Bernardes, São Paulo 1999 Leaf Sweet orange citri A PRJNA779375 SAMN23028287 This study 1
21 FDC1118 IBSBF 1453 Brazil Botucatu, São Paulo 1999 Leaf Sweet orange citri A PRJNA779375 SAMN23028288 This study 1
22 FDC1120 IBSBF 1484 Brazil Clementina, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028289 This study 1
23 FDC1121 IBSBF 1485 Brazil Luiziânia, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028290 This study 1
24 FDC1125 IBSBF 1491 Brazil Sud Mennucci, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028291 This study 1
25 FDC1129 IBSBF 1518 Brazil Guzolândia, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028292 This study 1
26 FDC1139 IAPAR 12426 Brazil Araraquara, São Paulo 1999 Leaf Sweet orange citri A PRJNA779375 SAMN23028293 This study 1
27 FDC1142 Brazil Leaf Sweet orange citri A PRJNA779375 SAMN23028294 This study 1
28 FDC1143 IAPAR 12778 Brazil Avaré, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028295 This study 1
29 FDC1144 IAPAR 12822 Brazil Guaimbê, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028296 This study 1
30 FDC1145 IAPAR 12989 Brazil Marília, São Paulo 2000 Leaf Sweet orange citri A PRJNA779375 SAMN23028297 This study 1
31 FDC1148 IAPAR 12991 Brazil Tarumã, São Paulo 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028298 This study 1
32 FDC1150 IAPAR 12001 Brazil Salto Grande, São Paulo 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028299 This study 1
33 FDC1182 Brazil Ourizona, Paraná 2005 Leaf Sweet orange citri A PRJNA779375 SAMN23028300 This study 1
34 FDC122 China Hong Kong Leaf Sweet orange citri A PRJNA779375 SAMN23028301 This study 1
35 FDC1227 Brazil Adolfo, São Paulo 2005 Leaf Sweet orange citri A PRJNA779375 SAMN23028302 This study 1
36 FDC124 Japan Leaf Sweet orange citri A PRJNA779375 SAMN23028303 This study 1
37 FDC1252 Brazil Sales, São Paulo 2005 Leaf Sweet orange citri A PRJNA779375 SAMN23028304 This study 1
38 FDC126 Philippines Leaf Sweet orange citri A PRJNA779375 SAMN23028305 This study 1
39 FDC1277 Brazil Marinópolis, São Paulo 2005 Leaf Sweet orange citri A PRJNA779375 SAMN23028306 This study 1
40 FDC129 France Reunion Leaf Sweet orange citri A PRJNA779375 SAMN23028307 This study 1
41 FDC1291 Brazil Rubiácea, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028308 This study 1
42 FDC130 China Leaf Sweet orange citri A PRJNA779375 SAMN23028309 This study 1
43 FDC131 Thailand Leaf Sweet orange citri A PRJNA779375 SAMN23028310 This study 1
44 FDC133 Mauritius Leaf Sweet orange citri A PRJNA779375 SAMN23028311 This study 1
45 FDC1387 Brazil Urânia, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028312 This study 1
46 FDC1424 Brazil Suzanápolis, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028313 This study 1
47 FDC1488 Brazil Urânia, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028314 This study 1
48 FDC15 Argentina Entre Rios Leaf Sweet orange citri A PRJNA779375 SAMN23028315 This study 1
49 FDC1531 Brazil Pereira Barreto, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028316 This study 1
50 FDC1533 Brazil Palmeira d’Oeste, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028317 This study 1
51 FDC1539 Brazil Ilha Solteira, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028318 This study 1
52 FDC1580 Brazil Boa Esperança do Sul, São Paulo 2007 Leaf Sweet orange citri A PRJNA779375 SAMN23028319 This study 1
53 FDC1666 Brazil Rondon, Paraná 2011 Leaf Sweet orange citri A PRJNA779375 SAMN23028320 This study 1
54 FDC1681 Brazil Matão, São Paulo 2012 Leaf Sweet orange citri A PRJNA779375 SAMN23028321 This study 1
55 FDC1705 Brazil Paranavaí, Paraná 2013 Leaf Sweet orange citri A PRJNA779375 SAMN23028322 This study 1
56 FDC1707 Brazil Alto Paraná, Paraná 2013 Leaf Sweet orange citri A PRJNA779375 SAMN23028323 This study 1
57 FDC1733 Brazil Guairaçã, Paraná 2014 Leaf Sweet orange citri A PRJNA779375 SAMN23028324 This study 1
58 FDC2 Brazil Lins, São Paulo 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028325 This study 1
59 FDC24 Paraguay Leaf Sweet orange citri A PRJNA779375 SAMN23028326 This study 1
60 FDC4167 Unknown Leaf Sweet orange citri A PRJNA779375 SAMN23028327 This study 1
61 FDC46 New Zealand New Plymouth Leaf Sweet orange citri A PRJNA779375 SAMN23028328 This study 1
62 FDC49 Japan Leaf Sweet orange citri A PRJNA779375 SAMN23028329 This study 1
63 FDC50 Fiji Vanua Levu Leaf Sweet orange citri A* PRJNA779375 SAMN23028330 This study 2
64 FDC502 New Zealand Leaf Sweet orange citri A PRJNA779375 SAMN23028331 This study 1
65 FDC512 Brazil Iacri, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028332 This study 1
66 FDC52 India New Delhi Leaf Sweet orange citri A PRJNA779375 SAMN23028333 This study 1
67 FDC53 Iran Leaf Sweet orange citri A* PRJNA779375 SAMN23028334 This study 2
68 FDC54 Australia Darwin Leaf Sweet orange citri A PRJNA779375 SAMN23028335 This study 1
69 FDC544 Brazil Mira Estrela, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028336 This study 1
70 FDC55 Taiwan Leaf Sweet orange citri A PRJNA779375 SAMN23028337 This study 1
71 FDC550 Brazil Rubinéia, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028338 This study 1
72 FDC551 Brazil Ibitinga, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028339 This study 1
73 FDC553 Brazil Avaré, São Paulo 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028340 This study 1
74 FDC559 Brazil Cafelândia, São Paulo Leaf Sweet orange citri A PRJNA779375 SAMN23028341 This study 1
75 FDC560 Brazil Urupês, São Paulo 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028342 This study 1
76 FDC562 Brazil Terra Roxa 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028343 This study 1
77 FDC565 Brazil Barbosa, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028344 This study 1
78 FDC575 Brazil Barbosa, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028345 This study 1
79 FDC601 IBSBF 1989 Brazil Rio Grande do Sul 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028346 This study 1
80 FDC603 IBSBF 1990 Brazil Mariana Moro, Rio Grande do Sul Leaf Sweet orange citri A PRJNA779375 SAMN23028347 This study 1
81 FDC7 Brazil Bataguassu, Mato Grosso do Sul Leaf Sweet orange citri A PRJNA779375 SAMN23028348 This study 1
82 FDC704 Brazil Nova Canaã, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028349 This study 1
83 FDC705 Brazil Nova Canaã, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028350 This study 1
84 FDC714 Brazil Jales, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028351 This study 1
85 FDC718 Brazil Borborema, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028352 This study 1
86 FDC719 Brazil Parapuã, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028353 This study 1
87 FDC724 Brazil Urupês, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028354 This study 1
88 FDC748 Brazil Osvaldo Cruz, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028355 This study 1
89 FDC749 Brazil Aparecida do Oeste, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028356 This study 1
90 FDC75 ISBSF 1421 Brazil Casa Branca, São Paulo 1998 Leaf Sweet orange citri A PRJNA779375 SAMN23028357 This study 1
91 FDC755 Brazil Caiuá, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028358 This study 1
92 FDC764 Brazil Sandovalina, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028359 This study 1
93 FDC769 Brazil Narandiba, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028360 This study 1
94 FDC782 Brazil Ibitinga, São Paulo 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028361 This study 1
95 FDC8 Brazil Corbélia, Paraná Leaf Sweet orange citri A PRJNA779375 SAMN23028362 This study 1
96 FDC806 Brazil Boa Vista, Roraima 2002 Leaf Sweet orange citri A PRJNA779375 SAMN23028363 This study 1
97 FL71 USA Florida Leaf Sweet orange citri A PRJNA779375 SAMN23028364 This study 1
98 FL72 USA Florida Leaf Sweet orange citri A PRJNA779375 SAMN23028365 This study 1
99 FL75 USA Leaf Sweet orange citri A PRJNA779375 SAMN23028366 This study 1
100 LM199 Argentina 2015 Leaf Sweet orange citri A PRJNA779375 SAMN23028367 This study 1
101 FDC4 Brazil Cuiabá-Mato Grosso 2001 Leaf Sweet orange citri A PRJNA779375 SAMN23028368 This study 1
102 12_2 Thailand Nakhon Ratchasima 1991 Leaf Soybean glycines PRJNA323439 SAMN05179543 3
103 1017 South Korea Suwon 1997 Unknown glycines PRJNA556098 SAMN12340733 3
104 1018 South Korea Hwaseong 1997 Unknown glycines PRJNA556099 SAMN12340735 3
105 1045 South Korea Hwaseong 1997 Unknown glycines PRJNA556109 SAMN12340804 3
106 1157 South Korea Pocheon 1997 Unknown glycines PRJNA556102 SAMN12340780 3
107 1566 Brazil Unknown aurantifolii PRJNA273983 SAMN03835495 4
108 4834-R France Beaucouzé 1998 Unknown Common bean fuscans PRJNA176873 SAMEA3283112 25 5
109 5208 USA Florida 2002 Unknown citri PRJNA255042 SAMN02911840 26 1
110 12609 Taiwan 2015 Leaf glycines PRJNA344018 SAMN05818161 3
112 AR81009 Argentina 1981 Unknown malvacearum PRJNA396899 SAMN07447516 6
113 AS8 Saudi Arabia Unknown citri A* PRJNA255042 SAMN02911853 26 2
114 AS9 Saudi Arabia Unknown citri A* PRJNA255042 SAMN02911854 26 2
115 AS270 Saudi Arabia 1988 Unknown citri A* PRJNA255042 SAMN02911852 26 2
116 AW13 USA Florida 2003 Unknown citri Aw PRJNA255042 SAMN02911848 26 7
117 AW14 USA Florida 2005 Unknown citri Aw PRJNA255042 SAMN02911849 26 7
118 AW15 USA Florida 2005 Unknown citri Aw PRJNA255042 SAMN02911850 26 7
119 AW16 USA Florida 2005 Unknown citri Aw PRJNA255042 SAMN02911851 26 7
120 Aw12879 USA Florida Unknown citri Aw PRJNA81931 SAMN02603165 27 7
121 BL18 USA Florida 2011 Unknown citri PRJNA255042 SAMN02911845 26 1
122 CCRMXCV-80 Brazil Pernambuco 2015 Unknown viticola PRJNA407795 SAMN07664206 8
123 CFBP6988 Réunion 2000 Unknown fuscans PRJNA212252 SAMN02645882 28 9
124 CFBP1815 Greece 1978 Unknown Common bean fuscans PRJEB23080 SAMEA104357164 28 5
125 CFBP2526 Sudan 1956 Unknown Soybean glycines PRJNA212247 SAMN02469936 29 3
126 CFBP2913 Brazil 1974 Unknown anacardii PRJNA232107 SAMN07766623 30 10
127 CFBP4884 France 1998 Leaf fuscans PRJNA254240 SAMN02902416 31 5
128 CFBP4885 France 1998 Unknown fuscans PRJNA384182 SAMN06829861 28 5
129 CFBP6165 Canada 1957 Unknown fuscans PRJNA384145 SAMN06829306 28 11
130 CFBP6166 South Africa 1963 Unknown fuscans PRJNA384183 SAMN06829862 28 5
131 CFBP6167 USA 1954 Unknown fuscans PRJNA384187 SAMN06829865 28 5
132 CFBP6960 Réunion 2000 Unknown Common bean fuscans PRJEB23080 SAMEA104357193 28 5
133 CFBP6970 USA 1990 Unknown Common bean fuscans PRJEB23080 SAMEA104357194 28 5
134 CFBP6975 France 1994 Unknown fuscans PRJNA384188 SAMN06829867 28 5
135 CFBP6988R Réunion 2000 Unknown fuscans PRJNA384160 SAMN06829542 28 9
136 CFBP6989 Réunion 2000 Unknown fuscans PRJNA384161 SAMN06829585 28 9
137 CFBP6990 Réunion 2000 Unknown fuscans PRJNA384163 SAMN06829587 28 9
138 CFBP6991 Réunion 2000 Unknown fuscans PRJNA384178 SAMN06829858 28 9
139 CFBP6992 Réunion 2000 Unknown fuscans PRJNA384177 SAMN06829854 28 13
140 CFBP6994 Tanzania 1990 Unknown Common bean fuscans PRJEB23080 SAMEA104357310 28 Singleton
141 CFBP6994R Tanzania 1990 Unknown fuscans PRJNA384179 SAMN06829859 28 13
142 CFBP6996 Réunion 2000 Leaf Common bean fuscans PRJNA212255 SAMN02645883 13
143 CFBP6996R Réunion 2000 Unknown fuscans PRJNA384180 SAMN06829860 13
144 CFBP7111 USA Texas 1942 Leaf vignicola PRJNA390891 SAMN07252112 Singleton
145 CFBP7113 Sudan 1966 Unknown vignicola PRJNA390890 SAMN07251989 Singleton
146 CFBP7119 Brazil 1981 Unknown Soybean glycines PRJNA212249 SAMN02469937 29 3
147 CFBP7764 Brazil Petrolina 2012 Stem viticola PRJNA422087 SAMN08163769 8
148 CFBP7766 Cameroon 2009 Unknown Common bean fuscans PRJEB23080 SAMEA104357197 28 5
149 CFBP7767 Cameroon 2009 Unknown Common bean fuscans PRJEB23080 SAMEA104357198 28 5
150 EB08 USA Central Iowa 2008 Unknown glycines PRJNA431457 SAMN08391414 32 3
151 FB19 USA Florida 2011 Unknown citri PRJNA255042 SAMN02911846 26 1
152 FDC535 Brazil Sao Paulo 2000 Unknown aurantifolii PRJNA273983 SAMN03317023 Singleton
153 FDC628 Brazil Santa Catarina 2001 Unknown citri PRJNA273983 SAMN03317028 1
154 FDC636 Brazil Paraná 1996 Unknown citri PRJNA273983 SAMN03317019 1
155 FDC654 Brazil Rio Grande do Sul 1999 Unknown citri PRJNA273983 SAMN03317029 1
156 FDC763 Brazil São Paulo 1981 Unknown aurantifolii PRJNA273983 SAMN03317030 17
157 FDC828 Brazil São Paulo 1997 Unknown citri PRJNA273983 SAMN03317018 1
158 FDC867 Brazil São Paulo 2002 Unknown aurantifolii PRJNA273983 SAMN03317026 17
159 FDC1559 Brazil São Paulo 1981 Unknown aurantifolii PRJNA273983 SAMN03317020 17
160 FDC1561 Argentina 1981 Unknown aurantifolii PRJNA273983 SAMN03317022 4
161 FDC1609 Brazil Sao Paulo 2009 Unknown aurantifolii PRJNA273983 SAMN03317027 Singleton
162 FDC1662 Brazil Paraná 2011 Unknown citri PRJNA273983 SAMN03317017 1
163 FDC1682 Oman 1986 Unknown citri A* PRJNA273983 SAMN03317024 2
164 GD2 China Guangdong 2011 Unknown citri PRJNA255042 SAMN02911834 26 1
165 GD3 China Guangdong 2011 Unknown citri PRJNA255042 SAMN02911835 26 1
166 GSPB1386 Nicaragua 1986 Unknown malvacearum PRJNA78127 SAMN02469610 18
167 GSPB2388 Sudan Unknown malvacearum PRJNA79081 SAMN02469611 Singleton
168 HD-1 China Leaf malvacearum PRJNA587534 SAMN13193097 6
169 IBSBF2579 Brazil 2009 Unknown anacardii PRJNA416784 SAMN07964563 10
170 ICPB10535 Brazil Unknown Mexican lime aurantifolii PRJNA18835 SAMN02472096 33 Singleton
171 ICPB11122 Argentina Unknown aurantifolii PRJNA18837 SAMN02472095 33 Singleton
172 ISO12C3 Canada Ontario Unknown Common bean fuscans PRJNA289080 SAMN03842216 5
173 ISO118C1 Canada Ontario Unknown Common bean fuscans PRJNA289080 SAMN03842217 5
174 ISO118C5 Canada Ontario Unknown Common bean fuscans PRJNA289080 SAMN03842218 5
175 JJ10-1 Mauritius Rodrigues Island 1985 Unknown citri A PRJEB7180 SAMEA2844848 4 1
176 JK4-1 China 1985 Unknown citri A PRJEB7186 SAMEA2844844 4 1
177 JK48 Saudi Arabia 1988 Unknown citri A* PRJEB7195 SAMEA2827235 4 Singleton
178 JK143-9 Thailand 1990 Unknown citri A* PRJEB7200 SAMEA2827230 4 Singleton
179 JK143-11 Thailand 1990 Unknown citri A* PRJEB7184 SAMEA2844846 4 2
180 JM35-2 Saudi Arabia 1992 Unknown citri A* PRJEB7189 SAMEA2827561 4 2
181 JS581 Iran 1997 Unknown citri A* PRJEB7190 SAMEA2827560 4 2
182 JS582 Iran 1997 Unknown citri A* PRJEB7203 SAMEA2827226 4 Singleton
183 jx-6 China Jiangxi 2014 Unknown citri PRJNA286060 SAMN03765509 1
184 JX4 China Jiangxi 2011 Unknown citri PRJNA255042 SAMN02911836 26 1
185 JX5 China Jiangxi 2011 Unknown citri PRJNA255042 SAMN02911837 26 1
186 K2 South Korea Danyang 2017 Unknown glycines PRJNA556107 SAMN12340803 3
187 LB100-1 Seychelles 2005 Unknown citri A PRJEB7185 SAMEA2844845 4 1
188 LB302 USA Florida 2002 Unknown citri Aw PRJEB7197 SAMEA2827233 4 Singleton
189 LE3-1 Ethiopia 2008 Unknown citri A* PRJEB7194 SAMEA2827236 4 Singleton
190 LE116-1 Mali Key lime 2008 Unknown citri A PRJEB7201 SAMEA2827228 4 Singleton
191 LG56-10 Réunion 2009 Unknown Mango mangiferaeindicae PRJNA232105 SAMN07766891 20
192 LG81-27 Réunion 2009 Unknown Mango mangiferaeindicae PRJNA232105 SAMN07766892 20
193 LG97 Bangladesh 2006 Unknown citri A PRJEB7196 SAMEA2827234 4 Singleton
194 LG98 Bangladesh 2006 Unknown citri A PRJEB7183 SAMEA2844847 4 1
195 LG102 Bangladesh 2006 Unknown citri A PRJEB7198 SAMEA2827232 4 Singleton
196 LG115 India 2007 Unknown citri Aw PRJEB7187 SAMEA2827563 4 7
197 LG117 Bangladesh 2009 Unknown citri A PRJEB7188 SAMEA2827562 4 1
198 LH37-1 Senegal 2010 Unknown Grapefruit citri A PRJEB7192 SAMEA2827558 4 21
199 LH201 Réunion 2010 Leaf citri PRJNA344031 SAMN05823148 1
200 LH276 Réunion 2010 Leaf citri PRJNA344031 SAMN05823145 1
201 LJ207-7 Réunion 2012 Leaf citri PRJNA344031 SAMN05823144 1
202 LL074-4 Martinique 2014 Leaf citri PRJNA344031 SAMN05823143 1
203 LM180 Argentina 2003 Leaf citri PRJNA344031 SAMN05823141 1
204 LMG941 India 1948 Unknown mangiferaeindicae PRJEA86101 SAMEA2272013 34 20
205 LMG965 India 1969 Unknown Grape viticola PRJEB5493 SAMEA3139044 35 8
206 LMG712 Sudan 1956 Unknown glycines PRJNA298608 SAMN04145201 3
207 LMG761 Sudan 1958 Unknown malvacearum PRJNA298617 SAMN04145210 6
208 LMG826 Belgium Merelbeke 2014 Unknown Common bean fuscans PRJNA254368 SAMN02903172 13 Singleton
209 LMG859 India 1959 Unknown punicae PRJNA73081 SAMEA3138416 36 Singleton
210 LMG7399 Belgium Merelbeke 2014 Unknown dieffenbachiae PRJNA254467 SAMN02903999 Singleton
211 LMG9322 USA Florida 1915 Unknown citri A PRJNA338819 SAMN05571463 Singleton
212 mf20 USA Florida 2011 Unknown citri PRJNA255042 SAMN02911847 26 1
213 MN10 USA Florida 2005 Unknown citri PRJNA255042 SAMN02911841 26 1
214 MN11 USA Florida Unknown citri PRJNA255042 SAMN02911842 26 1
215 MN12 USA Florida 1997 Unknown citri PRJNA255042 SAMN02911843 26 1
216 MS14003 USA Wilzone, MS 2014 Leaf malvacearum PRJNA396899 SAMN07447517 18
217 MSCT USA Mississippi 2011 Leaf malvacearum PRJNA299817 SAMN05595756 18
218 NCPPB1402 Uganda 1962 Unknown Common bean fuscans PRJNA264810 SAMN03142810 5
219 NCPPB381 Canada 1957 Unknown Common bean fuscans PRJNA264772 SAMN03142398 11
220 NCPPB670 Uganda 1958 Unknown Common bean fuscans PRJNA263153 SAMN03097370 5
221 NCPPB1056 Ethiopia 1961 Unknown Common bean fuscans PRJNA264809 SAMN03142809 5
222 NCPPB1058 Ethiopia 1961 Unknown Common bean fuscans PRJNA264815 SAMN03142815 5
223 NCPPB1433 Hungary 1956 Unknown Common bean fuscans PRJNA264773 SAMN03142399 5
224 NCPPB1654 South Africa 1963 Unknown Common bean fuscans PRJNA264775 SAMN03142402 5
225 NCPPB2665 Italy 1973 Unknown Common bean fuscans PRJNA264776 SAMN03142403 5
226 NCPPB3607 India 1988 Unknown citri A* PRJEB7191 SAMEA2827559 4 2
227 NCPPB3610 India 1988 Unknown citri A PRJEB7199 SAMEA2827231 4 Singleton
228 NCPPB3612 India Key lime 1988 Unknown citri A PRJEB7193 SAMEA2827237 4 21
230 NCPPB3660 Brazil 1975 Unknown Common bean fuscans PRJNA264814 SAMN03142811 13 5
231 NIGEB-88 Iran Hashtbandi 2009 Unknown citri A* PRJNA283400 SAMN03649471 2
232 NIGEB-386 Iran Nik Shahr 2009 Unknown citri A* PRJNA261284 SAMN03070127 2
233 NT17 USA Florida 2011 Unknown citri PRJNA255042 SAMN02911844 26 1
234 TAQ18 Brazil Leaf anacardii PRJNA416788 SAMN07964753 24
235 TAQ13 Brazil 2009 Leaf anacardii PRJNA416789 SAMN07964775 24
236 TX160042 USA Rancho Viejo, TX 2015 Leaf citri Aw PRJNA381640 SAMN06685652 7
237 TX160149 USA Rancho Viejo, TX 2015 Leaf citri Aw PRJNA381640 SAMN06685654 Singleton
238 TX160197 USA Rancho Viejo, TX 2015 Leaf citri Aw PRJNA381640 SAMN06685696 7
239 UI6 China Guangxi 2011 Unknown citri PRJNA255042 SAMN02911838 26 1
240 UI7 China Guangxi 2011 Unknown citri PRJNA255042 SAMN02911839 26 1
241 WHRI5232 Sudan 1959 Unknown malvacearum PRJNA438827 SAMN08729580 37 6
242 03-1638-1-1 Argentina 2003 Unknown citri PRJNA401937 SAMN07611881 38 1
243 x8ra South Korea Suwon 1999 Unknown glycines PRJNA556081 SAMN12340633 3
244 X18 Burkina Faso Unknown Cotton malvacearum PRJNA172044 SAMN02469929 39 18
245 X20 Burkina Faso Unknown Cotton malvacearum PRJNA172045 SAMN02469930 39 6
246 X621 South Africa 1995 Unknown Common bean fuscans PRJNA272380 SAMN03281080 5
247 Xcc29 China Jiangxi 2010 Unknown citri PRJNA407058 SAMN07665076 1
248 Xcc49 China Chongqing 2010 Unknown citri PRJNA407058 SAMN07638001 1
249 XcmH1005 USA Oklahoma 1968 Unknown malvacearum PRJNA298765 SAMN04166563 6
250 XcmN1003 Burkina Faso 1967 Unknown malvacearum PRJNA298770 SAMN04166615 6
251 XCP631 Colombia Quilichao 2004 Unknown Common bean fuscans PRJNA272630 SAMN03284618 5
252 Xff49 Brazil Pelotas 2017 Unknown fuscans PRJNA400313 SAMN07563171 Singleton
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a

NA, not applicable.

FIG 1.

FIG 1

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Neighbor-joining tree based on 43 concatenated rRNA gene sequences generated on the PubMLST website. The phylogeny was generated using the iTOL (24) plug-in on the PubMLST website (https://pubmlst.org/). The scale bar represents the genetic distance.

cgMLST.

A total of 1,618 core genes (present in >99% of isolates) were found among 250 X. citri isolate genomes. These genes were numbered XCIT00001 to XCIT01618. Allele calling of the subset of the initial 100 records (from study isolates) resulted in isolates having between 99.4% and 100% of their loci with alleles designated. Core-genome MLST (cgMLST) groupings of the 250 genomes uploaded to the PubMLST website were made based on the number of allelic mismatches. This resulted in 171 groups of genomes with 5 or fewer mismatches (isolates tagged as Xc_cgc_5 on the PubMLST website), 113 with 10 or fewer mismatches (Xc_cgc_10), 53 with 50 or fewer mismatches (Xc_cgc_50), 39 with 100 or fewer mismatches (Xc_cgc_100), and 25 with 200 or fewer mismatches (Xc_cgc_200).

cgMLST groupings.

The groupings of 250 isolates/genomes with fewer than 200 mismatches are shown in Table 1. Group 1 contained 132 X. citri subsp. citri isolates comprising 104 pathotype A isolates (pathotype data are missing for 28 isolates in this group); group 2 contained 12 X. citri subsp. citri genomes, all of which were isolates of pathotype A*; group 3 comprised 12 X. citri pv. glycines isolates; group 4 contained 2 X. citri pv. aurantifolii isolates; group 5 contained 24 X. citri pv. fuscans isolates; group 6 contained 7 X. citri subsp. malvacearum isolates; group 7 contained 8 X. citri pv. citri isolates with pathotype Aw; group 8 contained 3 X. citri pv. viticola isolates; group 9 contained 5 X. citri pv. fuscans isolates; group 10 contained 2 X. citri pv. anacardii isolates; group 11 contained 2 X. citri pv. fuscans isolates; group 13 contained 4 X. citri pv. fuscans isolates; group 17 contained 3 X. citri pv. aurantifolii isolates; group 18 contained 4 X. citri subsp. malvacearum isolates; group 20 contained 3 X. citri pv. mangiferaeindicae isolates; group 21 contained 2 X. citri pv. citri isolates of an unknown pathotype; and group 24 contained 2 X. citri pv. anacardii isolates. Twenty-three isolates had no close matches using any of the allelic mismatch groupings described above, and these are referred to as singleton isolate genomes in Table 1. Other cgMLST groupings (and all other genomic data and metadata) can be found in Table 1.

X. citri phylogeny.

A neighbor-joining tree of concatenated MLST allelic sequences of the 250 X. citri isolates is shown in Fig. 2. This phylogeny was generated using the Interactive Tree of Life (iTOL) plug-in on the PubMLST website. It clearly distinguishes individual X. citri pathovars (colored), with the genomes of isolates belonging to the same pathovar being grouped, although some subgroupings are evident. This is most marked for X. citri pv. fuscans isolate genomes, which are represented by three clades that include isolates from previous studies by Alavi et al. (12) and Aritua et al. (13). These correspond to isolates from three lineages originally named X. citri pv. phaseoli and X. citri pv. phaseoli GL 1 and X. citri pv. phaseoli GL fuscans GL2 and GL3. Genomes from isolates of X. citri pv. aurantifolii resolve as two main clades, with the smaller group showing more genetic similarity to members of the X. citri pv. anacardii group than to other members of X. citri pv. aurantifolii. Overall, this phylogeny displays a high degree of congruence with a neighbor-joining phylogeny based on core-genome SNPs, with the same groupings of genomes and only superficial differences in the tree structure (Fig. S1). A minimum-spanning tree generated using GrapeTree based on cgMLST allele data shows groupings identical to those made using both methods (Fig. 3).

FIG 2.

FIG 2

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Neighbor-joining tree based on 250 concatenated core-genome MLST allele sequences of Xanthomonas citri. Isolates are colored according to their original pathovar/subspecies designations and X. citri subsp. citri pathotype. The phylogeny was generated using the iTOL (24) plug-in on the PubMLST website (https://pubmlst.org/). The scale bar represents the genetic distance.

FIG 3.

FIG 3

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Minimum-spanning tree based on 250 core-genome allelic profiles of Xanthomonas citri. Isolates/groups are colored according to pathovar/subspecies. Pathotype A*, pathotype Aw, and the divergent pathotype A clade containing five genomes, including that of isolate LH37-1, are shown. The phylogeny was generated using the GrapeTree (23) plug-in on the PubMLST website (https://pubmlst.org/).

The times taken to generate phylogenies based on core-genome SNPs using kSNP3 and MEGA 11 (36 h), concatenated allelic sequences using iTOL (15 min), and MLST allele data using GrapeTree (4 min) varied considerably. All tests were run on a 2020 3.6-GHz 10-Core Intel Core i9 iMac with 16 GB RAM.

X. citri subsp. citri pathotype phylogenies.

The genomes of pathotype A strains of X. citri pv. citri represented the largest group in this study, and these isolate genomes resolve as a discrete group in an NJ phylogeny based on concatenated cgMLST allele data (Fig. 2 and 3), with the exception of five isolates whose genomes were listed in the nucleotide submission information as being of pathotype A. These five isolates correspond to a separate lineage of pathotype A isolates examined in a previous study by Gordon et al. (4), which were isolated from grapefruit, key lime, and citrus species. Pathotype A* and Aw isolate genomes also form distinct clades using all three tree-building methods (Fig. 2). This can be seen in Fig. 2 and 3 but is clearer in Fig. 4 and Fig. S1, which include only X. citri subsp. citri isolate genomes.

FIG 4.

FIG 4

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Minimum-spanning tree based on 127 Xanthomonas citri pv. citri core-genome allelic profiles, colored according to pathotype. The phylogeny was generated using the GrapeTree (23) plug-in on the PubMLST website (https://pubmlst.org/).

DISCUSSION

We have developed a cgMLST scheme for the study of Xanthomonas citri. Through its implementation on the PubMLST website, this scheme can be used to rapidly and robustly infer the pathovar (or subspecies) and, in the case of X. citri subsp. citri, pathotype designation(s) based on genetic similarity to uploaded and curated genomic sequence data with their associated metadata. The database currently contains 250 isolate genomes and their associated metadata, including 100 novel X. citri subsp. citri isolates sequenced in this study.

A neighbor-joining phylogeny based on concatenated allele sequences generated using the iTOL plug-in on the PubMLST website had a structure very similar to that of a core-genome SNP-based NJ tree but was generated within several minutes, compared to the >36 h required to generate an SNP-based phylogeny that depended on multiple-whole-genome alignment. This is important because as the number of sequenced X. citri genomes deposited in public databases increases, the computational resources required to generate SNP phylogenies de novo will become greater.

We used GrapeTree, implemented at PubMLST, to generate and display minimum-spanning trees of study data, and this plug-in can quickly and clearly display phylogenies colored according to metadata such as country of origin, date, host species, pathovar, and X. citri subsp. citri pathotype. The phylogenies generated using each of the methods used here, SNP-based NJ and iTOL and GrapeTree phylogenies based on concatenated cgMLST locus sequences, were largely congruent, with very similar groupings (see Fig. S1 in the supplemental material). However, GrapeTree, with its ability to easily and quickly display very large genomic data sets such as those present in EnteroBase, is eminently scalable as data sets grow, unlike core-SNP-based methods, which are more computationally intensive and time-consuming.

Neighbor-joining phylogenies based on concatenated rRNA gene sequences were generated in this study from genomes uploaded to the PubMLST website. This tree delineated the 21 different Xanthomonas species and 4 other more distantly related ones, including E. coli and P. aeruginosa. The rRNA gene is automatically applied to PubMLST genome data and serves as a further check of species designations for uploaded genome data.

The cgMLST scheme implemented on the PubMLST website for X. citri will, we hope, be an increasingly useful tool for the study of the epidemiology and evolution of the major cause of citrus canker, X. citri subsp. citri, but should also be of benefit for the study of other plant-pathogenic X. citri subspecies and pathovars included in this study as well as those not yet included in the database.

MATERIALS AND METHODS

Bacterial isolates.

A total of 101 X. citri subsp. citri isolates were obtained from Fundecitrus, Araraquara, São Paulo, Brazil, an association maintained by citrus growers and juice manufacturers from the State of São Paulo to conduct research, education, and implementation of citrus crop protection. Isolate 306, corresponding to the previously sequenced genome of strain 306 (14), was resequenced as part of this study, resulting in the sequencing of 100 novel isolates. These were sampled from citrus plants from 15 different countries and included 75 isolates from Brazil; 4 from South Korea; 3 each from Argentina and the United States; 2 each from China, New Zealand, and Paraguay; and 1 each from Australia, Fiji, France, India, Iran, Mauritius, Taiwan, Thailand, and Uruguay. One isolate’s country of origin is unknown. Details of the isolates are shown in Table 1. These isolates were all pathotype A isolates from sweet orange, with the exception of two pathotype A* isolates from key lime. Study bacteria were isolated between 1979 and 2015. Data on the year of isolation were not available for 31 of the 100 isolates.

Genomic DNA sequencing.

Genomic sequencing was performed by MicrobesNG (University of Birmingham) from pure culture material stabilized in DNA/RNA Shield buffer (Zymo Research, CA, USA). Genomic DNA libraries were prepared using Nextera XT library prep kits (Illumina, San Diego, CA, USA). Libraries were sequenced using Illumina sequencers (HiSeq), using a 250-bp paired-end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 (15) and scanned using Kraken (11) to confirm species identity. De novo assembly was performed on samples using SPAdes version 3.7 (16).

A further 150 X. citri genome sequences, downloaded from the European Nucleotide Archive (ENA), were included for analysis, including 65 X. citri subsp. citri isolates (comprising 12 pathotype A, 14 pathotype A*, and 10 pathotype Aw isolates according to their cited literature sources), 9 X. citri pv. aurantifolii isolates, 37 X. citri pv. fuscans isolates, 12 X. citri pv. glycines isolates, 12 X. citri subsp. malvacearum isolates, 3 X. citri pv. mangiferaeindicae isolates, 3 X. citri pv. viticola isolates, 2 X. citri pv. vignicola isolates, and 1 isolate each of X. citri pv. dieffenbachiae and pv. punicae. Details of all X. citri genomes included in this study are shown in Table 1. In addition, 24 genomes representing single examples of 20 different Xanthomonas spp. and single examples of Stenotrophomonas maltophilia, Escherichia coli, Xylella fastidiosa, and Pseudomonas aeruginosa were downloaded from GenBank. Details of these isolates are shown in Table S1 in the supplemental material.

Core-genome MLST.

Complete coding sequences were identified in the finished genome assembly of strain 306 (14) using Prokka (17) with default settings. These were used in Roary (18) to identify 1,618 genes found in all 250 genomes. A BIGSdb database for X. citri was set up on the PubMLST website (19), with loci being defined for each of the identified core genes and named using an XCIT prefix and a five-digit identifier, ranging from XCIT00001 to XCIT01618. The database was seeded with the coding sequence found in strain 306 for each of these loci defined as allele 1. Allelic variants found in the 100-isolate locally sequenced data set were then identified using the BIGSdb allele caller, with thresholds of 98% identity over 98% of the alignment length compared to reference alleles. A further round of allele calling using the same parameters and all previously identified alleles as references was performed, followed by manual scanning to identify more variable alleles containing small indels. The database was then expanded to include all 250 isolates and alleles identified as described above. Start codon positions were adjusted in nine loci as the codon identified in the reference genome was not found consistently across the data set, whereas an alternate consensus start codon was identified nearby. Core-genome sequence types (cgSTs) were defined automatically by BIGSdb for profiles with fewer than 50 missing loci. Single-linkage cluster schemes were set up within the database to identify related isolates using a range of locus mismatch thresholds (200, 100, 50, 25, 10, and 5 locus mismatches).

Ribosomal MLST (rMLST) (20), implemented on the PubMLST website, confirmed the species identity of all 274 study isolates. It was also used to generate concatenated rRNA gene sequences for phylogenetic analysis. As rMLST examines allelic variation at 53 universal rRNA genes, it is ideally suited for the rapid phenotypic analysis of genomes of different species.

Phylogenetic trees.

In common with previous studies of X. citri evolution and epidemiology, we generated phylogenies based on core-genome SNPs using a reference genome. We used the finished genome of X. citri pv. citri strain 306 (14) as a reference and kSNP3 v3.12 (21) to generate fasta nucleotide files of SNPs, which were used in MEGA 11 (22) to generate NJ trees. Genomic DNA sequence data and their associated metadata can be analyzed using a variety of methods implemented on the PubMLST website. Here, we generated minimum-spanning trees from allelic profiles using GrapeTree (23). Neighbor-joining trees based on concatenated nucleotides of cgMLST loci were generated using Interactive Tree of Life (iTOL) (24). Both the GrapeTree and iTOL plug-ins are implemented on the PubMLST website (https://pubmlst.org/). A neighbor-joining tree was constructed for the 250 X. citri study isolates and 24 other species listed in Table S1. This was generated from the 53 concatenated rRNA gene sequences used in the rMLST scheme using the iTOL plug-in as described above.

ACKNOWLEDGMENTS

This work was funded by BBSRC/Newton Fund (https://www.ukri.org/councils/bbsrc/ https://www.newton-gcrf.org/newton-fund/) awards BB/R022720/1 and BB/S018891/1 to M.C.E., CONFAP (https://confap.org.br/) research mobility award 2019/05497-7 to M.C.E., and FAPESP awards 2017/50454-9 and 2018/21164-5 to H.F.

None of the authors have any financial, personal, or professional interests that could be construed to have influenced the work.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download aem.02101-22-s0001.pdf, PDF file, 0.2 MB (244.6KB, pdf)

Contributor Information

M. C. Enright, Email: m.enright@mmu.ac.uk.

Charles M. Dozois, INRS Armand-Frappier Sante Biotechnologie Research Centre

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