long-lasting interaction among Pss and mango led us to search for effective control methods to allow farmers to deal with BAN symptoms. The efficacy of the alternative treatment silicon gel compared to the spray of copper compound BM has been demonstrated, and silicon gel has finally been registered for its commercial use in mango crops in Spain to combat BAN disease.

Given all of this, the future directions of this research are actually being targeted in two aims: (1) to unravel signaling mechanisms of Pss in interactions with other bacterial members of the mango microbiome by analysis of the transcript-level expression using *in vitro* and *in vivo* approaches; and (2) comparative genomics and evolutionary history analysis. In spite of the massive development of genomic sequencing technologies, there is a lack of information regarding genomic data from Pss strains isolated from mango trees. Thus, a great effort is currently being carried out to perform a major genome sequencing project involving a number of different strains to unravel the evolutionary processes that have occurred in mango populations from different geographical regions, separated in time. Phylogenetic and evolutionary approaches will open new windows of research that allow us to better understand why this phytopathogenic bacterium is so peculiar.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## REFERENCES

* Adetuyi, F. C., Isogai, A., Di Giorgio, D., Ballio, A., and Takemoto, J. Y. (1995). Saprophytic *Pseudomonas syringae* strain M1 of wheat produces cyclic lipodepsipeptides. *FEMS Microbiol. Lett.* 131, 63–67. doi: 10.1111/j.1574-6968.1995.tb07755.x

* Almeida, N. F., Yan, S., Cai, R., Clarke, C. R., Morris, C. E., Schaad, N. W., et al. (2010). PAMDB, a multilocus sequence typing and analysis database and website for plant associated microbes. *Phytopathology* 100, 208–215. doi: 10.1094/PHYTO-100-3-0208

* Alva, A. K., and Graham, J. H. (1991). The role of copper in citriculture. *Adv. Agron.* 1, 145–170.

* Andersen, G. L., Menkissoglou, O., and Lindow, S. E. (1991). Occurrence and properties of copper-tolerant strains of *Pseudomonas syringae* isolated from fruit trees in California. *Phytopathology* 81, 648–656. doi: 10.1094/Phyto-81-648

* Anonymous (2002). *Commission Regulation (EC) No 472/2002. Official Journal of the European Communities*. Available at: https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=O:L:2002:075:0018:0020:EN:PDF

* Anonymous (2018). Available at: https://www.diariosur.es/economia/agroalimentacion/cosecha-mango-batira-20180806211309-nt.html

* Arnold, D. L., Jackson, R. W., Fillingham, A. J., Goss, S. C., Taylor, J. D., Mansfield, J. W., et al. (2001). Highly conserved sequences flank avirulence genes: isolation of novel avirulence genes from *Pseudomonas syringae* pv.pisi. *Microbiology* 147, 1171–1182. doi: 10.1099/00221287-147-5-1171

* Arrebola, E., Carrión, V. J., Cazorla, F. M., Pérez-García, A., Murillo, J., and de Vicente, A. (2012). Characterisation of the *mgo* operon in *Pseudomonas syringae* pv. syringae UMAF0158 that is required for mangotoxin production. *BMC Microbiol.* 12:10. doi: 10.1186/1471-2180-12-10

* Arrebola, E., Carrión, V. J., Gutiérrez-Barranquero, J. A., Pérez-García, A., Rodríguez-Palenzuela, P., Cazorla, F. M., et al. (2015). Cellulose production in *Pseudomonas syringae* pv. syringae: a compromise between epiphytic and pathogenic lifestyles. *FEMS Microbiol. Ecol.* 91:fiv071. doi: 10.1093/femsec/fiv071

## FUNDING

This work was supported by grants from CICE-Junta de Andalucía, Proyecto de Excelencia (P12-AGR-1473) co-financed by FEDER (EU). JG-B was supported by a Postdoctoral Fellowship from the Research Own Plan of the University of Malaga "Ayuda de Incorporación de Doctores 2017".

## ACKNOWLEDGMENTS

We are very grateful to all the people who were directly involved in the development of this research: José María Farré, José María Hermoso, Emilio Guirado, David Sarmiento, Alejandro Pérez-García, Juan C. Codina, Eva Arrebola, Victor J. Carrión, and Jesús Murillo. We would like to thank SAT 2803 TROPS and all collaborating farmers. We also extend a special thanks to Irene Linares for her collaboration and technical support. This work is especially dedicated to the memory of our colleague Juan A. Torés Montosa, who sadly passed away in July 2018. He was one of the original researchers responsible for the discovery of BAN disease, and the development of this research line in our laboratory.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00570/full#supplementary-material

* Arrebola, E., Cazorla, F. M., Codina, J. C., Gutiérrez-Barranquero, J. A., Pérez-García, A., and de Vicente, A. (2009). Contribution of mangotoxin to the virulence and epiphytic fitness of *Pseudomonas syringae* pv. syringae. *Int. Microbiol.* 12, 87–95. doi: 10.2436/20.1501.01.85

* Arrebola, E., Cazorla, F. M., Durán, V. E., Rivera, E., Olea, F., Codina, J. C., et al. (2003). Mangotoxin: a novel antimetabolite toxin produced by *Pseudomonas syringae* inhibiting ornithine/ arginine biosynthesis. *Physiol. Mol. Plant Pathol.* 63, 117–127. doi: 10.1016/j.pmpp.2003.11.003

* Arrebola, E., Cazorla, F. M., Pérez-García, A., and de Vicente, A. (2011a). Chemical and metabolic aspects of antimetabolite toxins produced by *Pseudomonas syringae* pathovars. *Toxins* 3, 1089–1110. doi: 10.3390/toxins3091089

* Arrebola, E., Cazorla, F. M., Pérez-García, A., and de Vicente, A. (2011b). Genes involved in the production of antimetabolite toxins by *Pseudomonas syringae* pathovars. *Genes* 2, 640–660. doi: 10.3390/genes2030640

* Arrebola, E., Cazorla, F. M., Romero, D., Pérez-García, A., and de Vicente, A. (2007). A nonribosomal peptide synthetase gene (*mgoA*) of *Pseudomonas syringae* pv. syringae is involved in mangotoxin biosynthesis and is required for full virulence. *Mol. Plant Microbe Interact.* 20, 500–509. doi: 10.1094/MPMI-20-5-0500

* Bachmann, A. S., Matile, P., and Slusarenko, A. J. (1998). Inhibition of ornithine decarboxylase activity by phaseolotoxin: Implications for symptom production in halo blight of French bean. *Physiol. Mol. Plant Pathol.* 53, 287–299. doi: 10.1006/pmpp.1998.0183

* Ballio, A., Barra, D., Bossa, F., Collina, A., Grgurina, I., Marino, G., et al. (1991). Syringopeptins, new phytotoxic lipodepsipeptides of *Pseudomonas syringae* pv. syringae. *FEBS Lett.* 291, 109–112. doi: 10.1016/0014-5793(91)81115-0

* Baltrus, D. A., McCann, H. C., and Guttman, D. S. (2017). Evolution, genomics and epidemiology of *Pseudomonas syringae*: challenges in bacterial molecular plant pathology. *Mol. Plant Pathol.* 18, 152–168. doi: 10.1111/mpp.12506
Baltrus, D. A., Nishimura, M. T., Romanchuk, A., Chang, J. H., Mukhtar, M. S., Cherkis, K., et al. (2011). Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 *Pseudomonas syringae* isolates. *PLoS Pathog.* 7:e1002132. doi: 10.1371/journal.ppat.1002132

Beattie, G., and Lindow, S. E. (1995). The secret life of foliar bacterial pathogens on leaves. *Annu. Rev. Phytopathol.* 33, 145–172. doi: 10.1146/annurev.py.33.090195.001045

Becerra, L. (1995). "Enfermedades del cultivo del mango," in *Producción del Mango en México*, eds I. Mata and R. Mosqueda ((México: Noriega editores), 84–86.

Bélanger, R. R., Benhamou, N., and Menzies, J. G. (2003). Cytological evidence of an active role of silicon in wheat resistance to powdery mildew (*Blumeria graminis* f. sp. tritici). *Phytopathology* 93, 402–412. doi: 10.1094/PHYTO.2003.93.4.402

Bender, C., and Cooksey, D. (1986). Indigenous plasmids in *Pseudomonas syringae* pv. tomato: conjugative transfer and role in copper resistance. *J. Bacteriol.* 165, 534–541. doi: 10.1128/jb.165.2.534-541.1986

Bender, C. L., Alarcón-Chaidez, F., and Gross, D. C. (1999). *Pseudomonas syringae* phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases. *Microbiol. Mol. Biol. Rev.* 63, 266–292.

Bender, C. L., and Cooksey, D. (1987). Molecular cloning of copper resistance genes from *Pseudomonas syringae* pv. tomato. *J. Bacteriol.* 169, 470–474. doi: 10.1128/jb.169.2.470-474.1987

Berge, O., Monteil, C. L., Bartoli, C., Chandeysson, C., Guilbaud, C., Sands, D. C., et al. (2014). A user's guide to a data base of the diversity of *Pseudomonas syringae* and its application to classifying strains in this phylogenetic complex. *PLoS One* 9:e105547. doi: 10.1371/journal.pone.0105547

Block, A., and Alfano, J. R. (2011). Plant targets for *Pseudomonas syringae* type III effectors: virulence targets or guarded decoys? *Curr. Opin. Microbiol.* 14, 39–46. doi: 10.1016/j.mib.2010.12.011

Bradbury, J. F. (1986). *Guide to Plant Pathogenic Bacteria*. Slough: CAB International Mycological Institute.

Bull, C. T., Clarke, C. R., Cai, R., Vinatzer, B. A., Jardini, T. M., and Koike, S. T. (2011). Multilocus sequence typing of *Pseudomonas syringae* sensu lato confirms previously described genospecies and permits rapid identification of *P. syringae* pv. coriandricola and *P. syringae* pv. apii causing bacterial leaf spot on parsley. *Phytopathology* 101, 847–858. doi: 10.1094/PHYTO-11-10-0318

Cánovas, D., Cases, I., and de Lorenzo, V. (2003). Heavy metal tolerance and metal homeostasis in *Pseudomonas putida* as revealed by complete genome analysis. *Environ. Microbiol.* 5, 1242–1256. doi: 10.1111/j.1462-2920.2003.00463.x

Carrión, V. J., Arrebola, E., Cazorla, F. M., Murillo, J., and de Vicente, A. (2012). The *mbo* operon is specific and essential for biosynthesis of mangotoxin in *Pseudomonas syringae*. *PLoS One* 7:e36709. doi: 10.1371/journal.pone.0036709

Carrión, V. J., Gutiérrez-Barranquero, J. A., Arrebola, E., Bardaji, L., Codina, J. C., de Vicente, A., et al. (2013). The mangotoxin biosynthetic operon (*mbo*) is specifically distributed within *Pseudomonas syringae* Genospecies 1 and was acquired only once during evolution. *Appl. Environ. Microbiol.* 79, 756–767. doi: 10.1128/AEM.03007-12

Carrión, V. J., van der Voort, M., Arrebola, E., Gutiérrez-Barranquero, J. A., de Vicente, A., Raaijmakers, J. M., et al. (2014). Mangotoxin production of *Pseudomonas syringae* pv. syringae is regulated by MgoA. *BMC Microbiol.* 14:46. doi: 10.1186/1471-2180-14-46

Cazorla, F. M., Arrebola, E., Olea, F., Velasco, L., Hermoso, J. M., Pérez-García, A., et al. (2006). Field evaluation of treatments for the control of the bacterial apical necrosis of mango (*Mangifera indica*) caused by *Pseudomonas syringae* pv. syringae. *Eur. J. Plant Pathol.* 116, 279–288. doi: 10.1007/s10658-006-9059-7

Cazorla, F. M., Arrebola, E., Sesma, A., Pérez-García, A., Codina, J. C., Murillo, J., et al. (2002). Copper resistance in *Pseudomonas syringae* strains isolated from mango is encoded mainly by plasmids. *Phytopathology* 92, 909–916. doi: 10.1094/PHYTO.2002.92.8.909

Cazorla, F. M., Codina, J. C., Abad, C., Arrebola, E., Torés, J. A., Murillo, J., et al. (2008). 62-kb plasmids harboring *rulAB* homologues confer UV-tolerance and epiphytic fitness to *Pseudomonas syringae* pv. syringae mango isolates. *Microbiol. Ecol.* 56, 283–291. doi: 10.1007/s00248-007-9346-7

Cazorla, F. M., Olalla, L., Torés, J. A., Pérez-García, A., Codina, J. C., and de Vicente, A. (1995). A method for estimation of population densities of ice nucleating active *Pseudomonas syringae* in buds and leaves of mango. *J. Appl. Bacteriol.* 79, 341–346. doi: 10.1111/j.1365-2672.1995.tb03146.x

Cazorla, F. M., Torés, J. A., de Vicente, A., and Farré, J. M. (1992). Procesos necróticos de mango: aislamiento de *Pseudomonas syringae*. *Bol. Cultivos Trop.* 92:8.

Cazorla, F. M., Torés, J. A., Olalla, L., Pérez-García, A., Farré, J. M., and de Vicente, A. (1998). Bacterial apical necrosis of mango in Southern Spain: a disease caused by *Pseudomonas syringae* pv. syringae. *Phytopathology* 88, 614–620. doi: 10.1094/PHYTO.1998.88.7.614

Clarke, C. R., Cai, R., Studholme, D. J., Guttman, D. S., and Vinatzer, B. A. (2010). Pseudomonas syringae strains naturally lacking the classical *P. syringae* hrp/hrc locus are common leaf colonizers equipped with an atypical type III secretion system. *Mol. Plant Microbe Interact.* 23, 198–210. doi: 10.1094/MPMI-23-2-0198

Collmer, A., Badel, J. L., Charkowski, A. O., Deng, W.-L., Fouts, D. E., Ramos, A. R., et al. (2000). *Pseudomonas syringae* Hrp type III secretion system and effector proteins. *Proc. Natl. Acad. Sci. U.S.A.* 97, 8770–8777. doi: 10.1073/pnas.97.16.8770

Cooksey, D. A. (1987). Characterization of a copper resistance plasmid conserved in copper-resistant strains of *Pseudomonas syringae* pv. tomato. *Appl. Environ. Microbiol.* 53, 454–456.

Crespo, M., Cazorla, F. M., Hermoso, J. M., Guirado, E., Maymon, M., Torés, J. A., et al. (2012). First report of mango malformation disease caused by *Fusarium mangiferae* in Spain. *Plant Dis.* 96, 286–287. doi: 10.1094/PDIS-07-11-0599

Cunnac, S., Chakravarthy, S., Kvitko, B. H., Russell, A. B., Martin, G. B., and Collmer, A. (2011). Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in *Pseudomonas syringae*. *Proc. Natl. Acad. Sci. U.S.A.* 108, 2975–2980. doi: 10.1073/pnas.1013031108

Denny, T. P., Gilmour, M. N., and Selander, R. K. (1988). Genetic diversity and relationships of two pathovars of *Pseudomonas syringae*. *J. Gen. Microbiol.* 134, 1949–1960. doi: 10.1099/0022187-134-7-1949

Dillion, M. M., Thakur, S., Almeida, R. N. D., Wang, P. W., Weir, B. S., and Guttman, D. S. (2019). Recombination of ecologically and evolutionarily significant loci maintains genetic cohesion in the *Pseudomonas syringae* species complex. *Genome Biol.* 20:3. doi: 10.1186/s13059-018-1606-y

Diogo, R., and Wydra, K. (2007). Silicon-induced basal resistance in tomato against *Ralstonia solanacearum* is related to modification of pectic cell wall polysaccharide structure. *Physiol. Mol. Plant Pathol.* 70, 120–129. doi: 10.1016/j.pmpp.2007.07.008

Dye, D. W., Bradbury, J. F., Goto, M., Hayward, A. C., Lelliott, R. A., and Schroth, M. N. (1980). International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathotypes. *Rev. Plant Pathol.* 59, 153–168.

Egan, F., Barret, M., and O'Gara, F. (2014). The SPI-1-like type III secretion system: more roles than you think. *Front. Plant Sci.* 5:34. doi: 10.3389/fpls.2014.00034

Endert, E., and Ritchie, D. F. (1984). Overwintering and survival of *Pseudomonas syringae* pv. syringae and symptom development in peach trees. *Plant Dis.* 68, 468–470. doi: 10.1094/PD-69-468

English, H., DeVay, J. E., and Ogawa, J. M. (1980). *Bacterial Canker and Blast of Deciduous Fruits*. Geneva: WHO, 2155.

Fauteux, F., Rémus-Bore, W., Menzies, J. G., and Bélanger, R. R. (2005). Silicon and plant disease resistance against pathogenic fungi. *FEMS Microbiol. Lett.* 249, 1–6. doi: 10.1016/j.femsle.2005.06.034

Fouts, D. E., Abramovitch, R. B., Alfano, J. R., Baldo, A. M., Buell, C. R., Cartinhour, S., et al. (2002). Genome wide identification of *Pseudomonas syringae* pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor. *Proc. Natl. Acad. Sci. U.S.A.* 99, 2275–2280. doi: 10.1073/pnas.032514099

Franke, S., Grass, G., Rensing, C., and Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of *Escherichia coli*. *J. Bacteriol.* 185, 3804–3812. doi: 10.1128/JB.185.13.3804-3812.2003

Freeman, B. C., Chen, C., Yu, X., Nielsen, L., Peterson, K., and Beattie, G. A. (2013). Physiological and transcriptional responses to osmotic stress of two *Pseudomonas syringae* strains that differ in epiphytic fitness and osmotolerance. *J. Bacteriol.* 195, 4742–4752. doi: 10.1128/JB.00787-13

Gagnevin, L., and Pruvost, O. (2001). Epidemiology and control of mango bacterial black spot. *Plant Dis.* 85, 928–935. doi: 10.1094/PDIS.2001.85.9.928

Gal, M., Preston, G. M., Massey, R. C., Spiers, A. J., and Rainey, P. B. (2003). Genes encoding a cellulosic polymer contribute toward the ecological success of *Pseudomonas fluorescens* SBW25 on plant surfaces. *Molec. Ecol.* 12, 3109–3121. doi: 10.1046/j.1365-294X.2003.01953.x
Galán-Saúco, V. (2015). Current situation and future prospects of worldwide mango production and market. *Acta Hortic.* 1066, 69-84. doi: 10.17660/ActaHortic.2015.1066.7

Gardan, L., Bollet, C., Abu Ghorrah, M., Grimont, F., and Grimont, P. A. D. (1992). DNA relatedness among the pathovar strains of *Pseudomonas syringae* subsp. savastanoi Janse (1982) and proposal of *Pseudomonas savastanoi* sp. nov. *Int. J. Syst. Bacteriol.* 42, 606-612. doi: 10.1099/00207713-42-4-606

Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F., and Grimont, P. A. D. (1999). DNA relatedness among the pathovars of *Pseudomonas syringae* and description of *Pseudomonas tremae* sp. nov. and *Pseudomonas cannabina* sp. nov. (ex Sutic and Dowson 1959). *Int. J. Syst. Bacteriol.* 49, 469-478. doi: 10.1099/00207713-49-2-469

Gasic, K., Pavlovic, Z., Santander, R., Meredith, C., and Aćimović, S. (2018). First report of *Pseudomonas syringae* pv. syringae associated with bacterial blossom blast on apple (Malus pumila) in USA. *Plant Dis.* 102:1848. doi: 10.1094/PDIS-01-18-0184-PDN

Gazi, A. D., Sarris, P. F., Fadouloglou, V. E., Charova, S. N., Mathioudakis, N., Panopoulos, N., et al. (2012). Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among *Pseudomonas syringae* strains. *BMC Microbiol.* 12:188. doi: 10.1186/1471-2180-12-188

Gomila, M., Busquets, A., Mulet, M., García-Valdés, E., and Lalucat, J. (2017). Clarification of taxonomic status within the *Pseudomonas syringae* species group based on a phylogenomic analysis. *Front. Microbiol.* 8:2422. doi: 10.3389/fmicb.2017.02422

Green, S., Studholme, D. J., Laue, B. E., Dorati, F., Lovell, H., Arnold, D., et al. (2010). Comparative genome analysis provides insights into the evolution and adaptation of *Pseudomonas syringae* pv. aesculi on *Aesculus hippocastanum*. *PLoS One* 5:e10224. doi: 10.1371/journal.pone.0010224

Gross, D. C., and DeVay, J. E. (1977). Production and purification of syringomycin, a phytotoxin produced by *Pseudomonas syringae*. *Physiol. Plant. Pathol.* 11, 13-28. doi: 10.1016/0048-4059(77)90083-2

Guével, M. H., Menzies, J. G., and Bélanger, R. R. (2007). Effect of root and foliar applications of soluble silicon on powdery mildew control and growth of wheat plants. *Eur. J. Plant Pathol.* 119, 429-436. doi: 10.1007/s10658-007-9181-1

Gutiérrez-Barranquero, J. A., Arrebola, E., Bonilla, N., Sarmiento, D., Cazorla, F. M., and de Vicente, A. (2012). Environmentally friendly treatment alternatives to Bordeaux mixture for controlling bacterial apical necrosis (BAN) of mango. *Plant Pathol.* 61, 665-676. doi: 10.1111/j.1365-3059.2011.02559.x

Gutiérrez-Barranquero, J. A., Cazorla, F. M., de Vicente, A., and Sundin, G. W. (2017a). Complete sequence and comparative genomic analysis of eight native *Pseudomonas syringae* plasmids belonging to the pPT23A family. *BMC Genomics* 18:365. doi: 10.1186/s12864-017-3763-x

Gutiérrez-Barranquero, J. A., Cazorla, F. M., Guirado, E., Sarmiento, D., Torés, J. A., and de Vicente, A. (2017b). Cómo manejar la necrosis apical y la malformación, las dos principales enfermedades que afectan al mango en España. *Phytoma* 287, 16-21.

Gutiérrez-Barranquero, J. A., Cazorla, F. M., Torés, J. A., and de Vicente, A. (2019). *Pantoea agglomerans* as a new etiological agent of a bacterial necrotic disease of mango trees. *Phytopathology* 109, 17-26. doi: 10.1094/PHYTO-06-18-0186-R

Gutiérrez-Barranquero, J. A., Carrión, V. J., Murillo, J., Arrebola, E., Arnold, D. L., and Cazorla, F. M. (2013a). A *Pseudomonas syringae* diversity survey reveals a differentiated phylotype of the pathovar syringae associated with the mango host and mangotoxin production. *Phytopathology* 103, 1115-1129. doi: 10.1094/PHYTO-04-13-0093-R

Gutiérrez-Barranquero, J. A., de Vicente, A., Carrión, V. J., Sundin, G. W., and Cazorla, F. M. (2013b). Recruitment and rearrangement of three different genetic determinants into a conjugative plasmid increase copper resistance in *Pseudomonas syringae*. *Appl. Environ. Microbiol.* 79, 1028-1033. doi: 10.1128/AEM.02644-12

Hirano, S. S., and Upper, C. D. (1995). "Ecology of ice nucleation-active bacteria," in *Biological Ice Nucleation and Its Applications*, eds R. E. Lee, G. J. Warren, and L. V. Gusta (St. Paul, MN: APS Press), 41-61.

Hirano, S. S., and Upper, C. D. (2000). Bacteria in the leaf ecosystem with emphasis on *Pseudomonas syringae*-a pathogen, ice nucleus, and epiphyte. *Microbiol. Mol. Biol. Rev.* 64, 624-653. doi: 10.1128/MMBR.64.3.624-653. 2000

Huang, H. C., He, S. Y., Bauer, D. W., and Collmer, A. (1992). The *Pseudomonas syringae* pv. syringae 61 *hrpH* product, an envelope protein required for elicitation of the hypersensitive response in plants. *J. Bacteriol.* 174, 6878-6885. doi: 10.1128/jb.174.21.6878-6885.1992

Hulin, M. T., Armitage, A. D., Vicente, J. G., Holub, E. B., Baxter, L., Bates, H. J., et al. (2018). Comparative genomics of *Pseudomonas syringae* reveals convergent gene gain and loss associated with specialization onto cherry (*Prunus avium*). *New Phytol.* 219, 672-696. doi: 10.1111/nph.15182

Hwang, M. S., Morgan, R. L., Sarkar, S. F., Wang, P. W., and Guttman, D. S. (2005). Phylogenetic characterization of virulence and resistance phenotypes of *Pseudomonas syringae*. *Appl. Environ. Microbiol.* 71, 5182-5191. doi: 10.1128/AEM.71.9.5182-5191.2005

Iacobellis, N. S., Lavermicocca, P., Grgurina, I., Simmaco, M., and Ballio, A. (1992). Phytotoxic properties of *Pseudomonas syringae* pv. syringae toxins. *Physiol. Mol. Plant Pathol.* 40, 107-116. doi: 10.1016/0885-5765(92)90038-W

Iannotta, N., Belfiore, T., Brandmaya, P., Noce, M. E., and Scalercio, S. (2007). Evaluation of the impact on entomococenosis of active agents allowed in organic olive farming against *Bactrocera oleae* (Gmelin, 1790). *J. Environ. Sci. Health B* 42, 783-788. doi: 10.1080/03601230701551020

Ichinose, Y., Taguchi, F., and Mukaihara, T. (2013). Pathogenicity and virulence factors of *Pseudomonas syringae*. *J. Gen. Plant Pathol.* 79, 285-296. doi: 10.1007/s10327-013-0452-8

Ivanović, Ž., Perović, T., Popović, T., Blagojević, J., Trkulja, N., and Hrnčić, S. (2017). Characterization of *Pseudomonas syringae* pv. syringae, causal agent of citrus blast of mandarin in Montenegro. *Plant Pathol. J.* 33, 21-33. doi: 10.5423/PPJ.OA.08.2016.0161

Jackson, R. W., Athanassopoulos, E., Tsiamis, G., Mansfield, J. W., Sesma, A., Arnold, D. L., et al. (1999). Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen *Pseudomonas syringae* pathovar phaseolicola. *Proc. Natl. Acad. Sci. U.S.A.* 96, 10875-10880. doi: 10.1073/pnas.96.19.10875

Jacobs, J. L., and Sundin, G. W. (2001). Effect of solar UV-B radiation on a phyllosphere bacterial community. *Appl. Environ. Microbiol.* 67, 5488-5496. doi: 10.1128/AEM.67.12.5488-5496.2001

Janse, J. D., Rossi, P., Angelucci, L., Scortichini, M., Derks, J. H. J., Akkermans, A. D. L., et al. (1996). Reclassification of *Pseudomonas syringae* pv. avellanae as *Pseudomonas avellanae* (spec. nov.), the bacterium causing canker of hazelnut (*Corylus avellana* L.). *Syst. Appl. Microbiol.* 19, 589-595. doi: 10.1016/S0723-2020(96)80030-0

Jenul, C., Sieber, S., Daeppen, C., Mathew, A., Lardi, M., Pessi, G., et al. (2018). Biosynthesis of fragin is controlled by a novel quorum sensing signal. *Nat. Commun.* 9:1297. doi: 10.1038/s41467-018-03690-2

Kabata-Pendias, A. (2001). *Trace Elements in Soils and Plants*. New York, NY: CRC Press.

Kairu, G. M., Nyangena, C. M. S., and Crosse, J. E. (1985). The effect of copper sprays on bacterial blight and coffee berry disease in Kenya. *Plant Pathol.* 34, 207-213. doi: 10.1111/j.1365-3059.1985.tb01351.x

Kasapis, S., Morris, E. R., Gross, M., and Rudolph, K. (1994). Solution properties of levan polysaccharide from *Pseudomonas-syringae* pv. phaseolicola, and its possible primary role as a blocker of recognition during pathogenesis. *Carbohydr. Polym.* 23, 55-64. doi: 10.1016/0144-8617(94)90090-6

Kennelly, M. M., Cazorla, F. M., de Vicente, A., Ramos, C., and Sundin, G. W. (2007). *Pseudomonas syringae* diseases of fruit trees. Progress toward understanding and control. *Plant Dis.* 91, 4-17. doi: 10.1094/PD-91-0004

Kim, J. J., and Sundin, G. W. (2000). Regulation of the *rulAB* mutagenic DNA repair operon of *Pseudomonas syringae* by UV-B (290 to 320 nanometers) radiation and analysis of *rulAB*-mediated mutability in vitro and in planta. *J. Bacteriol.* 182, 6137-6144. doi: 10.1128/JB.182.21.6137-6144.2000

Kim, S. G., Kim, K. W., Park, E. W., and Choi, D. (2002). Silicon-induced cell wall fortification of rice leaves: a possible cellular mechanism of enhanced host resistance to blast. *Phytopathology* 92, 1095-1103. doi: 10.1094/PHYTO.2002.92.10.1095

Kretsch, A. M., Morgan, G. L., Tyrrell, J., Mevers, E., Vallet-Gély, I., and Li, B. (2018). Discovery of (dihydro)pyrazine N-oxides via genome mining in *Pseudomonas. Org. Lett.* 20, 4791-4795. doi: 10.1021/acs.orglett.8b01944

Kvitko, B. H., Park, D. H., Velásquez, A. C., Wei, C. F., Russell, A. B., Martin, G. B., et al. (2009). Deletions in the repertoire of *Pseudomonas syringae* pv. tomato DC3000 type III secretion effector genes reveal functional overlap
among effectors. *PLoS Pathog.* 5:e1000388. doi: 10.1371/journal.ppat.100388

Lamichhane, J. R., Osdaghi, E., Behlau, F., Köhl, J., Jones, J. B., and Aubertot, J.-N. (2018). Thirteen decades of antimicrobial copper compounds applied in agriculture. A review. *Agron. Sustain. Dev.* 38:28. doi: 10.1007/s13593-018-0503-9

Lamichhane, J. R., Varvaro, L., Parisi, L., Audergon, J.-M., and Morris, C. E. (2014). Disease and frost damage of woody plants caused by *Pseudomonas syringae*: seeing the forest for the trees. *Adv. Agron.* 126, 235–295. doi: 10.1016/B978-0-12-800132-5.00004-3

Laue, H., Schenk, A., Li, H., Lambertsen, L., Neu, T. R., Molin, S., et al. (2006). Contribution of alginate and levan production to biofilm formation by *Pseudomonas syringae*. *Microbiology* 152, 2909–2918. doi: 10.1099/mic.0.28875-0

Lavermicocca, P., Iacobellis, N., Simmaco, M., and Graniti, A. (1997). Biological properties and spectrum of activity of *Pseudomonas syringae* pv. *syringae* toxins. *Physiol. Mol. Plant Pathol.* 50, 129–140. doi: 10.1006/ppm.1996.0078

Lee, J., Teitzel, G. M., Munkvold, K., del Pozo, O., Martin, G. B., Michelmore, R. W., et al. (2012). Type III secretion and effectors shape the survival and growth pattern of *Pseudomonas syringae* on leaf surfaces. *Plant Physiol.* 158, 1803–1818. doi: 10.1104/pp.111.190686

Leedjärv, A., Ivask, A., and Virta, M. (2008). Interplay of different transporters in the mediation of divalent heavy metal resistance in *Pseudomonas putida* KT2440. *J. Bacteriol.* 190, 2680–2689. doi: 10.1128/JB.01494-07

Lelliott, R. A., and Stead, D. E. (1987). *Methods in Plant Pathology, Vol. 2: Methods for the Diagnosis of Bacterial Diseases of Plants*. Oxford: Blackwell Scientific Publications.

Lim, C. K., and Cooksey, D. A. (1993). Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of *Pseudomonas syringae*. *J. Bacteriol.* 175, 4492–4498. doi: 10.1128/jb.175.14.4492-4498.1993

Lindeberg, M., Cunnac, S., and Collmer, A. (2012). *Pseudomonas syringae* type III effector repertoires: last words in endless arguments. *Trends Microbiol.* 20, 199–208. doi: 10.1016/j.tim.2012.01.003

Lindeberg, M., Myers, C. R., Collmer, A., and Schneider, D. J. (2008). Roadmap to new virulence determinants in *Pseudomonas syringae*: insights from comparative genomics and genome organization. *Mol. Plant Microbe Interac.* 21, 685–700. doi: 10.1094/MPMI-21-6-0685

Lindgren, P. B., Peet, R. C., and Panopoulos, N. J. (1986). Gene cluster of *Pseudomonas syringae* pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity on nonhost plants. *J. Bacteriol.* 168, 512–522. doi: 10.1128/jb.168.2.512-522.1986

Lindow, S. E., Arny, D. C., and Upper, C. D. (1978). *Erwinia herbicola*: a bacterial ice nucleus active in increasing frost injury to corn. *Phytopathology* 68, 523–527.

Lindow, S. E., Arny, D. C., and Upper, C. D. (1982). Bacterial ice nucleation: a factor in frost injury to plants. *Plant physiol.* 70, 1084–1089. doi: 10.1104/pp.70.4.1084

Lindow, S. E., and Brandl, M. T. (2003). Microbiology of the phyllosphere. *Appl. Environ. Microbiol.* 69, 1875–1883. doi: 10.1128/AEM.69.4.1875-1883.2003

Lindow, S. E., and Connell, J. H. (1984). Reduction of frost injury to almond by control of ice nucleation active bacteria. *J. Am. Soc. Hort. Sci.* 109, 48–53.

Lo, T., Koulena, N., Seto, D., Guttman, D. S., and Desveaux, D. (2017). The HopF family of *Pseudomonas syringae* type III secreted effectors. *Mol. Plant Pathol.* 18, 457–468. doi: 10.1111/mpp.12412

Lovell, H. C., Mansfield, J. W., Godfrey, S. A. C., Jackson, R. W., Hancock, J. T., and Arnold, D. L. (2009). Bacterial evolution by genomic island transfer occurs via DNA transformation in planta. *Curr. Biol.* 19, 1586–1590. doi: 10.1016/j.cub.2009.08.018

Ma, Z., Smith, J. J., Zhao, Y., Jackson, R. W., Arnold, D. L., Murillo, J., et al. (2007). Phylogenetic analysis of the pPT23A plasmid family of *Pseudomonas syringae*. *Appl. Environ. Microbiol.* 73, 1287–1295. doi: 10.1128/AEM.01923-06

Mansfield, J. W., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., et al. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. *Mol. Plant Pathol.* 13, 614–629. doi: 10.1111/j.1364-3703.2012.00804.x

Mansvelt, E. L., and Hattingh, M. J. (1986). Bacterial blister bark and blight of fruit spurs of apple in South Africa caused by *Pseudomonas syringae* pv. *syringae*. *Plant Dis.* 70, 403–405. doi: 10.1094/PD-70-403

Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997). Modular peptide synthetases involved in nonribosomal peptide synthesis. *Chem. Rev.* 97, 2651–2674. doi: 10.1021/cr960029e

Martínez-García, P. M., López-Solanilla, E., Ramos, C., and Rodríguez-Palenzuela, P. (2016). Prediction of bacterial associations with plants using a supervised machine-learning approach. *Environ. Microbiol.* 18, 4847–4861. doi: 10.1111/1462-2920.13389

Martínez-García, P. M., Rodríguez-Palenzuela, P., Arrebola, E., Carrión, V. J., Gutiérrez-Barranquero, J. A., Pérez-García, A., et al. (2015). Bioinformatics analysis of the complete genome sequence of the mango tree pathogen *Pseudomonas syringae* pv. *syringae* UMAF0158 reveals traits relevant to virulence and epiphytic lifestyle. *PLoS One* 10:e0136101. doi: 10.1371/journal.pone.0136101

Matas, I. M., Castañeda-Ojeda, M. P., Aragón, I. M., Antúnez-Lamas, M., Murillo, J., Rodríguez-Palenzuela, P., et al. (2014). Translocation and functional analysis of *Pseudomonas savastanoi* pv. savastanoi NCPPB 3335 type III secretion system effectors reveals two novel effector families of the *Pseudomonas syringae* complex. *Mol. Plant Microbe Interact.* 27, 424–436. doi: 10.1094/MPMI-07-13-0206-R

McCann, H. C., Rikkerink, E. H. A., Bertels, F., Fiers, M., Lu, A., Rees-George, J., et al. (2013). Genomic analysis of the kiwifruit pathogen *Pseudomonas syringae* pv. actinidiaea provides insight into the origins of an emergent plant disease. *PLoS Pathog.* 9:e1003503. doi: 10.1371/journal.ppat.1003503

Medini, D., Donati, C., Tettelin, H., Masignani, V., and Rappouli, R. (2005). The microbial pangenome. *Curr. Opin. Genet. Dev.* 15, 589–594. doi: 10.1016/j.gde.2005.09.006

Mellano, M. A., and Cooksey, D. A. (1988). Nucleotide sequence and organization of copper resistance genes from *Pseudomonas syringae* pv. tomato. *J. Bacteriol.* 170, 2879–2883. doi: 10.1128/jb.170.6.2879-2883.1988

Mergeay, M., Monchy, S., Vallaeyts, T., Auquier, V., Benotmane, A., Bertin, P., et al. (2003). *Ralstonia metallidurans*, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. *FEMS Microbiol. Rev.* 27, 385–410. doi: 10.1016/S0168-6445(03)00045-7

Mirik, M., Baloglu, S., Aysan, Y., Cetinkaya-Yildiz, R., Kusek, M., and Sahin, F. (2005). First outbreak and occurrence of citrus blast disease, caused by *Pseudomonas syringae* pv. *syringae*, on orange and mandarin trees in Turkey. *Plant Pathol.* 54:238. doi: 10.1111/j.1365-3059.2005.01134.x

Mitchell, R. E. (1991). Implications of toxins in the ecology and evolution of plant pathogenic microorganisms: bacteria. *Experientia* 47, 791–803. doi: 10.1007/BF01922459

Monteil, C. L., Yahara, K., Studholme, D. J., Mageiros, L., Méric, G., Swingle, B., et al. (2016). Population-genomic insights into emergence, crop adaptation and dissemination of *Pseudomonas syringae* pathogens. *Microb. Genom.* 2:e000089. doi: 10.1099/mgen.0.000089

Montesinos, E., and Vilardell, P. (1991). Relationships among population levels of *Pseudomonas syringae*, amount of ice nuclei, and incidence of blast dormant flower in commercial pear orchards in Catalunya. Spain. *Phytopathology* 81, 113–119. doi: 10.1094/Phyto-81-113

Morris, C. E., Sands, D. C., Vinatzer, B. A., Glaux, C., Guilbaud, C., Buffière, A., et al. (2008). The life history of the plant pathogen *Pseudomonas syringae* is linked to the water cycle. *ISME J.* 2, 321–334. doi: 10.1038/ismej.2007.113

Norman, A., Hansen, L. H., and Sørensen, S. J. (2009). Conjugative plasmids: vessels of the communal gene pool. *Philos. Trans. R. Soc. Lond. B* 364, 2275–2289. doi: 10.1098/rstb.2009.0037

O'Brien, R. D., and Lindow, S. E. (1988). Effect of plant species and environmental conditions on ice nucleation activity of *Pseudomonas syringae* on leaves. *Appl. Environ. Microbiol.* 54, 2281–2286.

Oh, H. S., Park, D. H., and Collmer, A. (2010). Components of the *Pseudomonas syringae* type III secretion system can suppress and may elicit plant innate immunity. *Mol. Plant Microbe Interact.* 23, 727–739. doi: 10.1094/MPMI-23-6-0727

Palleroni, N. J., Ballard, R. W., Ralston, E., and Doudoroff, M. (1972). Deoxyribonucleic acid homologies among some *Pseudomonas* species. *J. Bacteriol.* 110, 1–11.

Patil, S. S., Tam, L. Q., and Sakai, W. S. (1972). Mode of action of the toxin from *Pseudomonas phaseolicola*. I. Toxin specificity, chlorosis and ornithine accumulation. *Plant Physiol.* 49, 803–807. doi: 10.1104/pp.49.5.803
Pecknold, P. C., and Grogan, R. G. (1973). Deoxyribonucleic acid homology groups among phytopathogenic *Pseudomonas* species. *Int. J. Syst. Syst. Bacteriol.* 23, 111-121. doi: 10.1099/00207713-2-3-111

Pérez-Martínez, I., Rodríguez-Moreno, L., Lambertsen, L., Matas, I. M., Murillo, J., Tegli, S., et al. (2010). Fate of a *Pseudomonas savastanoi* pv. savastanoi type III secretion system mutant in olive plants (*Olea europaea* L.). *Appl. Environ. Microbiol.* 76, 3611-3619. doi: 10.1128/AEM.00133-10

Pérez-Mendoza, D., Aragón, I. M., Prada-Ramírez, H. A., Romero-Jiménez, L., Ramos, C., Gallegos, M. T., et al. (2014). Responses to elevated c-di-GMP levels in mutualistic and pathogenic plant-interacting bacteria. *PLoS One* 9:e91645. doi: 10.1371/journal.pone.0091645

Pfeilmeier, S., Caly, D. L., and Malone, J. G. (2016). Bacterial pathogenesis of plants: future challenges from a microbial perspective: challenges in bacterial molecular plant pathology. *Mol. Plant Pathol.* 17, 1298-1313. doi: 10.1111/mpp.12427

Prada-Ramírez, H. A., Pérez-Mendoza, D., Felipe, A., Martínez-Granero, F., Rivilla, R., Sanjuán, J., et al. (2016). AmrZ regulates cellulose production in *Pseudomonas syringae* pv. tomato DC3000. *Mol. Microbiol.* 99, 960-977. doi: 10.1111/mmi.13278

Rainey, P. B., and Travisano, M. (1998). Adaptive radiation in a heterogeneous environment. *Nature* 394, 69–72. doi: 10.1038/27900

Reinhardt, J. A., Baltrus, D. A., Nishimura, M. T., Jeck, W. R., Jones, C. D., and Dangl, J. L. (2009). De novo assembly using low coverage short read sequence data from the rice pathogen *Pseudomonas syringae* pv. oryzae. *Genome Res.* 19, 294–305. doi: 10.1101/gr.083311.108

Rodgers-Gray, B. S., and Shaw, M. W. (2004). Effects of straw and silicon soil amendments on some foliar and stem-base diseases in pot-grown winter wheat. *Plant Pathol.* 53, 733-740. doi: 10.1111/j.1365-3059.2004.01102.x

Rodrigues, F. A., Benhamou, N., Datnoff, L. E., Jones, J. B., and Bélanger, R. R. (2003). Ultrastructural and cytochemical aspects of silicon mediated rice blast resistance. *Phytopathology* 93, 535-546. doi: 10.1094/PHYTO.2003.93.5.535

Rogers, J. S., Clark, E., Cirvilleri, G., and Lindow, S. E. (1994). Cloning and characterization of genes conferring copper resistance in epiphytic ice nucleation-active *Pseudomonas syringae* strains. *Phytopathology* 84, 891-897. doi: 10.1094/Phyto-84-891

Römling, U. (2002). Molecular biology of cellulose production in bacteria. *Res. Microbiol.* 153, 205–212. doi: 10.1016/S0923-2508(02)01316-5

Römling, U., and Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. *Trends Microbiol.* 23, 545–557. doi: 10.1016/j.tim.2015.05.005

Römling, U., Galperin, M. Y., and Gomelsky, M. (2013). Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. *Microbiol. Mol. Biol. Rev.* 77, 1–52. doi: 10.1128/MMBR.00043-12

Salmond, G. P. (1994). Secretion of extracellular virulence factors by plant pathogenic bacteria. *Annu. Rev. Phytopathol.* 32, 181–200. doi: 10.1146/annurev.py.32.090194.001145

Samson, J. A. (1986). *Tropical Fruits*, 2nd Edn. Essex: Logman Scientific and Technical.

Sarkar, S. F., and Guttman, D. S. (2004). Evolution of the core genome of *Pseudomonas syringae*, a highly clonal, endemic plant pathogen. *Appl. Environ. Microbiol.* 70, 1999-2012. doi: 10.1128/AEM.70.4.1999-2012.2004

Schechter, L. M., Vencato, M., Jordan, K. L., Schneider, S. E., Schneider, D. J., and Collmer, A. (2006). Multiple approaches to a complete inventory of *Pseudomonas syringae* pv. tomato DC3000 type III secretion system effector proteins. *Mol. Plant Microbe Interact.* 19, 1180-1192. doi: 10.1094/MPMI-19-1180

Scheck, H. J., and Pscheidt, J. W. (1998). Effect of copper bactericides on copper-resistant and-sensitive strains of *Pseudomonas syringae* pv. syringae. *Plant Dis.* 82, 397-406. doi: 10.1094/PDIS.1998.82.4.397

Scholz-Schroeder, B. K., Hutchison, M. L., Grgurina, I., and Gross, D. C. (2001). The contribution of syringopeptin and syringomycin to virulence of *Pseudomonas syringae* pv. syringae strain B301D on the basis of *sypA* and *syrB1* biosynthesis mutant analysis. *Mol. Plant Microbe Interact.* 14, 336-348. doi: 10.1094/MPMI.2001.14.3.336

Semsa, A., Murillo, J., and Sundin, G. W. (2000). Phylogeny of the replication regions of pPT23A-like plasmids from *Pseudomonas syringae*. *Microbiology* 146, 2375-2384. doi: 10.1099/00221287-146-10-2375

Semsa, A., Sundin, G. W., and Murillo, J. (1998). Closely related plasmid replicons in the phytopathogen *Pseudomonas syringae* show a mosaic organization of

the replication region and an altered incompatibility behavior. *Appl. Environ. Microbiol.* 64, 3948-3953.

Silby, M. W., Winstanley, C., Godfrey, S. A. C., Levy, S. B., and Jackson, R. W. (2011). *Pseudomonas* genomes: diverse and adaptable. *FEMS Microbiol. Rev.* 35, 652-680. doi: 10.1111/j.1574-6976.2011.00269.x

Sorensen, K. N., Kim, K. H., and Takemoto, J. Y. (1998). PCR detection of cyclic lipodepsinopeptide-producing *Pseudomonas syringae* pv. syringae and similarity of strains. *Appl. Environ. Microbiol.* 64, 226-230.

Spiers, A. J., Bohannon, J., Gehrig, S., and Rainey, P. B. (2003). Biofilm formation at the air-liquid interface by the *Pseudomonas fluorescens* SBW25 wrinkly spreader requires an acetylated form of cellulose. *Mol. Microbiol.* 50, 15-27. doi: 10.1046/j.1365-2958.2003.03670.x

Spiers, A. J., Kahn, S. G., Travisano, M., Bohannon, J., and Rainey, P. B. (2002). Adaptive divergence in experimental populations of *Pseudomonas fluorescens*. *I. Genetic and phenotypic bases of wrinkly spreader fitness*. *Genetics* 161, 33–46.

Stein, T., and Vater, J. (1996). Amino acid activation and polymerization at modular multienzymes in nonribosomal peptide biosynthesis. *Amino Acids* 10, 201–227. doi: 10.1007/BF00807324

Studholme, D. J., Ibanez, S. G., MacLean, D., Dangl, J. L., Chang, J. H., and Rathjen, J. P. (2009). A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of *Pseudomonas syringae* pathovar tabaci 11528. *BMC Genomics* 10:395. doi: 10.1186/1471-2164-10-395

Studholme, D. J., Rachel, H., and Glover, N. B. (2011). Application of high-throughput DNA sequencing in phytopathology. *Annu. Rev. Phytopathol.* 49, 87-105. doi: 10.1146/annurev-phyto-072910-095408

Sun, W., Zhang, J., Fan, Q., Xue, G., Li, Z., and Liang, Y. (2010). Silicon enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrier. *Eur. J. Plant Pathol.* 128, 39-49. doi: 10.1007/s10658-010-9625-x

Sundin, G. W. (2007). Genomic insights into the contribution of phytopathogenic bacterial plasmids to the evolutionary history of their hosts. *Annu. Rev. Phytopathol.* 45, 129-151. doi: 10.1146/annurev.phyto.45.062806.094317

Sundin, G. W., and Bender, C. L. (1993). Ecological and genetic analysis of copper and streptomycin resistance in *Pseudomonas syringae* pv. syringae. *Appl. Environ. Microbiol.* 59, 1018-1024.

Sundin, G. W., and Bender, C. L. (1996). Molecular analysis of closely related copper- and streptomycin-resistance plasmids in *Pseudomonas syringae* pv. syringae. *Plasmid* 35, 98-107. doi: 10.1006/plas.1996.0012

Sundin, G. W., and Jacobs, J. L. (1999). Ultraviolet radiation (UVR) sensitivity analysis and UVR survival strategies of a bacterial community from the phyllosphere of field-grown peanut (*Arachis hypogaea* L.). *Microb. Ecol.* 38, 27-38. doi: 10.1007/s002489900152

Sundin, G. W., Jacobs, J. L., and Murillo, J. (2000). Sequence diversity of *rulA* among natural isolates of *Pseudomonas syringae* and effect on function of *rulAB*-mediated UV radiation tolerance. *Appl. Environ. Microbiol.* 66, 5167-5173. doi: 10.1128/AEM.66.12.5167-5173.2000

Sundin, G. W., Jones, A. L., and Fulbright, D. W. (1989). Copper resistance in *Pseudomonas syringae* pv. syringae from cherry orchards and its associated transfer in vitro and in planta with a plasmid. *Phytopathology* 79, 861-865. doi: 10.1094/Phyto-79-861

Sundin, G. W., Kidambi, S. P., Ulrich, M., and Bender, C. L. (1996). Resistance to ultraviolet light in *Pseudomonas syringae*: sequence and functional analysis of the plasmid-encoded *rulAB* genes. *Gene* 177, 77-81. doi: 10.1016/0378-1119(96)00273-9

Sundin, G. W., and Murillo, J. (1999). Functional analysis of the *Pseudomonas syringae* *rulAB* determinant in tolerance to ultraviolet B (290-320 nm) radiation and distribution of *rulAB* among *P. syringae* pathovars. *Environ. Microbiol.* 1, 75-88. doi: 10.1046/j.1462-2920.1999.00008.x

Thakur, S., Weir, B. S., and Guttman, D. S. (2016). Phytopathogen genome announcement: draft genome sequences of 62 *Pseudomonas syringae* type and pathotype strains. *Mol. Plant Microbe Interac.* 29, 243-246. doi: 10.1094/MPMI-01-16-0013-TA

Turner, J. G., and Debbage, J. M. (1982). Tabtoxin-induced symptoms are associated with accumulation of ammonia formed during photorespiration. *Physiol. Plant Pathol.* 20, 223-233. doi: 10.1016/0048-4059(82)90087-X
Ude, S., Arnold, D. A., Moon, C. D., Timmis-Wilson, T., and Spiers, A. J. (2006). Biofilm formation and cellulose expression among diverse environmental *Pseudomonas* isolates. *Environ. Microbiol.* 8, 1997–2011. doi: 10.1111/j.1462-2920.2006.01080.x

Vallet-Gely, I., Opota, O., Boniface, A., Novikov, A., and Lemaitre, B. (2010). A secondary metabolite acting as a signalling molecule controls *Pseudomonas entomophila* virulence. *Cell. Microbiol.* 12, 1666–1679. doi: 10.1111/j.1462-5822.2010.01501.x

Vassilev, V., Lavermicocca, P., Di Giorgio, C., and Iacobellis, N. (1996). Production of syringomycins and syringopeptins by *Pseudomonas syringae* pv. atrofaciens. *Plant Pathol.* 45, 316–322. doi: 10.1046/j.1365-3059.1996.d01-126.x

Vinatzer, B. A., Teitzel, G. M., Lee, M. W., Jelenska, J., Hotton, S., Fairfax, K., et al. (2006). The type III effector repertoire of *Pseudomonas syringae* pv. syringae B728a and its role in survival and disease on host and non-host plants. *Mol. Microbiol.* 62, 26–44. doi: 10.1111/j.1365-2958.2006.05350.x

Vivian, A., Murillo, J., and Jackson, R. W. (2001). The roles of plasmids in phytopathogenic bacteria: mobile arsenals? *Microbiology* 147, 763–780. doi: 10.1099/00221287-147-4-763

Volksch, B., and Weingart, H. (1998). Toxin production by pathovars of *Pseudomonas syringae* and their antagonistic activities against epiphytic microorganisms. *J. Basic Microbiol.* 38, 135–145.

Von Rozycki, T., and Nies, D. H. (2009). *Cupriavidus metallidurans*: evolution of a metal-resistant bacterium. *Antonie Van Leeuwenhoek* 96, 115–139. doi: 10.1007/s10482-008-9284-5

Wang, M. J. (1997). Land application of sewage sludge in China. *Sci. Total Environ.* 197, 149–160. doi: 10.1016/S0048-9697(97)05426-0

Wenneker, M., Meijer, H., Maas, F. M., de Bruine, A., Vink, P., and Pham, K. (2013). Bacterial canker of plum trees (prunus domestica), caused by *Pseudomonas syringae* pathovars, in the Netherlands. *Acta Hortic.* 985, 235–239. doi: 10.17660/ActaHortic.2013.985.30

Xin, X.-F., Kvitko, B., and He, S. Y. (2018). *Pseudomonas syringae*: what it takes to be a pathogen. *Nat. Rev. Microbiol.* 16, 316–328. doi: 10.1038/nrmicro.2018.17

Xiong, Z. (1998). Heavy metal contamination of urban soils and plants in relation to traffic in Wuhan City, China. *Toxicol. Environ. Chem.* 65, 31–39. doi: 10.1080/02772249809358555

Xiong, Z., and Wang, H. (2005). Copper toxicity and bioaccumulation in Chinese cabbage (*Brassica pekinensis* Rupr.). *Environ. Toxicol.* 20, 188–194. doi: 10.1002/tox.20094

Xu, L. H., Xie, G. L., Li, B., Zhu, B., Xu, F. S., and Qian, J. (2008). First report of pear blossom blast caused by *Pseudomonas syringae* pv. syringae in China. *Plant Dis.* 92:832. doi: 10.1094/PDIS-92-5-0832C

Young, J. M. (2010). Taxonomy of *Pseudomonas syringae*. *J. Plant Pathol.* 92, S5–S14. doi: 10.4454/jpp.v92i1sup.2501

Yu, J., Penaloza-Vazquez, A., Chakrabarty, A. M., and Bender, C. L. (1999). Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of *Pseudomonas syringae* pv. syringae. *Mol. Microbiol.* 33, 712–720. doi: 10.1046/j.1365-2958.1999.01516.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Gutiérrez-Barranquero, Cazorla and de Vicente. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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# Analyses of Seven New Genomes of *Xanthomonas citri* pv. *aurantifolii* Strains, Causative Agents of Citrus Canker B and C, Show a Reduced Repertoire of Pathogenicity-Related Genes

OPEN ACCESS

**Edited by:**

Dawn Arnold,
University of the West of England,
United Kingdom

**Reviewed by:**

Jeffrey Jones,
University of Florida, United States
David J. Studholme,
University of Exeter, United Kingdom

*Correspondence:

Leandro Marcio Moreira
Immorei@gmail.com
João C. Setubal
setubal@iq.usp.br

† These authors have contributed
equally to this work

**Specialty section:**

This article was submitted to
Plant Microbe Interactions,
a section of the journal
Frontiers in Microbiology

**Received:** 01 March 2019

**Accepted:** 27 September 2019

**Published:** 11 October 2019

**Citation:**

Fonseca NP, Patané JSL,
Varani AM, Felestrino ÉB,
Caneschi WL, Sanchez AB,
Cordeiro IF, Lemes CGdC,
Assis RdAB, Garcia CCM,
Belasque J Jr, Martins J Jr,
Facincani AP, Ferreira RM, Jaciani FJ,
Almeida NFd, Ferro JA, Moreira LM
and Setubal JC (2019) Analyses
of Seven New Genomes
of Xanthomonas citri pv. aurantifolii
Strains, Causative Agents of Citrus
Canker B and C, Show a Reduced
Repertoire of Pathogenicity-Related
Genes. Front. Microbiol. 10:2361.
doi: 10.3389/fmicb.2019.02361

Natasha Peixoto Fonseca<fnref n="1" />†, José S. L. Patané²†, Alessandro M. Varani<fnref n="3" />, Érica Barbosa Felestrino<fnref n="1" />, Washington Luiz Caneschi<fnref n="1" />, Angélica Bianchini Sanchez<fnref n="1" />, Isabella Ferreira Cordeiro<fnref n="1" />, Camila Gracyelle de Carvalho Lemes<fnref n="1" />, Renata de Almeida Barbosa Assis<fnref n="1" />, Camila Carrião Machado Garcia<fnref n="1" />, José Belasque Jr.<fnref n="4" />, Joaquim Martins Jr.⁵, Agda Paula Facincani<fnref n="3" />, Rafael Marini Ferreira<fnref n="3" />, Fabricio José Jaciani<fnref n="6" />, Nalvo Franco de Almeida⁷, Jesus Aparecido Ferro<fnref n="3" />, Leandro Marcio Moreira<fnref n="1" />,⁸* and João C. Setubal⁵*

*Xanthomonas citri* pv. *aurantifolii* pathotype B (XauB) and pathotype C (XauC) are the causative agents respectively of citrus canker B and C, diseases of citrus plants related to the better-known citrus canker A, caused by *Xanthomonas citri* pv. *citri*. The study of the genomes of strains of these related bacterial species has the potential to bring new understanding to the molecular basis of citrus canker as well as their evolutionary history. Up to now only one genome sequence of XauB and only one genome sequence of XauC have been available, both in draft status. Here we present two new genome sequences of XauB (both complete) and five new genome sequences of XauC (two complete). A phylogenomic analysis of these seven genome sequences along with 24 other related *Xanthomonas* genomes showed that there are two distinct and well-supported major clades, the XauB and XauC clade and the *Xanthomonas citri* pv. *citri* clade. An analysis of 62 Type III Secretion System effector genes showed that there are 42 effectors with variable presence/absence or pseudogene status among the 31 genomes analyzed. A comparative analysis of secretion-system and surface-structure genes showed that the XauB and XauC genomes lack several key genes

<footnote n="1">¹Programa de Pós-graduação em Biotecnologia, Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Brazil, ²Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, Brazil,</footnote>

<footnote n="3">³Departamento de Tecnologia, Universidade Estadual Paulista, UNESP, Campus de Jaboticabal, Jaboticabal, Brazil,</footnote>

<footnote n="4">⁴Departamento de Fitopatologia e Nematologia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, Brazil, ⁵Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil,</footnote>

<footnote n="6">⁶Fundo de Defesa da Citricultura (FUNDECITRUS), São Paulo, Brazil, ⁷Faculdade de Computação, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil, ⁸Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Brazil</footnote>
in pathogenicity-related subsystems. These subsystems, the Types I and IV Secretion Systems, and the Type IV pilus, therefore emerge as important ones in helping explain the aggressiveness of the A type of citrus canker and the apparent dominance in the field of the corresponding strain over the B and C strains.

**Keywords:** effectors, adaptation, virulence, Xanthomonas evolution, genome sequencing

# INTRODUCTION

Citrus is an important worldwide crop (FAS, 2019) that has been threatened by various diseases over many decades. Even though citrus Huanglongbin (greening) is today the major citrus threat (Wang et al., 2017), citrus canker (Goto, 1992; Schubert and Miller, 1996; Schubert et al., 2001) still is an important disease (CAB-International, 2019), especially in Brazil (Mendonça et al., 2017).

Three species of bacteria of the genus *Xanthomonas* are associated with citrus canker diseases in citrus: *Xanthomonas citri* subsp. *citri* (Xcc) pathotypes A, A\* and A\*w, X. citri subsp. *aurantifolii* pathotypes B and C (XauB and XauC), and X. alfalfae subsp. *citrumelonis* (Xacm). Xcc, XauB and XauC are respectively the causative agents of citrus canker A, B, and C, which cause small necrotic raised lesions surrounded by a water-soaked margin (Civerolo, 1984). Citrus canker A, the most aggressive, remains a concern in all citrus growing regions in Asia and South America (CAB-International, 2019). XauB strains are less aggressive, and XauC strains have a more restricted host range, when compared with symptoms and host range of Xcc, respectively. Canker B is currently known to be present only in Argentina, Paraguay, and Uruguay (Civerolo, 1984); moreover, XauB may have been eradicated even from this restricted region by competition from Xcc (Chiesa et al., 2013). Canker C is limited to the state of São Paulo, Brazil (Malavolta Júnior et al., 1984); the most recent field report dates to 2009 (Jaciani et al., 2009). Xacm is the causal agent of citrus bacterial spot, which induces symptoms very similar to those of canker, but the lesions are flat and not raised.

Sequencing of the *X. citri* subsp. *citri* strain A306 genome (A306) allowed the characterization of important properties of this more aggressive pathotype (da Silva et al., 2002). Following that work, genomes of the other pathotypes were sequenced and compared with each other (Jalan et al., 2013; Bodnar et al., 2017).

Given the phylogenetic relatedness of the causal pathogens of cankers A, B, and C, the comparative study of XauB and XauC strains at the genomic level offers the opportunity of achieving a better understanding of citrus canker disease in general. Up to now, only one XauB and only one XauC strain genome have been sequenced (Moreira et al., 2010). We therefore decided to sequence the genomes of additional strains of XauB and XauC. The newly sequenced isolates were selected because they showed significant differences in pathogenicity and aggressiveness when inoculated in different citrus genotypes and/or had different genetic characteristics (Jaciani, 2012; **Table 1**). The isolates XauB 1561 and XauB

1566 showed less virulence with respect to the other isolates and absence of clear infection symptoms, suggesting a probable loss of pathogenicity, besides being genetically different by AFLP and ERIC-PCR (Jaciani, 2012). The selection of XauC strains was based on the ability of some isolates to produce dark pigment when cultivated in NB or NA culture media (NB: 0.5% peptone, 0.3% beef extract; NA: 0.5% peptone, 0.3% beef extract, 1.5% agar), also observed in *X. citri* pv. *fuscans* and *X. campestris* pv. *vignicola* (Schaad et al., 2005; Schaad et al., 2006). XauC 535 and XauC 1609 cause lesions only in Mexican lime [C. aurantifolia (Christm.) Swingle] and Swingle citrumelo [C. paradisi Macfad. × *Poncirus trifoliata* (L.) Raf.], and in both hosts with mild symptoms. XauC 1609 was found to infect Swingle citrumelo under natural conditions (Jaciani et al., 2009), despite the fact that prior work suggested that only Mexican lime was susceptible to XauC (Malavolta Júnior et al., 1984). Additionally, XauC 535 and XauC 1609 were also differentiated by AFLP and BOX-PCR (Jaciani, 2012). The isolates XauC 763, XauC 867, and XauC 1559, which do not produce pigment, were distinguished in terms of pathogenicity and aggressiveness. XauC 763 and XauC 1559, which are also Mexican lime pathogens, caused injuries in Swingle citrumelo and Cravo mandarin (*C. reticulata* Blanco), and when inoculated in high concentration they infected Rangpur lime (*C. limonia* Osbeck), Persian lime [C. latifolia (Yu. Tanaka) Tanaka], lemon [*Citrus limon* (L.) Burm. f.], Grapefruit (*C. paradisi* Macfad.), and Cleopatra mandarin (*C. reshni* hort. ex Tanaka) (Jaciani, 2012). Finally, XauC 1559 was slightly more aggressive than XauC 763 when inoculated in Cravo mandarin, and XauC 867 presented a slightly more restricted pathogenicity and lower aggressiveness in Mexican lime compared to XauC 763 and XauC 1559 (Jaciani, 2012).

Altogether, based on the information above, we have sequenced two new XauB and five new XauC genomes, with the aim of achieving a better understanding of the genomic basis of citrus canker and the evolutionary history of these strains. Together with 24 other public and closely related genomes, this allowed us to carry out a phylogenomic analysis as well as an investigation of selected gene families relevant in bacteria-plant interactions in general and in citrus canker in particular (Ryan et al., 2011), which we present here.

A note on taxonomic nomenclature: *Xanthomonas* species that are pathogenic to citrus were described in this study using names as proposed by Schaad et al. (2006), since this classification is adopted for all cited references found until the present. The other *Xanthomonas* species were described as proposed by Bui Thi Ngoc et al. (2010) and Constantin et al. (2016).
<table><caption>TABLE 1 | Phenotype characteristics of eight Xanthomonas citri strains (Jaciani, 2012).</caption><thead><tr><th>Strain (short name)</th><th>Year</th><th>Origin</th><th>Pathogenicity on: Host</th><th>Pigment production</th><th>Swingle citrumelo</th><th>Hamlin sweet orange</th><th>Mexican lime</th><th>Persian lime</th><th>Rangpur lime</th><th>Lemon</th><th>Grapefruit</th><th>Cleopatra mandarin</th><th>Cravo mandarin</th><th>Ponkan mandarin</th></tr></thead><tbody><tr><td>XauB 1561</td><td>1981</td><td>Argentina</td><td>Lemon</td><td>No</td><td>-</td><td>-</td><td>±</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>±</td></tr><tr><td>XauB 1566</td><td>1990</td><td>Argentina</td><td>Lemon</td><td>No</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>±</td><td>-</td><td>-</td><td>-</td></tr><tr><td>XauC 535</td><td>2000</td><td>Brazil</td><td>Mexican lime</td><td>Yes</td><td>+</td><td>+</td><td>++</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>±</td></tr><tr><td>XauC 763</td><td>1981</td><td>Brazil</td><td>Mexican lime</td><td>No</td><td>++</td><td>±</td><td>+++</td><td>+</td><td>+</td><td>+</td><td>±</td><td>-</td><td>±</td><td>±</td></tr><tr><td>XauC 867</td><td>2002</td><td>Brazil</td><td>Mexican lime</td><td>No</td><td>++</td><td>±</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>±</td><td>±</td></tr><tr><td>XauC 1559</td><td>1981</td><td>Brazil</td><td>Mexican lime</td><td>No</td><td>++</td><td>±</td><td>+</td><td>+++</td><td>-</td><td>-</td><td>-</td><td>-</td><td>++</td><td>+</td></tr><tr><td>XauC 1609</td><td>2009</td><td>Brazil</td><td>Swingle citrumelo</td><td>Yes</td><td>+</td><td>±</td><td>+</td><td>+++</td><td>+</td><td>+</td><td>+++</td><td>+++</td><td>++</td><td>+++</td></tr><tr><td>Xac306 or A306</td><td>1997</td><td>Brazil</td><td>Sweet orange</td><td>No</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td><td>+++</td></tr></tbody></table>

–, non-pathogenic (absence of symptom); ±, not aggressive (slight eruption at the wound site and no necrosis or water-soaking); +, weakly aggressive (limited necrosis and no water-soaking); ++, moderately aggressive (small necrosis and limited water-soaking); +++, highly aggressive (extensive necrosis surrounded by a water-soaking margin).

# MATERIALS AND METHODS

## Media and Culture Conditions

The new genomes presented here were sequenced from strains stored both in autoclaved tap water at room temperature and at –80°C in NB medium (3 g/L meat extract, 5 g/L peptone) containing 25% glycerol. Each one of the strains was recovered from a –80°C stock, streaked on solid NA medium (3 g/L meat extract, 5 g/L peptone and 15g/L agar) and cultivated for 48 h at 29°C. For each strain, colonies were inoculated into 10 mL of liquid NB medium in a sterile 50 mL Falcon conical centrifuge tube and incubated at 29°C in a rotary shaker at 180 rpm for 16 h (final OD600 nm ~1.0).

## DNA Extraction and Quantification

A volume of 2 mL of the culture was centrifuged at 16,000 g for 10 min at 4°C in a refrigerated benchtop microcentrifuge. The supernatant was discarded and the cell pellet was resuspended in 600 µL of Nuclei Lysis Solution supplied by Promega Wizard Genomic DNA purification kit (Promega Corporation, Madison, United States). Total DNA extraction was performed using Promega Wizard Genomic DNA purification kit according to manufacturer instructions. DNA quantity and quality were determined using NanoDrop ND-1000 spectrophotometer (NanoDrop Tech, Wilmington, DE, United States), Qubit 2.0 fluorometer (Invitrogen, Life Technologies, CA, United States) and 0.8% agarose gel electrophoresis. Each extraction yielded at least 5 µg of high-quality genomic DNA.

## Genome Sequencing and Assembly

The genomes of XauC 535, XauC 763, and XauC 867 strains were sequenced using the Illumina HiScanSQ platform at NGS Soluções Genômicas (Piracicaba, Brazil). An average of ~20 M reads (2 × 100 bp) for each genome was generated. The raw reads were trimmed with seqclean software<fnref n="1" />, using minimum phred value of 23, minimum read length of 30 bp, and removing custom Illumina TruSeq adapters. Genome assembly was carried out with SPAdes v3.8.1 (Bankevich et al., 2012) with default parameters. Contig sequences were assigned to plasmids using plasmidSPAdes v3.8.1 (Antipov et al., 2016).

The genomes of XauB1561, XauB1566, XauC1559, XauC1609 strains were sequenced using PacBio single molecule real-time (SMRT) technology at the Duke Center for Genomic and Computational Biology (United States). One SMRT library was sequenced for each sample using P6-C4 chemistry, generating reads with average length of 10–15 Kb, thus yielding ~150X coverage of each genome. De novo assembly was conducted using SMRT® Analysis Server v2.3.0². Raw PacBio reads were mapped against the resulting contigs using the blasR aligner, and SNP corrections were conducted with variant-caller software using the quiver algorithm (both part of the Analysis Server).

The rationale for having some genomes sequenced using PacBio technology and some using Illumina technology was as

<footnote n="1">¹https://bitbucket.org/izhbannikov/seqyclean</footnote>

<footnote n="2">²https://www.pacb.com/documentation/smrt-analysis-software-installation-v2-3-0/</footnote>
follows. We wanted to ensure that we could provide complete genomes for both XauB and XauC, given that prior to this work only draft genomes were available for these pathotypes (Moreira et al., 2010). On the other hand our budget was limited, and we could afford PacBio sequencing for only four genomes. Under these constraints, the choice of which genomes to sequence by PacBio was arbitrary.

All assembled genomes were verified with CheckM (Parks et al., 2015), resulting in 100% completeness and 0% contamination for all of them.

## Genome Selection

For the purposes of phylogenomic analyses, we searched for genomes in NCBI/GenBank using "Xanthomonas citri" as a keyword, then looked at the automatic dendrogram generated by genomic distances on the NCBI website<fnref n="3" />, which reveals all genomes within this group, including all subspecies/lineages/varieties available as reference sequences (RefSeq). After downloading this tree, we searched for all different lineages, and then downloaded up to three genomes from each such lineage, if available, and preferentially (if possible) drawing from separate clades where the lineage appears in NCBI's dendrogram, to avoid pseudoreplication (i.e., avoiding picking two closely related genomes). This led to a final dataset of 31 genomes.

## Phylogenomic Reconstruction

In order to generate comparable sets of gene families, Prokka (Seemann, 2014) (with default parameters) was employed for annotation of each genome. Get_Homologues (Contreras-Moreira and Vinuesa, 2013) was used for multiple local BLAST-directed comparisons among all genes (of all genomes), and these were further clustered by the OMCL method which drives the OrthoMCL algorithm (Li et al., 2003) within Get_Homologues. Subsequently compare clusters.pl (a script within the same software) was used for retrieval of the set of orthologous genes uniquely present in all genomes (hereafter denominated the unicopy set). Mafft (Katoh and Standley, 2013) was used for multiple alignment of each unicopy gene, and then concatenation of all genes was done using FASconCAT (Kück and Meusemann, 2010). IQTree (Nguyen et al., 2015) was used for maximum likelihood (ML) estimation, with model choice employed before tree search, and branch support computed by UFBoot (Hoang et al., 2017).

## Effector Analysis

The aminoacid sequences of 62 effectors were retrieved from the Xanthomonas.org site (AvrBs2, AvrXccA1, AvrXccA2, HpaA, HrpW, XopA, XopAA, XopAB, XopAC, XopAD, XopAE, XopAF1, XopAF2, XopAG, XopAH, XopAI, XopAJ, XopAK, XopAL1, XopAL2, XopAM, XopAP, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopB, XopC1, XopC2, XopD, XopE1, XopE2, XopE3, XopF1, XopF2, XopG1, XopG2, XopH1, XopI1, XopJ1, XopJ2, XopJ3, XopJ4, XopJ5, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, XopU, XopV,

XopW, XopX, XopY, and XopZ1) (Supplementary Table S1), to infer their evolution across the 31 genomes. For each genome, we assessed whether each effector without a premature stop codon had a frameshift or not. In order to do so, we performed local tBLASTn searches within the Blast + suite (Camacho et al., 2009) with an e-value of 1e-50 (a threshold obtained by trial-and-error, that minimized the number of extra hits bearing indels and mismatches without compromising detection of supposedly functional copies), discarding alignments in which the subject sequences aligned to less than 60% of the query length or with less than 60% identity. After tBLASTn runs, multiple alignments were generated by an in-house python script for each effector, each of which was manually checked in Aliview (Larsson, 2014) for detection of frameshifts and premature stop codons. Optimization of character evolution for each effector along the ML tree (i.e., presence, frameshifts without premature stop codons, sequences with premature stop codons, and absence) was obtained by the ace function within the R library phytools (Revell, 2012).

## Additional Gene Analyses

Additional gene families were investigated based on OrthoMCL clustering (Li et al., 2003) and STRING (Snel et al., 2000). OrthoMCL was run with default parameters, and results were then processed in the OrthologSorter pipeline (Setubal et al., 2018). Additionally, we created an Ortholog Alignment using gene families provided by OrthoMCL, with the A306 strain as anchor and all the XauB and XauC genomes, plus X. citri pv. fuscans 4384. This alignment is useful to visualize syntenic regions among genomes. The parts of this alignment that were used in reporting results in this work are shown in a simplified version in Supplementary Table S2. In the case of STRING, for each family of interest, the relevant genes as present in A306 were used as queries.

## Gum Production Assay

The xanthan gum production assays were performed as described by Moreira et al. (2010), without modification.

## Biofilm Production Assay

Biofilm production assays were performed following O'Toole (O'Toole, 2011), with a few modifications. The bacterial isolates were grown in liquid LB or XVM2 medium at 28°C. Bacterial density was standardized for all the isolates in OD600nm equal to 1.0. The samples were diluted 1:10 in liquid LB and 100 µL of each sample were placed in the 96-well plate for growth during 96 h at 28°C. After the incubation period, the plate was washed with distilled water to remove the cells and left drying for 2 h. Subsequently, 125 µL of crystal violet solution 0.1% (CV) were transferred to each well, which were left resting for 45 min. After the incubation period the plate was washed again with distilled water and left drying once more. Next, 125 µL of 95% ethanol were added to each well, which were left to rest for 45 min to complete CV dissolution. The absorbance reading was done at OD550nm. For each bacterial isolate 6 replicates were performed.

<footnote n="3">³https://www.ncbi.nlm.nih.gov/projects/treeview/</footnote>
## Autoaggregation Assay

The autoaggregation assay was adapted from Alamuri et al. (2010), with modifications. Cultures of different bacterial isolates were grown at 28°C in liquid LB medium or XVM2: 1.16 g/L NaCl, 1.32 g/L (NH₄)₂SO₄, 0.021 g/L KH₂PO₄, 0.055 g/L K₂HPO₄, 0.0027 g/L FeSO₄·7H₂O, 1.8 g/L fructose, 3.423 g/L sucrose, 5 mMgSO₄, 1 mM CaCl₂, 0.03% Casamino acid (pH 6.7), in triplicate. Samples with 10 mL of each culture were placed in a sterile 20 mL tube. Initially all cultures were vigorously shaken for 15 s and the tubes remained static throughout the experiment. Aliquots containing 100 µL were removed from approximately 1 cm of the top of the culture of each tube over time and optical density was measured at OD600 nm every hour.

## RESULTS

Information about the genomes that were sequenced for this work is given in Table 2. The additional genomes listed there were included in the analysis of pathogenicity-related genes.

### Phylogenomic Analyses

For the phylogenomic analyses we used 31 genomes (Table 3). Gene family computation resulted in 2,449 single-copy shared families, leading to a concatenated alignment of 2,516,841 bp. The best ML model was GTR + G + R2 (where R2 means a mixed model of rate variation with two rate classes), with most nodes with support ≥ 95%. The resulting phylogeny is shown in Figure 1.

### Xanthan Gum, Biofilm, and Autoaggregation Analysis

In an attempt to understand which physiological factors could contribute to the induction of the respective virulence phenotypes of the investigated strains, xanthan gum production, biofilm, and cell self-aggregation were analyzed (Supplementary Table S3). As expected, A306 is the strain with the highest production of xanthan gum by bacterial mass. On the other hand, XauC 535 and XauC 1609 showed respectively the highest biofilm production and self-aggregation capacity in virulence-inducing medium (XVM2).

### Type III Secretion System Effector Analysis

Out of the 62 effectors investigated, four were present in all genomes, 16 were absent from all genomes, for a total of 42 effectors with variable presence/absence across lineages (Figure 1). For 11 effectors we observed interesting patterns of presence, absence, or pseudogenization. For these effectors we inferred their evolutionary history in terms of gains, losses or pseudogenization (Supplementary Figure S1).

### Other Pathogenicity-Related Genes

Individual genes or genes that collectively encode proteins that compose cell complexes involved in virulence and adaptation were analyzed in all genomes listed in Table 2. The virulence

and adaptation genes were grouped into two broad categories: (1) secretion systems (other than Type III effectors); (2) surface structure. The analysis framework we have adopted is as follows. The A306 strain has several genes in each of the categories analyzed (da Silva et al., 2002). On the other hand, as will be seen, the XauB and XauC genomes that we have analyzed lack many or all of the genes in some of these categories. In order to better understand the potential impact that the lack of these genes may have in the pathogenicity and/or survival capabilities of the XauB and XauC strains, for each category in which XauB and XauC lack genes we first describe the A306 gene content. We then note the differences exhibited by XauB and XauC (as given by the Ortholog Alignment of 11 genomes, with A306 as anchor, as described in section Materials and Methods), followed by a network analysis based on the A306 genes, using the tool STRING (Snel et al., 2000).

## Secretion Systems

We verified that all the analyzed genomes retain all orthologous genes belonging to the two gene clusters associated with synthesis of the type II secretion system (T2SS, XAC0694-XAC0705, and XAC3534-3544), all the genes involved in structuring the type III secretion system apparatus (T3SS, XAC0393-XAC0417), all the genes associated with the type VI secretion system (T6SS, XAC4119-20-24, XAC4139-40-45), as well as complete Sec and Tat secretion systems. The main differences observed are related with the type I secretion system (T1SS), the type IV secretion system (T4SS) and effectors of the type III secretory system (T3SS). Results for T3SS effectors were already presented above.

### XauB and XauC Lack Key Genes in the Type I Secretion System

The T1SS corresponds to an ABC transporter system and it is basically composed of two proteins, HlyD – an ABC transporter, and HlyB – a membrane fusion protein, whose main function, together with TolC, is to promote the secretion of toxins (Koronakis et al., 2004). In A306 two copies of the gene encoding the toxin presumably secreted by this system, hemolysin (type-calcium, XAC2197-98), are upstream of the genes *hlyDB* (XAC2201-02), separated by two hypothetical proteins (XAC2199-2200). These gene families (XAC2197-2202) were not found in the XauB genomes. The XauC genomes on the other hand do not have orthologs of XAC2197-98, but they do have *hlyB* and *hlyD*. Other genes associated with synthesis and regulation of hemolysin in these genomes were also analyzed. All genomes have orthologs of XAC4303 and XAC1668 (cryptic hemolysin transcriptional regulator), XAC3043 and XAC0079 (hemolysin III, *hly3*), and XAC1709 (hemolysin, *tlyC*). However, in XauB and XauC strains we did not find orthologs for the genes XAC1814 (outer membrane hemolysin activator protein) and XAC1918 (hemolysin-like protein).

Analysis of possible interactions of the products of genes *hlyB* and *hlyD* (Figure 2) revealed two well-defined interaction networks for the A306 *hlyB* gene used as query to STRING. One of these groups, in orange background, is the genes/proteins
<table><caption>**TABLE 2** | List of genomes used in the comparative analysis section, including information about the seven newly sequenced XauB and XauC genomes.</caption><thead><tr><th>Strain</th><th>Short name</th><th>Source</th><th>Replicons</th><th>Status</th><th>Length (bp)</th><th>CDS</th><th>Accession</th></tr></thead><tbody><tr><td>X. citri pv. aurantifolii FDC 535</td><td>XauC 535</td><td>This work</td><td>Chromosome</td><td>372 contigs – N50 37-kb</td><td>5,226,212</td><td>4,364</td><td>LAUH00000000.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 763</td><td>XauC 763</td><td>This work</td><td>Chromosome</td><td>314 contigs – N50 35-kb</td><td>4,984,643</td><td>4,045</td><td>LAUI00000000.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 867</td><td>XauC 867</td><td>This work</td><td>Chromosome</td><td>303 contigs – N50 41-kb</td><td>5,014,602</td><td>4,085</td><td>LAUJ00000000.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1559</td><td>XauC 1559</td><td>This work</td><td>Chromosome</td><td>complete</td><td>5,191,653</td><td>4,225</td><td>CP011160.1</td></tr><tr><td></td><td></td><td></td><td>pXfc38</td><td></td><td>37,980</td><td>40</td><td>CP011162.1</td></tr><tr><td></td><td></td><td></td><td>pXfc43</td><td></td><td>43,013</td><td>36</td><td>CP011161.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1609</td><td>XauC 1609</td><td>This work</td><td>Chromosome</td><td>complete</td><td>5,164,518</td><td>4,175</td><td>CP011163.1</td></tr><tr><td></td><td></td><td></td><td>pXfc32</td><td></td><td>32,021</td><td>34</td><td>CP011165.1</td></tr><tr><td></td><td></td><td></td><td>pXfc37</td><td></td><td>37,089</td><td>42</td><td>CP011164.1</td></tr><tr><td></td><td></td><td></td><td>pXfc46</td><td></td><td>46,049</td><td>38</td><td>CP011166.1</td></tr><tr><td>X. citri pv. aurantifolii ICPB 10535</td><td>XauC 10535</td><td>NCBI</td><td>Chromosome</td><td>352 contigs – N50 29-kb</td><td>5,012,633</td><td>4,034</td><td>ACPY00000000.1</td></tr><tr><td>X. citri pv. aurantifolii ICPB 11122</td><td>XauB 11122</td><td>NCBI</td><td>Chromosome</td><td>244 contigs – N50 38-kb</td><td>4,879,662</td><td>3,918</td><td>ACPX00000000.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1561</td><td>XauB 1561</td><td>This work</td><td>Chromosome</td><td>complete</td><td>4,993,063</td><td>4,048</td><td>CP011250.1</td></tr><tr><td></td><td></td><td></td><td>pXfb33</td><td></td><td>33,022</td><td>42</td><td>CP011251.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1566</td><td>XauB 1566</td><td>This work</td><td>Chromosome</td><td>complete</td><td>4,926,567</td><td>3,983</td><td>CP012002.1</td></tr><tr><td></td><td></td><td></td><td>pXfb35</td><td></td><td>35,521</td><td>44</td><td>CP012003.1</td></tr><tr><td>X. citri pv. fuscans str. 4834-R</td><td>Xfus 4834</td><td>NCBI</td><td>Chromosome</td><td>complete</td><td>4,981,995</td><td>3,977</td><td>FO681494.1</td></tr><tr><td></td><td></td><td></td><td>pla</td><td></td><td>45,224</td><td>51</td><td>FO681495.1</td></tr><tr><td></td><td></td><td></td><td>plb</td><td></td><td>19,514</td><td>22</td><td>FO681496.1</td></tr><tr><td></td><td></td><td></td><td>plc</td><td></td><td>41,950</td><td>48</td><td>FO681497.1</td></tr><tr><td>X. citri pv. citri str. 306</td><td>Xac 306 or A306</td><td>NCBI</td><td>Chromosome</td><td>complete</td><td>5,175,554</td><td>4,321</td><td>AE008923.1</td></tr><tr><td></td><td></td><td></td><td>pXAC33</td><td></td><td>33,700</td><td>42</td><td>AE008924.1</td></tr><tr><td></td><td></td><td></td><td>pXAC64</td><td></td><td>64,920</td><td>73</td><td>AE008925.1</td></tr></tbody></table>

associated with T1SS composition and functionality. The other network (green background) is composed primarily of membrane genes/proteins, essentially ABC transporters. Eight genes/proteins represented by the nodes of the network composing the T1SS apparatus correspond to the same genes in the cluster discussed above, including the gene encoding the lytic enzyme (XAC0466) present in the XauC10535 genome. We observed that the genomes of XauB strains do not have any of these genes. However, the loss of a single gene of hemolysin in XauC strains would have a small effect, since this loss could be compensated by paralogous genes in their genomes. Concerning the cluster of membrane proteins, three of the ABC transporters are associated with resistance to acriflavin (XAC3994-95 and XAC3850) and two have the hlyD domain (PF00529), involved with secretion of toxins. Some of the genes in this network were not found in the genomes of strains XauC 1609 and XauC 535.

## XauB and XauC Genomes Lack the Chromosomal Copy of Type IV Secretion System Genes

The genes encoding the T4SS in A306 are found in two similar gene clusters, one in the chromosome (XAC2607-2623) and another in the plasmid (XACb0036-b0047) (da Silva et al., 2002). The genomes of XauB and XauC have only the plasmidial cluster (Supplementary Table S2). Note that in the cases of XauB

11122 and XauC 10535, whose contigs are not distinguished as belonging to the chromosome or to a plasmid, it is our inference based on synteny that the T4SS genes actually belong to a plasmid (Supplementary Table S2).

## XauB and XauC Genomes Lack Key Genes in the Synthesis and Regulation of Type IV Pilus

A306 has at least four clusters of genes involved with synthesis and regulation of type IV pilus (T4p). One of these clusters, *pilE-Y1-X-W-V-fimT* (XAC2664-2669) is found between a set of prophage genes upstream and a transposase downstream, suggesting possible horizontal gene transfer. We observed that two of these genes (*pilX-pilV*) are missing in the XauB and XauC genomes (Supplementary Table S2). In the case of cluster *pilS-R-B-A-A-C-D* (XAC3237-3243) (Yang et al., 2004), the XauB and XauC genomes lack the two copies of *pilA*. *PilA* encodes pilin, an essential T4p component that contributes to twitching motility and biofilm development in A306 (Dunger et al., 2014; Petrocelli et al., 2016). Another gene whose product has a function related to T4p is *pilL* (XAC2253). In A306 this gene is found in a large genomic island (XAC2176 to XAC2286), but is absent from the XauB and XauC genomes.

We carried out an analysis of predicted interactions of *pilA* (XAC3240) (Figure 3). In orange background we observe that
<table><caption>TABLE 3 | List of 31 genomes used in the phylogenomic analysis.</caption><thead><tr><th>Strain name</th><th>NCBI name</th><th>NCBI assembly</th></tr></thead><tbody><tr><td>X. citri pv. anacardii CFBP 2913</td><td>X. citri pv. anacardii CFBP 2913</td><td>GCF_002688625.1</td></tr><tr><td>X. citri pv. anacardii IBSBF2579</td><td>X. citri pv. anacardii IBSBF2579</td><td>GCF_002837255.1</td></tr><tr><td>X. citri pv. anacardii TAQ18</td><td>X. citri pv. anacardii TAQ18</td><td>GCF_002898415.1</td></tr><tr><td>X. citri pv. aurantifolii ICPB 11122</td><td>X. fuscans subsp. aurantifolii ICPB 11122</td><td>GCF_000175135.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1561</td><td>X. fuscans subsp. aurantifolii FDC 1561</td><td>GCF_002079965.1</td></tr><tr><td>X. citri pv. aurantifolii 1566</td><td>X. fuscans subsp. aurantifolii 1566</td><td>GCF_001610915.1</td></tr><tr><td>X. citri pv. aurantifolii ICPB 10535</td><td>X. fuscans subsp. aurantifolii ICPB 10535</td><td>GCF_000175155.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1559</td><td>X. fuscans subsp. aurantifolii FDC 1559</td><td>GCF_001610795.1</td></tr><tr><td>X. citri pv. aurantifolii FDC 1609</td><td>X. fuscans subsp. aurantifolii FDC 1609</td><td>GCF_001610815.1</td></tr><tr><td>X. citri pv. aurantifolii XauC535</td><td>X. citri pv. aurantifolii XauC535</td><td>GCF_004329265.1</td></tr><tr><td>X. citri pv. aurantifolii XauC763</td><td>X. citri pv. aurantifolii XauC763</td><td>GCF_004329275.1</td></tr><tr><td>X. citri pv. aurantifolii XauC867</td><td>X. citri pv. aurantifolii XauC867</td><td>GCF_004329295.1</td></tr><tr><td>X. citri pv. bilvae NCPPB 3213</td><td>X. citri pv. bilvae NCPPB 3213</td><td>GCF_001497855.1</td></tr><tr><td>X. citri pv. citri 306</td><td>X. axonopodis pv. citri 306</td><td>GCF_000007165.1</td></tr><tr><td>X. citri pv. citri AS8</td><td>X. citri pv. citri AS8</td><td>GCF_000950875.1</td></tr><tr><td>X. citri pv. citri Aw12879</td><td>X. citri subsp. citri Aw12879</td><td>GCF_000349225.1</td></tr><tr><td>X. citri pv. fuscans 4834-R</td><td>X. fuscans subsp. fuscans 4834-R</td><td>GCF_000969685.2</td></tr><tr><td>X. citri pv. fuscans CFBP6992</td><td>X. citri pv. phaseoli var. fuscans CFBP6992</td><td>GCF_002759335.1</td></tr><tr><td>X. citri pv. fuscans Xff49</td><td>X. citri pv. fuscans Xff49</td><td>GCF_002309515.1</td></tr><tr><td>X. citri pv. glycines 12-2</td><td>X. citri pv. glycines 12-2</td><td>GCF_002163775.1</td></tr><tr><td>X. citri pv. glycines CFBP 2526</td><td>X. axonopodis pv. glycines CFBP 2526</td><td>GCF_000495275.1</td></tr><tr><td>X. citri pv. glycines CFBP 7119</td><td>X. axonopodis pv. glycines CFBP 7119</td><td>GCF_000488895.1</td></tr><tr><td>X. citri pv. malvacearum GSPB2388</td><td>X. citri pv. malvacearum GSPB2388</td><td>GCF_000309925.1</td></tr><tr><td>X. citri pv. malvacearum X18</td><td>X. citri pv. malvacearum X18</td><td>GCF_000454505.1</td></tr><tr><td>X. citri pv. malvacearum X20</td><td>X. citri pv. malvacearum X20</td><td>GCF_000454525.1</td></tr><tr><td>X. citri pv. mangiferaindicae LG56-10</td><td>X. citri pv. mangiferaindicae LG56-10</td><td>GCF_002920975.1</td></tr><tr><td>X. citri pv. mangiferaindicae LG81-27</td><td>X. citri pv. mangiferaindicae LG81-27</td><td>GCF_002926255.1</td></tr><tr><td>X. citri pv. mangiferaindicae LMG 941</td><td>X. citri pv. mangiferaindicae LMG 941</td><td>GCF_000263335.1</td></tr><tr><td>X. citri pv. punicae LMG 859</td><td>X. axonopodis pv. punicae LMG 859</td><td>GCF_000285775.1</td></tr><tr><td>X. citri pv. vignicola CFBP7112</td><td>X. citri pv. vignicola CFBP7112</td><td>GCA_002218265.1</td></tr><tr><td>X. citri pv. vignicola CFBP7113</td><td>X. citri pv. vignicola CFBP7113</td><td>GCF_002218285.1</td></tr></tbody></table>

XAC3240 interconnects five other networks and that the pilin subunits (XAC3240 and XAC3241) are connected to one another, and connect to another *pilA* (XAC3805). As expected, one of the networks starting from pilins refers to genes/proteins associated with the pilus structure and with the T2SS (cyan background), as is known that both are evolutionarily related (Peabody et al., 2003). Moreover, three genes share the same genomic region of pilins in the chromosome of A306 (1, 4, and 5). Close to the pilin network (purple background) there is a network involving genes associated with quorum sensing (*rpf*), gum synthesis (*gum*) and the plant tissue degrading enzyme polygalacturonase (*pglA*), known to be virulence-related (Wang et al., 2008). Likewise, this network reflects the interaction profiles of DSF production mediated by *rpf* genes, which act as signaling molecules of gum synthesis and consequently of the production of plant cell wall degrading enzymes, as is the case for PglA mediated by T2SS (Vojnov et al., 2001; An et al., 2013). In addition, another network expands from *rpfc*. Indeed, this network (pale green background), associated with chemotaxis-related genes, includes *phoB*, which is involved in phosphate regulons, essential for adaptation and virulence induction in members of the genus *Xanthomonas* (Pegos et al., 2014; Moreira et al., 2015).

## XauB and XauC Genomes Lack an Alginate Biosynthesis Gene

In A306, the first gene downstream of *pilE-Y1-X-W-V-FimT*, XAC2670, encodes an alginate biosynthesis protein, which is absent from the XauB and XauC genomes (Supplementary Table S2). The A306, XauB, and XauC genomes encode another gene whose product is involved with the metabolism of alginate, alginate lyase: *algL* (XAC4349).

Analysis of predicted interactions of the gene XAC2670 with other genes/proteins revealed two distinct clusters (Figure 4). The first one on blue background contains seven nodes whose genes/proteins are directly related to synthesis and regulation of T4p (*pil* genes previously described). In this group, excepting *pilO* (XAC3383), all other genes are present in a cluster (XAC2664-XAC2669) downstream of a transposase and a phage insertion (numbers 1–6), and upstream of the gene XAC2670. The second cluster, on yellow background, contains 12 nodes, with most genes/proteins related to regulatory functions, especially *algZR* (XAC0620-21), encoding a two-component system, respectively for sensor and regulatory proteins (Okkotsu et al., 2014), and *algC* (phosphomannomutase) (Davies and Geesey, 1995), all described as essential to alginate synthesis. Another two-component system, *lytST* (XAC2142-2141, sensor-regulator), is also present in this network. *LytT*, as well as *rpfD*, also present in the yellow background network (XAC1874) and member of the *rpf* gene cluster, exhibit the *lytR* domain, also present in proteins such as AlgR with DNA binding function (Nikolskaya and Galperin, 2002). Finally, *rpoE* (XAC1319) connects the two clusters (Figure 4), and therefore may be directly associated with both by regulating the EPS synthesis and/or by modulating T4p expression.
<figure><img src="image_81.png" /><figcaption>**FIGURE 1** | Maximum likelihood phylogeny of 31 Xanthomonas citri strains and table of presence, absence, and pseudogene status of 46 effector genes.</figcaption></figure>

## XauB and XauC Lack Several Genes Related to Hemagglutinin and Hemolysin Synthesis

A further set of genes with significant differences in terms of presence and absence in the analyzed genomes is related to hemagglutinin and hemolysin synthesis. These genes are located in two regions in the genome of A306 (XAC4112-XAC4125 and XAC1810-XAC1819). The first region is flanked by genes that are part of the T6SS, both downstream and upstream. All genes of this region are present in all genomes of XauB and XauC. However, the genes in the second region (XAC1810-XAC1819) are totally absent in the XauB and XauC genomes. Among these genes we highlight *fhaC* (XAC1814), which codes for an outer membrane hemolysin activator, *fhaB* (XAC1815), which codes for a filamentous hemagglutinin, XAC1816, which codes for a hemagglutinin/hemolysin-related protein, XAC1818, which codes for hemagglutinin, and the genes in the operon *HmsHFR-hp* (XAC1813-1810).

Analysis of predicted interactions of the gene *fhaB* (XAC1815) allowed the characterization of two major interaction networks (Figure 5). One of these networks (pink background) is associated with adhesion, whereas the other network (gray background) basically contains hypothetical genes/proteins. Furthermore, other genes/proteins in the adhesion network are located in the two regions related to hemagglutinin and hemolysin synthesis mentioned in the previous paragraph.

Using *hmsF* (XAC1812), we obtained an interaction network made of three clusters, two of which seem to be functionally related (Figure 5). One of the clusters (green background) contains genes/proteins associated with carbohydrate

metabolism. The other cluster (orange background) contains the *hmsFHR* genes, related to biofilm formation.

A summary of these results is presented in Figure 6, which includes some additional pathogenicity-related genes also lacking in the XauB and XauC genomes: *vapBC*, a toxin-antitoxin module in *Acidovorax citrulli* (Shavit et al., 2015); and *tspO*, which encodes a protein with a potential role in the oxidative stress response, iron homeostasis, and virulence expression in *Pseudomonas* (Leneveu-Jenvrin et al., 2014).

## DISCUSSION

Our results show that the three lineages inflicting citrus canker (A strains and XauB and XauC strains) can be robustly separated into two well defined clades, with A strains in one clade, which we call the Citri-citri (C-c) clade, and XauB and XauC in another clade, which we call the aurantifolii clade (Figure 1); furthermore, XauB and XauC were shown to be in a paraphyletic clade, with *X. citri* pv. *anacardii* being closer to XauB (Figure 1). It is noteworthy to observe that the C-c and aurantifolii clades contain strains that are pathogenic in taxonomically disparate plant hosts, such as citrus (*X. citri* pv. citri and *X. citri* pv. *aurantifolii*), leguminosae (*X. citri* pv. glycines and *X. citri* pv. *fuscans* emerging from more basal nodes), cashew (*X. citri* pv. *anacardii*), mango (*X. citri* pv. *mangiferaindicae*), and cotton (*X. citri* pv. glycines). Curiously, *X. citri* pv. *anacardii* (infecting cashew) apparently evolved within a citrus-associated clade, suggesting a possible host jump.

We have made an extensive analysis of the presence and absence of effectors in the 31 genomes we sampled to reconstruct
<figure><img src="image_82.png" /></figure>

**FIGURE 2** | Network of putative interactions between genes/proteins using as query the *hlyB* gene from strain A306, a key gene in the Type 1 Secretion System. The nodes represent the genes/proteins present in the A306 genome. The color codes on the lines is: red – fusion evidence; green – neighborhood evidence; blue – co-occurrence evidence; purple – experimental evidence; yellow – text mining evidence; light blue – database evidence; black – co-expression evidence. The network part of the figure was generated by the program STRING (Snel et al., 2000). Genomic context is shown at the bottom, based on a figure generated by the BioCyc resource (Karp et al., 2017). The arrows represent the genes and the gray background the transcriptional units. The blue numbers correlate the position of a given gene in the genomic context and the same gene on the network. The number in red has a similar purpose and simply highlights the query gene in the network inference.

the phylogeny. We now discuss the main results of this analysis. XopS was shown to be the only effector among the 62 investigated that is present in the C-c clade (although in some cases as a pseudogene) but absent in the aurantifolii clade (Figure 1). XopS is completely dependent on HpaB to be translocated; it contributes to disease symptoms and bacterial growth and suppresses pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression (Schulze et al., 2012). XopF1 was found to have the opposite pattern compared to xopS: present in the aurantifolii clade (in some cases as a pseudogene) and absent in the C-c clade. In Xanthomonas oryzae pv. oryzae, XopF1 has been shown to repress basal PAMP-triggered immunity response in rice (Mondal et al., 2015). A third interesting case is xopK, which is present in the C-c clade, but was found to be a pseudogene in all genomes of the aurantifolii clade. XopK has been shown to inhibit PAMP-triggered immunity upstream

of mitogen-activated protein kinase cascades in Xanthomonas oryzae pv. oryzae (Qin et al., 2018). **Figure 1** makes clear that there are many other differences in effector repertoires among the 31 genomes analyzed; 11 of these other effectors have been studied in terms of their gains and losses across the evolution of the 31 strains (**Supplementary Figure S1**). Because the pattern of gains, losses and pseudogenization is more intricate, additional studies are required to correlate these inferred histories to known phenotypic traits of the affected strains.

In addition to effectors, we have carefully analyzed the gene content in the broad categories of secretion systems-related genes and surface structure-related genes. Our main tool in this analysis, in addition to presence/absence results, was the prediction of possible interactions. These analyses resulted in several noteworthy differences of XauB and XauC strains when compared to the A306 genome.
<figure><img src="image_83.png" /><figcaption>**FIGURE 3** | Network of putative interactions between genes/proteins using as query the pilin gene XAC3240 from the A306 strain. For additional details see legend of **Figure 2**. The network figure was generated by the program STRING (Snel et al., 2000) and the genomic context figure was based on a figure generated by the BioCyc resource (Karp et al., 2017).</figcaption></figure>

## Type I Secretion System

The T1SS is responsible for secreting toxins, such as hemolysins in *E. coli* (Thomas et al., 2014). In the three XauB strains investigated, both genes coding for apparatus secretory proteins (*hlyB* and *hlyD*) and genes coding for hemolysins (*hlyA*) were not found. This absence might contribute to a decrease in the elicitation of the plant immune response as well as to decreased

competitive capability with other organisms due to the inability to secrete these toxins.

## Type IV Secretion System

The T4SS has multiple functions, including transport of a variety of substrates from DNA and protein-DNA complexes to proteins, and it plays fundamental roles in both bacterial
<figure><img src="image_84.png" /><figcaption>**FIGURE 4** | Network of putative interactions between genes/proteins using as query the A306 gene XAC2670, which encodes an alginate biosynthesis protein. For additional details see legend of **Figure 2**. The network figure was generated by the program STRING (Snel et al., 2000) and the genomic context figure was based on a figure generated by the BioCyc resource (Karp et al., 2017).</figcaption></figure>

pathogenesis and adaptation to the cellular milieu in which bacteria live (Darbari and Waksman, 2015). Jacob et al. (Jacob et al., 2014), reported that the T4SS in A306, unlike the T3SS, is not associated with virulence induction, but rather in cell-cell interactions. This finding was confirmed by Souza et al. (2015), who demonstrated the involvement of the chromosomal T4SS in bacterial killing, showing that this special class of T4SS is a mediator of both antagonistic and cooperative interbacterial interactions. We speculate that the lack of the T4SS chromosomal gene cluster in XauB and XauC genomes may have a consequence in the ability of these strains to

compete with other bacteria, in particular with A306 itself. If this speculation is correct, this may be an explanation for the apparent disappearance of XauB strains from the field (Chiesa et al., 2013).

## Synthesis and Regulation of Type IV Pilus

Among the protein complexes involved in biofilm formation is the type IV pilus (T4p) (Dunger et al., 2014). Besides actively participating in this matrix, the T4p is of fundamental importance in the adhesion process to the host tissue in the early stages of infection and independent flagellum displacement,
<figure><img src="image_85.png" /></figure>

**FIGURE 5** | Networks of putative interactions between genes/proteins using as queries the A306 genes *fhaB* (XAC1815) and *hmsF* (XAC1812). For additional details see legend of **Figure 2**. The network figure was generated by the program STRING (Snel et al., 2000) and the genomic context figure was based on a figure generated by the BioCyc resource (Karp et al., 2017).

<figure><img src="image_86.png" /></figure>

**FIGURE 6** | Summary of the comparative analyses of 11 *Xanthomonas citri* genomes listed in **Table 2** in terms of the pathogenicity-related genes and systems discussed in the text. The red X symbol denotes lack of the genes or system next to it.

called twitching motility (Mattick, 2002). The orthologous gene cluster *pilE-Y1-X-W-V-FimT* (XAC2664-2669) of A306 in *P. aeruginosa* has been shown to be involved in negative regulation of swarming motility (Giltner et al., 2010; Kuchma et al., 2012). In this same context, inactivation of *pilA* inserted

in the *pilS-R-B-A-A-C-D* cluster (XAC3238-3243) interfered with twitching motility, biofilm development, and adherence of XAC (Dunger et al., 2014). Thus, the lack of genes *pilX*, *pilV*, *pilA*, *pill*, and *fimT* genes in the XauB and XauC genomes, all involved with T4p apparatus structuring, seem
to explain at least in part the decreased production of biofilm and self-aggregation capability in some XauB and XauC strains (Supplementary Table S3). On the other hand, these same results show that XauC 535 and XauC 1609 presented, respectively, the highest biofilm production and self-aggregation capability in virulence-inducing media (XVM2) among all strains, even in the absence of the genes listed; this result requires further investigation.

## Alginate Biosynthesis

Alginate is an EPS related to biofilm formation and produced by bacteria of the genus *Pseudomonas* (Baker and Svanborg-Eden, 1989; Orgad et al., 2011). The function of alginate lyase is to hydrolyze bonds that hold the structured polymer, thereby enabling the bacterium to leave the biofilm structure, allowing its spreading by the colonized tissue (Boyd and Chakrabarty, 1994).

The intricate network we inferred for XAC2670 (which codes for an alginate biosynthesis protein in A306) may be depleted in XauB and XauC due to the lack of key genes/proteins in the composition of these clusters, as it is the case of *pilX* and *pilV*, and XAC2670 itself, which could impair the synthesis and regulation of T4p apparatus and EPS production. Importantly, there are no reports in the literature mentioning any *Xanthomonas* species as an alginate producer. However, it is interesting to notice the presence of at least nine genes in A306 that may be involved with synthesis and regulation of this polymer, from which four are present in the interaction networks described above.

## Hemagglutinin and Hemolysin Synthesis

The hemagglutinin gene (XAC1818) has been described as fundamental to the virulence process in many organisms, including *Xylella fastidiosa* (Caserta et al., 2010; Voegel et al., 2010), another plant pathogen of the Xanthomonadaceae family, and in A306 (Gottig et al., 2009). The *hmsHFR-hp* genes (XAC1813-1810) are involved in adaptation and virulence, and have been reported respectively to be homologous to *E. coli* genes *pgaABCD* (Wang et al., 2004). Mutations in genes from this operon in members of the genera *Chromobacterium*, *Yersinia*, and *Xanthomonas* have resulted in reduction of biofilm formation and consequent reduction in virulence induction (Becker et al., 2009; Abu Khweek et al., 2010; Wang et al., 2012). Therefore, the absence of XAC1810-XAC1819 in the genomes of strains XauB and XauC might contribute to less efficient tissue adhesion processes and biofilm formation, and reduce cell-to-cell aggregation dependent of adhesin and exopolysaccharides molecules; this in turn would lead to reduction in tissue colonization capabilities in these strains.

## CONCLUSION

Taken together, our results show that the XauB and XauC genomes lack many genes that are known to play a role in host infection, either in A306 or in other pathosystems. This result is consistent with the attenuated citrus canker phenotypes of the XauB and XauC strains. In addition, the

lack of recent reports about the presence of XauB and XauC strains in the field suggests a scenario in which A306 or other A strains may have outcompeted the XauB and XauC strains, possibly leading to their eradication. If so, this would be a process similar to what has taken place with *Candidatus Liberibacter americanus*, a causative agent of citrus huanglongbin, which has reportedly been eradicated, in South America, by *Candidatus Liberibacter asiaticus* (Wulff et al., 2014). It is to be hoped that such knowledge can be put to practical use in the efforts to eradicate from the field the A strains as well.

## DATA AVAILABILITY STATEMENT

Raw reads are available at the Short Read Archive at NCBI at the following URLs: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA273983; https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA273983.

## AUTHOR CONTRIBUTIONS

JS, LM, JF, JB, and FJ conceived the study. JB and FJ selected and prepared strains for sequencing. AF, RF, and JF did the genome sequencing. AV and NA assembled the genomes. NF, ÉF, WC, AS, IC, CL, RA, CG, JM, JP, LM, and JS analyzed the data and interpreted the results. JS, LM, and JP wrote the manuscript.

## FUNDING

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 (the BIGA project). NF was funded in part by grant Fundect-MS (007/2015 SIAFEM 025139). JS, LM, NF, and AV were funded in part by Researcher Fellowships from CNPq.

## ACKNOWLEDGMENTS

We thank Carlos Morais Pirupo for providing computational assistance.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02361/full#supplementary-material

**FIGURE S1** | Trees with the reconstruction of gains, losses, and pseudogenization events for 11 effector genes. The effector name is shown at the top of each tree frame.

**TABLE S1** | Type III Secretion System Effectors investigated.

**TABLE S2** | Representation of an alignment of ortholog genes having as anchor the Xac306 genome.

**TABLE S3** | Results of biochemical assays related to xanthan gum and biofilm production, and aggregation (left-had side).
REFERENCES

Abu Khweek, A., Fetherston, J. D., and Perry, R. D. (2010). Analysis of HmsH and its role in plague biofilm formation. *Microbiology* 156, 1424–1438. doi: 10.1099/mic.0.036640-0

Alamuri, P., Lower, M., Hiss, J. A., Himpsl, S. D., Schneider, G., and Mobley, H. L. T. (2010). Adhesion, invasion, and agglutination mediated by two trimeric autotransporters in the human uropathogen *Proteus mirabilis*. *Infect. Immun.* 78, 4882–4894. doi: 10.1128/IAI.00718-10

An, S. Q., Febrer, M., McCarthy, Y., Tang, D. J., Clissold, L., Kaithakottil, G., et al. (2013). High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis. *Mol. Microbiol.* 88, 1058–1069. doi: 10.1111/mmi. 12229

Antipov, D., Hartwick, N., Shen, M., Raiko, M., Lapidus, A., and Pevzner, P. A. (2016). plasmidSPAdes: assembling plasmids from whole genome sequencing data. *Bioinformatics* 32, 3380–3387.

Baker, N. R., and Svanborg-Eden, C. (1989). Role of alginate in the adherence of *Pseudomonas aeruginosa*. *Antibiot. Chemother.* 42, 72–79. doi: 10.1159/ 000417607

Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., et al. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. *J. Comput. Biol.* 19, 455–477. doi: 10.1089/cmb.2012. 0021

Becker, S., Soares, C., and Porto, L. M. (2009). Computational analysis suggests that virulence of *Chromobacterium violaceum* might be linked to biofilm formation and poly-NAG biosynthesis. *Genet. Mol. Biol.* 32, 640–644. doi: 10.1590/S1415- 47572009000300031

Bodnar, A. M., Santillana, G., Mavrodieva, V. A., Liu, Z., Nakhla, M. K., and Gabriel, D. W. (2017). Three complete genome sequences of novel *Xanthomonas citri* strains from Texas carry atypical PthA alleles and unusual large plasmids. *Phytopathology* 107:11.

Boyd, A., and Chakrabarty, A. M. (1994). Role of alginate lyase in cell detachment of *Pseudomonas aeruginosa*. *Appl. Environ. Microbiol.* 60, 2355–2359.

Bui Thi Ngoc, L., Vernière, C., Jouen, E., Ah-You, N., Lefeuvre, P., Chiroleu, F., et al. (2010). Amplified fragment length polymorphism and multilocus sequence analysis-based genotypic relatedness among pathogenic variants of *Xanthomonas citri* pv. citri and *Xanthomonas campestris* pv. *bilvae*. *Int. J. Syst. Evol. Microbiol.* 60, 515–525. doi: 10.1099/ijs.0.009514-0

CAB-International, (2019). *Xanthomonas citri* (*citrus canker*). Wallingford: Invasive Species Compendium.

Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al. (2009). BLAST+: architecture and applications. *BMC Bioinformatics* 10:421. doi: 10.1186/1471-2105-10-421

Caserta, R., Takita, M. A., Targon, M. L., Rosselli-Murai, L. K., de Souza, A. P., Peroni, L., et al. (2010). Expression of *Xylella fastidiosa* fimbrial and afimbrial proteins during biofilm formation. *Appl. Environ. Microbiol.* 76, 4250–4259. doi: 10.1128/AEM.02114-09

Chiesa, M. A., Siciliano, M. F., Ornella, L., Roeschlin, R. A., Favaro, M. A., Delgado, N. P., et al. (2013). Characterization of a variant of *Xanthomonas citri* subsp citri that triggers a host-specific defense response. *Phytopathology* 103, 555–564. doi: 10.1094/PHYTO-11-12-0287-R

Civerolo, E. L. (1984). Bacterial canker disease of citrus. *J. Rio Grande Valley Hortic. Soc.* 37, 127–145.

Constantin, E. C., Cleenwerck, I., Maes, M., Baeyen, S., Van Malderghem, C., De Vos, P., et al. (2016). Genetic characterization of strains named as *Xanthomonas axonopodis* pv. dieffenbachiae leads to a taxonomic revision of the X-axonopodis species complex. *Plant Pathol.* 65, 792–806.

Contreras-Moreira, B., and Vinuesa, P. (2013). GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis. *Appl. Environ. Microbiol.* 79, 7696–7701. doi: 10.1128/AEM.02411-13

da Silva, A. C., Ferro, J. A., Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B., et al. (2002). Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. *Nature* 417, 459–463.

Darbari, V. C., and Waksman, G. (2015). Structural biology of bacterial Type IV secretion systems. *Annu. Rev. Biochem.* 84, 603–629. doi: 10.1146/annurev- biochem-062911-102821

Davies, D. G., and Geesey, G. G. (1995). Regulation of the alginate biosynthesis gene algC in *pseudomonas*-aeruginosa during biofilm development in continuous-culture. *Appl. Environ. Microb.* 61, 860–867.

Dunger, G., Guzzo, C. R., Andrade, M. O., Jones, J. B., and Farah, C. S. (2014). *Xanthomonas citri* subsp citri Type IV pilus is required for twitching motility, biofilm development, and adherence. *Mol. Plant Microbe Int.* 27, 1132–1147. doi: 10.1094/MPMI-06-14-0184-R

FAS, (2019). *Citrus: World Market and Trade*. Washington, DC: Foreign Agricultural Service.

Giltner, C. L., Habash, M., and Burrows, L. L. (2010). *Pseudomonas aeruginosa* minor pilils are incorporated into type IV pili. *J. Mol. Biol.* 398, 444–461. doi: 10.1016/j.jmb.2010.03.028

Goto, M. (1992). "Citrus canker," in *Plant Diseases of International Importance*, Vol III, eds J. Kumer, et al. (Upper Saddle River NJ: Prentice Hall).

Gottig, N., Garavaglia, B. S., Garofalo, C. G., Orellano, E. G., and Ottado, J. (2009). A filamentous hemagglutinin-like protein of Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker, is involved in bacterial virulence. *PLoS One* 4:e4358. doi: 10.1371/journal.pone.0004358

Hoang, V., Medrek, S. K., Pendurthi, M., Kao, C., and Parulekar, A. D. (2017). Improving donor lung management and recipient selection in lung transplantation. *Am. J. Respir. Crit. Care Med.* 196, 782–784.

Jaciani, F. J. (2012). *Diversidade Genética de Xanthomonas citri* subsp. *Citri*, Caracterização biomolecular e patogênica. (de)Xanthomonas fuscans subsp. aurantifolii e detecção de Xanthomonas alfalfae em. (citrumelo)'Swingle' (*Citrus paradisi* Macf. x Poncirus trifoliataL. Raf. (.)no Brasil. Ph.D thesis, Universidade Estadual Paulista Julio de Mesquita Filho, Jaboticabal, SP, Brazil.

Jaciani, F. J., Destefano, S. A. L., Neto, J. R., and Belasque, J. (2009). Detection of a new bacterium related to Xanthomonas fuscans subsp aurantifolii Infecting Swingle Citrumelo in Brazil. *Plant Dis.* 93, 1074–1074. doi: 10.1094/PDIS-93- 10-1074B

Jacob, T. R., de Laia, M. L., Moreira, L. M., Goncalves, J. F., Carvalho, F. M., Ferro, M. I., et al. (2014). Type IV secretion system is not involved in infection process in citri. *Int. J. Microbiol.* 2014:763575. doi: 10.1155/2014/763575

Jalan, N., Kumar, D., Andrade, M. O., Yu, F., Jones, J. B., Graham, J. H., et al. (2013). Comparative genomic and transcriptome analyses of pathotypes of *Xanthomonas citri* subsp. citri provide insights into mechanisms of bacterial virulence and host range. *BMC Genomics* 14:551. doi: 10.1186/1471-2164-14- 551

Karp, P. D., Billington, R., Caspi, R., Fulcher, C. A., Latendresse, M., Kothari, A., et al. (2017). The BioCyc collection of microbial genomes and metabolic pathways. *Brief. Bioinform.* doi: 10.1093/bib/bbx085 [Epub ahead of print].

Katoh, K., and Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Mol. Biol. Evol.* 30, 772–780. doi: 10.1093/molbev/mst010

Koronakis, V., Eswaran, J., and Hughes, C. (2004). Structure and function of TolC: the bacterial exit duct for proteins and drugs. *Annu. Rev. Biochem.* 73, 467–489. doi: 10.1146/annurev.biochem.73.011303.074104

Kuchma, S. L., Griffin, E. F., and O'Toole, G. A. (2012). Minor pilils of the type IV pilus system participate in the negative regulation of swarming motility. *J. Bacteriol.* 194, 5388–5403. doi: 10.1128/JB.00899-12

Kück, P., and Meusemann, K. (2010). FASconCAT: convenient handling of data matrices. *Mol. Phylogenet. Evol.* 56, 1115–1118. doi: 10.1016/j.ympev.2010.04. 024

Larsson, A. (2014). AliView: a fast and lightweight alignment viewer and editor for large datasets. *Bioinformatics* 30, 3276–3278. doi: 10.1093/bioinformatics/ btu531

Leneveu-Jenvrin, C., Connil, N., Bouffartigues, E., Papadopoulos, V., Feuilloley, M. G., and Chevalier, S. (2014). Structure-to-function relationships of bacterial translocator protein (TSPO): a focus on *Pseudomonas*. *Front. Microbiol.* 5:631. doi: 10.3389/fmicb.2014.00631

Li, L., Stoeckert, C. J., and Roos, D. S. (2003). OrthoMCL: identification of ortholog groups for eukaryotic genomes. *Genome Res.* 13, 2178–2189. doi: 10.1101/gr. 1224503

Malavolta Júnior, V. A., Yamashiro, T., Nogueira, E. M. C., and Feichtenberger, E. (1984). Distribuição do tipo C de Xanthomonas campestris pv. citri no Estado de São Paulo. *Summa Phytopathol.* 10:11.

Mattick, J. S. (2002). Type IV pili and twitching motility. *Annu. Rev. Microbiol.* 56, 289–314. doi: 10.1146/annurev.micro.56.012302.160938
Mendonça, L. B. P., Zambolim, L., and Badel, J. L. (2017). Bacterial citrus diseases: major threats and recent progress. *J. Bacteriol.* **Mycol.** 5, 340–350.

Mondal, K. K., Verma, G., Junaid, M. A., and Mani, C. (2015). Rice pathogen Xanthomonas oryzae pv. oryzae employs inducible hrp-dependent XopF type III effector protein for its growth, pathogenicity and for suppression of PTI response to induce blight disease. *Eur. J. Plant Pathol.* 144, 311–323. doi: 10.1007/s10658-015-0768-7

Moreira, L. M., Almeida, N. F., Potnis, N., Digiampietri, L. A., Adi, S. S., Bortolossi, J. C., et al. (2010). Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii. *BMC Genomics* 11:238. doi: 10.1186/1471-2164-11-238

Moreira, L. M., Facincani, A. P., Ferreira, C. B., Ferreira, R. M., Ferro, M. I., Gozzo, F. C., et al. (2015). Chemotactic signal transduction and phosphate metabolism as adaptive strategies during citrus canker induction by *Xanthomonas citri*. *Funct. Integr. Genomics* 15, 197–210. doi: 10.1007/s10142-014-0414-z

Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., and Minh, B. Q. (2015). IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. *Mol. Biol. Evol.* 32, 268–274. doi: 10.1093/molbev/msu300

Nikolskaya, A. N., and Galperin, M. Y. (2002). A novel type of conserved DNA-binding domain in the transcriptional regulators of the AlgR/AgrA/LytR family. *Nucleic Acids Res.* 30, 2453–2459. doi: 10.1093/nar/30.11.2453

Okkotsu, Y., Little, A. S., and Schurr, M. J. (2014). The *Pseudomonas aeruginosa* AlgZR two-component system coordinates multiple phenotypes. *Front. Cell Infect. Microbiol.* 4:82. doi: 10.3389/fcimb.2014.00082

Orgad, O., Oren, Y., Walker, S. L., and Herzberg, M. (2011). The role of alginate in *Pseudomonas aeruginosa* EPS adherence, viscoelastic properties and cell attachment. *Biofouling* 27, 787–798. doi: 10.1080/08927014.2011.603145

O'Toole, G. A. (2011). Microtiter dish biofilm formation assay. *J. Vis. Exp.* 47:2437. doi: 10.3791/2437

Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P., and Tyson, G. W. (2015). CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. *Genome Res.* 25, 1043–1055. doi: 10.1101/gr.186072.114

Peabody, C. R., Chung, Y. J., Yen, M. R., Vidal-Ingigliardi, D., Pugsley, A. P., and Saier, M. H. Jr. (2003). Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. *Microbiology* 149, 3051–3072. doi: 10.1099/mic.0.26364-0

Pegos, V. R., Nascimento, J. F., Sobreira, T. J., Pauletti, B. A., Paes-Leme, A., and Balan, A. (2014). Phosphate regulated proteins of Xanthomonas citri subsp. citri: a proteomic approach. *J. Proteomics* 108, 78–88. doi: 10.1016/j.jprotot.2014.05.005

Petrocelli, S., Arana, M. R., Cabrini, M. N., Casabuono, A. C., Moyano, L., Beltramino, M., et al. (2016). Deletion of pilA, a minor pilin-like gene, from Xanthomonas citri subsp citri influences bacterial physiology and pathogenesis. *Curr. Microbiol.* 73, 904–914. doi: 10.1007/s00284-016-1138-1

Qin, J., Zhou, X., Sun, L., Wang, K., Yang, F., Liao, H., et al. (2018). The Xanthomonas effector XopK harbours E3 ubiquitin-ligase activity that is required for virulence. *New Phytol.* 220, 219–231. doi: 10.1111/nph.15287

Revell, L. J. (2012). phytools: an R package for phylogenetic comparative biology (and other things). *Methods Ecol. Evol.* 3, 217–223. doi: 10.1111/j.2041-210x.2011.00169.x

Ryan, R. P., Vorholter, F. J., Potnis, N., Jones, J. B., Van Sluys, M. A., Bogdanove, A. J., et al. (2011). Pathogenomics of Xanthomonas: understanding bacterium-plant interactions. *Nat. Rev. Microbiol.* 9, 344–355. doi: 10.1038/nrmicro 2558

Schaad, N. W., Postnikova, E., Lacy, G., Sechler, A., Agarkova, I., Stromberg, P. E., et al. (2006). Emended classification of xanthomonad pathogens on citrus. *Syst. Appl. Microbiol.* 29, 690–695. doi: 10.1016/j.syapm.2006.08.001

Schaad, N. W., Postnikova, E., Lacy, G. H., Sechler, A., Agarkova, I., Stromberg, P. E., et al. (2005). Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov. *Syst. Appl. Microbiol.* 28, 494–518. doi: 10.1016/j.syapm.2005.03.017

Schubert, T. S., and Miller, J. W. (1996). *Bacterial Citrus Canker*. Gainesville, FL: Florida Department of Agriculture and Consumer Services, Division of Plant Industry.

Schubert, T. S., Rizvi, S. A., Sun, X. A., Gottwald, T. R., Graham, J. H., and Dixon, W. N. (2001). Meeting the challenge of eradicating citrus canker in Florida - Again. *Plant Dis.* 85, 340–356. doi: 10.1094/pdis.2001.85.4.340

Schulze, S., Kay, S., Buttner, D., Egler, M., Eschen-Lippold, L., Hause, G., et al. (2012). Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity. *New Phytol.* 195, 894–911. doi: 10.1111/j.1469-8137.2012.04210.x

Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. *Bioinformatics* 30, 2068–2069. doi: 10.1093/bioinformatics/btu153

Setubal, J. C., Almeida, N. F., and Wattam, A. R. (2018). Comparative genomics for prokaryotes. *Methods Mol. Biol.* 1704, 55–78. doi: 10.1007/978-1-4939-7463-4-3

Shavit, R., Lebendiker, M., Pasternak, Z., Burdman, S., and Helman, Y. (2015). The vapB-vapC operon of acidovorax citrulli functions as a bona-fide toxin-antitoxin module. *Front. Microbiol.* 6:1499. doi: 10.3389/fmicb.2015.01499

Snel, B., Lehmann, G., Bork, P., and Huynen, M. A. (2000). STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. *Nucleic Acids Res.* 28, 3442–3444. doi: 10.1093/nar/28.18.3442

Souza, D. P., Oka, G. U., Alvarez-Martinez, C. E., Bisson-Filho, A. W., Dunger, G., Hobeika, L., et al. (2015). Bacterial killing via a type IV secretion system. *Nat. Commun.* 6:453. doi: 10.1038/ncomms7453

Thomas, S., Holland, I. B., and Schmitt, L. (2014). The Type I secretion pathway - the hemolysin system and beyond. *Biochim. Biophys. Acta* 1843, 1629–1641. doi: 10.1016/j.bbamcr.2013.09.017

Voegel, T. M., Warren, J. G., Matsumoto, A., Igo, M. M., and Kirkpatrick, B. C. (2010). Localization and characterization of *Xylella fastidiosa* haemagglutinin adhesins. *Microbiology* 156, 2172–2179. doi: 10.1099/mic.0.037564-0

Vojnov, A. A., Slater, H., Daniels, M. J., and Dow, J. M. (2001). Expression of the gum operon directing xanthan biosynthesis in *Xanthomonas campestris* and its regulation in planta. *Mol. Plant Microbe Interact.* 14, 768–774. doi: 10.1094/mpmi.2001.14.6.768

Wang, J., Yan, Q., and Wang, N. (2012). "hmsF Is a virulence factor of the citrus canker pathogen *Xanthomonas citri* subsp. citri 306," in *Proceedings of the 2012 APS annual meeting*, (Providebce, RI: APS), 546.

Wang, L. F., Rong, W., and He, C. Z. (2008). Two Xanthomonas extracellular polygalacturonases, PghAxc and PghBxc, are regulated by type III secretion regulators HrpX and HrpG and are required for virulence. *Mol. Plant Microbe Int.* 21, 555–563. doi: 10.1094/MPMI-21-5.0555

Wang, N., Pierson, E. A., Setubal, J. C., Xu, J., Levy, J. G., Zhang, Y., et al. (2017). The candidatus liberibacter-host interface: insights into pathogenesis mechanisms and disease control. *Annu. Rev. Phytopathol.* 55, 451–482. doi: 10.1146/annurev-phyto-080516-035513

Wang, X., Preston, J. F. III, and Romeo, T. (2004). The pgaABCD locus of *Escherichia coli* promotes the synthesis of a polysaccharide adhesin required for biofilm formation. *J. Bacteriol.* 186, 2724–2734. doi: 10.1128/jb.186.9.2724-2734.2004

Wulff, N. A., Zhang, S., Setubal, J. C., Almeida, N. F., Martins, E. C., Harakava, R., et al. (2014). The complete genome sequence of 'Candidatus Liberibacter americanus', associated with *Citrus huanglongbing*. *Mol. Plant Microbe Interact.* 27, 163–176. doi: 10.1094/MPMI-09-13-0292-R

Yang, Y. C., Chou, C. P., Kuo, T. T., Lin, S. H., and Yang, M. K. (2004). PiR enhances the sensitivity of Xanthomonas axonopodis pv. citri to the infection of filamentous bacteriophage Cf. *Curr. Microbiol.* 48, 251–261. doi: 10.1007/s00284-003-4191-5

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Fonseca, Patané, Varani, Felestrino, Caneschi, Sanchez, Cordeiro, Lemes, Assis, Garcia, Belasque, Martins, Facincani, Ferreira, Jaciani, Almeida, Ferro, Moreira and Setubal. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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