A metadata analysis of walnut associated Xanthomonas spp.: from population diversity to comparative genomics Camila Borges Fernandes Doutoramento em Biologia D Departamento de Biologia 2019 Orientador Fernando Manuel dos Santos Tavares, Professor Auxiliar Faculdade de Ciências, Universidade do Porto (FCUP). Coorientador Maria Leonor Pato da Cruz,Investigadora Auxiliar Instituto Nacional de Investigação Agrária e Veterinária, I. P. (INIAV). A metadata analysis of walnut associated Xanthomonas spp.: from population diversity to comparative genomics O trabalho apresentado nesta dissertação foi realizado no Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório associado (CiBIO-InBIO) e no Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), no âmbito da bolsa de doutoramento (SFRH/BD/95913/2013) financiada pela Fundação para a Ciência e a Tecnologia (FCT) com o projeto de doutoramento intitulado, “Xanthomonas arboricola pv. juglandis – unveiling the biogeographic distribution of emergent and acutely virulent Xaj genotypes to infer epidemiological patterns and improve phytosanitary control”. A metadata analysis of walnut associated Xanthomonas spp.: from population diversity to comparative genomics Os trabalhos apresentados também tiveram apoio dos projetos de investigação: - EVOXANT (PTDC/BIA-EVF/3635/2014, POCI-01-0145-FEDER-016600), financiado pelo European Structural & Investment Funds (ESIFs) através da Autoridade de Gestão do Programa Operacional Competitividade e Internacionalização (COMPETE 2020) e pela Fundação para a Ciência e a Tecnologia (FCT). - European Cooperation in Science and Technology (COST) action CA16107 EuroXanth – “Integrating science on Xanthomonadaceae for integrated plant disease management in Europe”. FCUP vii Acknowledgments Four years have passed since I started my PhD and hereby I would like to express my gratitude to all the people that contributed to this dissertation and made me a better scientist and a better person throughout these years: To my PhD supervisor, Professor Fernando Tavares, thank you for believing in me since the beginning, when we met at the interview for a fellowship. For always inspiring me, believing in the work we set out to accomplish and encouraging me to challenge myself. Thank you to open your lab for me, but most of all, to opening your home and always, together with Perpétua and Antónia, making me feel welcome! It has been an honour to be your PhD student and I hope our paths continue to cross. To my co-supervisor, Eng. Leonor Cruz, thank you for accepting me in your lab, where you generously received me with open arms and allowed me to perform a substantial amount of my work. For your help and expertise in phytopathology that were essential to my professional progress. Thank you to share your knowledge with me, for your constant availability and support in the last five years. To Dr. Pedro Albuquerque, for me my second co-supervisor, thank you for all your scientific explanations, leadership in the lab, for being a brilliant person and for teaching me a lot! Thank you for your availability since the beginning and for making sure you would be always present! To Dr. Paula Sá Pereira, thank you for sharing with me your enthusiasm for science! Thank you for your scientific discussions and helpful suggestions during my thesis. Thank you for cheer me up through the hard times and encouraging words. To my colleagues that turned friends for life, Ana Chegão, Joana Cruz and Márcia Santos, thank you for your continued support and encouragement through all these years. Thanks for your advices in all matters of life! To all the members of Microbial Diversity and Evolution group, to Cláudia Serra, Cristina Dias, Leonor Martins and Sofia Martins, thank you for all the support during my stay at Porto and friendship. A special acknowledgement is due to Leonor Martins for her important contributions to this thesis regarding the work developed in Chapter III and Chapter V. viii FCUP To Eng. Rui de Sousa, co-author in joint publications, thank you for your expertise that were essential to this work and support during surveys on walnut orchards. To Dr. Jochen Blom and Dr. Joël Pothier, both co-authors in joint publications, thank you for allowing us to take our work one step further. I would like to thank specially Dr. Joël Pothier for welcoming me in your lab in Switzerland and for your guidance in the results presented in Chapter V. To the team of the Seed Lab (INIAV), Eng. Lurdes Santos and Eng. Paulo Godinho, thank you for the work performed on walnut seedlings (Chapter IV), to Eng. Joaquim Trindade for your kind collaboration in taking the photos of pathogenicity results (Chapter IV and VI) and to all the walnut producers, thank you for the valuable collaborations. À Isaura Velez, obrigada por ser a nossa Isaura, que sempre está pronta a ajudar e a defender as suas meninas. Obrigada por toda a sua amizade e os momentos de descontração que partilhamos! À minha família maluca e a todos os meus amigos, obrigada pelo carinho constante. Aos meus primos, obrigada pelas nossas jurucas que me ajudaram a descontrair e pela vossa amizade de irmãos! #reisdasjurucas Aos meus queridos pais, obrigada por sempre acreditarem em mim e por apoiarem todas as minhas decisões e os meus sonhos académicos. Espero poder um dia retribuir tudo aquilo que fizeram por mim. Considero-me uma pessoa muito feliz e devo tudo o que sou a vocês! Amo- vos muito! Às minhas manas, nani e pilili, obrigada por serem minhas amigas e pela nossa união eterna. A mana mais velha estará sempre aqui para vocês! Amo-vos muito! Ao meu yuyu, obrigada por teres mudado a minha forma de estar na vida e por me teres tornado numa pessoa mais afetuosa. Ao meu marido, meu melhor amigo e bioquímico de todas as horas, Marco obrigada por todo o teu apoio e por estar sempre ao meu lado! Agradeço a tua compreensão e paciência nos momentos em que estive mais distante. A nossa vida ainda agora começou e acredito que seremos muito felizes! Te amo! ♪ “I found a love for me Darling just dive right in, And follow my lead…♫” This thesis is dedicated to my parents, my sisters, my husband and my dog. Thank you for everything, you are my reason for living. FCUP xi Abstract Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of the most damaging and widespread diseases affecting walnut trees-growing areas, namely walnut bacterial blight (WBB), brown apical necrosis (BAN) and vertical oozing canker (VOC). These diseases are responsible for major economic losses in the main walnut production regions, associated with the reduction of marketability of walnut fruits, the reduction of walnut orchard productivity and the increase in walnut nursery costs. Regardless the research efforts carried out in the last decade, there is still a poor understanding of Xaj biology, including pathogen diversity, population dynamics and adaptation fitness to host and environment. In a phytodiagnostics setting, detection of Xaj is still carried out mainly via culture-based methods, with no specific diagnostic tools available. Molecular identification of Xaj strains is limited to the species due to difficulties in finding reliable discriminatory genetic regions at pathovar level. Furthermore, the comprehensive assessment of genetic diversity within and between Xaj population is still missing, which delays the development of advanced tools for identification of particularly virulent strains, as well as the implementation of efficient control and prevention measures for Xaj. In this work, the selection and validation of nine taxa-specific DNA markers for Xaj (XAJ1 to XAJ9) was carried out using in silico approaches. These markers were shown to be useful for both hybridization and PCR-based detection and identification techniques. A multiplex PCR was validated as an efficient culture-independent method for detection of Xaj in naturally infected walnut samples, and a qPCR assay was optimized to determine the bacterial load of infected walnut tissues. Furthermore, a high throughput dot blot platform using the nine DNA markers allowed prompt identification of different Xaj lineages. Genetic diversity of Xanthomonas isolates obtained from walnut trees in Portugal was assessed, considering thermoclimatic and ecological variables thought to modulate the diseases caused by Xaj. The walnut-associated xanthomonads population observed to occur in Portugal was more complex than originally thought, including lineages of Xaj, lineages of non-juglandis X. arboricola and lineages of non-arboricola Xanthomonas sp. Among Xanthomonas sp. lineages, the evidence of a pathogenic strain diverging from the nonpathogenic phenotype and commensal lifestyle to which these atypical strains have been described was an interesting finding as a promising model to address pathoadaptations to walnut. Additionally, this work showed that co- colonization of the same plant host by distinct Xanthomonas strains is frequent, overcoming the phytopathology paradigm of a single pathogenic strain for each diseased plant. Moreover, the isolation of new X. arboricola strains from a new host species, Carya illinoinensis of the xii FCUP Juglandaceae family, and shown to be pathogenic in walnut, was particularly exciting, since calls for the need to review the host range of walnut infective Xanthomonas arboricola. The comparative genomics study carried out allowed to highlight the genomic differences between five Xanthomonas strains (CPBF 367, CPBF 424, CPBF 426, CPBF 427 and CPBF 1521) isolated from a single walnut host. Core genome phylogeny and average nucleotide identity of genomes of Xanthomonas spp. showed two distinct clusters regarding the five strains, one grouping CPBF 427 and CPBF 1521 with Xaj strains, and another grouping together CPBF367, CPBF424 and CPBF426, indicating that these strains belong to a new Xanthomonas species. The unfolded genomic differences between the two groups, could translate into different pathogenicity and virulence features that are still being addressed. Major differences regarding the repertoire of type III secretion system and associated effectors genes were observed among Xaj and Xanthomonas sp.