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30 Pág. Departamento de ​Protección Vegetal (INIA), Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis., J.C. (Joana Costa) acknowledges R&D Unit Center for Functional Ecology-Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC). V.C. acknowledges support of the University of Catania PIA.CE.RI. 2020–2022 Linea 2-Research Project MEDIT-ECO. J.C. acknowledges support from AEI by RTI2018-096018-R-C31 and FEDER. O.P. and I.R. acknowledge the European Union (ERDF, EAFRD) and Réunion regional council for support. J.F.P. acknowledges support from the Department of Life Sciences and Facility Management of the Zurich University of Applied Sciences (ZHAW) in Wädenswil. D.J.S. and J.G.V. also acknowledge joint funding by a grant from BBSRC, NERC, Defra, and the Scottish Government, under the Strategic Priorities Fund Plant Bacterial Diseases programme under the project ‘Xanthomonas plants diseases: mitigating existing, emerging and future threats to UK agriculture’ (BB/T010916/1 and BB/T010908/1). The APC was funded by COST (European Cooperation in Science and technology).

A thorough knowledge of genotypic and phenotypic variations (e.g., virulence, resistance to antimicrobial compounds) in bacteria causing plant disease outbreaks is key for optimizing disease surveillance and management. Using a comprehensive strain collection, tandem repeat-based genotyping techniques and pathogenicity assays, we characterized the diversity of X. citri pv. citri from the South West Indian Ocean (SWIO) region. Most strains belonged to the prevalent lineage 1 pathotype A that has a wide host range among rutaceous species. We report the first occurrence of genetically unrelated, nonepidemic lineage 4 pathotype A* (strains with a host range restricted to Mexican lime and related species) in Mauritius, Moheli and Réunion. Microsatellite data revealed that strains from the Seychelles were diverse, grouped in three different clusters not detected in the Comoros and the Mascarenes. Pathogenicity data suggested a higher aggressiveness of strains of one of these clusters on citron (Citrus medica). With the noticeable exception of the Comoros, there was no sign of recent interisland movement of the pathogen. Consistent with this finding, the copL gene, a marker for the plasmid-borne copLAB copper resistance that was recently identified in Réunion, was not detected in 568 strains from any islands in the SWIO region apart from Réunion.

National audience; Ralstonia solanacearum species complex (RSSC) is one of the most devastating bacterial plant pathogens causing bacterial wilt in a wide range of hosts including crops and wild plants. These soil-borne Gram-negative bacteria have a worldwide geographic distribution and significantly reduce crop yields especially in tropical regions such as the southwestern Indian Ocean. Due to the lack of efficient curative measures and the high persistence of RSSC, phage therapy represents a sustainable plant disease management strategy to be applied in agricultural settings. However, using phages as a biocontrol tool raises concerns about the potential of phage resistance evolution in plant pathogenic bacterial populations. To explore this question, we first evaluated the in vitro virulence of a local phage collection against the most prevalent strain of RSSC in Reunion Island. Then, we examined the temporal adaptation of the strain to these phages, and its consequences for bacterial fitness. Analysis of the virulence of the 10 phages tested showed that they were all able to effectively reduce bacterial growth. The emergence of bacterial resistance was reported after two or three days. Absence of clear lysis plaques were observed in evolved bacteria not only against the phage encountered but also against the entire phage collection, which included several phage species and genera. These results show high cross-resistance emergence, which resulted in a fitness cost for the bacteria, as measured by a reduction in growth rate and loss of swimming ability for 8 of 10 evolved bacteria exposed to the different phages. These results suggest that phylogenetically distant phages can be rapidly blocked in bacteria. Mechanistically different phages or complementary strategies may be needed to efficiently control phytopathogens. Conversely, fitness costs in bacteria point in the direction of a reduced virulence in planta. Further in vivo and genetic analysis will bring light to these questions.

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International audience; Copper-based compounds are widely used in integrated pest management (1PM) programs aiming at controlling agriculturally important plant bacterial pathogens and the latter have adapted in response to this selective pressure. Copper resistance of Xanthomonas citri pv. citri (Xcc), a major citrus pathogen worldwide causing Asiatic citrus canker, was first observed in Argentina two decades ago and subsequently reported as a coplAB-based, plasmid-encoded system. The emergence of resistant strains has since been reported in Réunion (South West lndian Ocean) and Martinique (Eastern Caribbean Sea). Disease severity was found markedly increased in groves established with susceptible cultivars and infected with copper-resistant Xcc. Using tandem repeat-based genotyping and coplAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of coplAB amplicons. ln order to investigate this pattern more closely, we sequenced six copper-resistant Xcc strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be straindependent with the novel identification in Xcc of copABCD and a eus heavy metal efflux resistancenodulation- division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. The mining of all bacterial genomes available from public databases suggested that the mobile elements containing copper resistance genes and their plasmid environments were primarily detected in the Xanthomonadaceae family.

Cassava is the most important vegetatively propagated food staple crop in Africa. Even if its vegetative propagation through stem cuttings has many advantages, it also represents its Achilles heel. Pathogens are passed from one generation to the next and can easily accumulate, threatening cassava production. Two viral diseases are currently considered to be the greatest global threat to cassava production in Africa. Faced with the gravity of this situation, 43 scientists from African national and regional institutions and international agricultural research centres met in Saint-Pierre, La Réunion Island from 10 to 13 June 2014, to contribute to the surveillance and control of cassava diseases and pests that threaten the food security and livelihoods of millions Africans. The main action was to consolidate and develop a global alliance against cassava diseases in Africa and a strategic action plan with several international initiatives for the coming years. Firstly, the establishment of a Pan-African Cassava Surveillance Network (PACSUN) with an interactive website and an International Cassava Transit Site (ICTS) in La Réunion. Secondly, the development of diagnostic technologies to better identifying viruses, bacteria and vectors, and a comprehensive educational and training plan to support all these activities. (Texte intégral)

Citrus bacterial canker (CBC) is a severe disease affecting citrus worldwide. Mediterranean area if free of CBC, ORPRAMed project aims at assessing the introduction risk of CBC through Citrus rutaceous relatives (CRR), especially those not covered by 2000/29EC Directive. Despite the high agricultural significance of CBC, there are not recent and in depth studies about the interaction between CRR and Xanthomonas citri subsp. citri (Xcc) and Xanthomonas fuscans subsp. aurantifolii (Xfa), causal agents of CBC. The project consortium includes Italian, Spanish, French and Turkish research centres and universities with expertise from agriculture economics to plant pathology and genomics. A selection of CRR will be assayed for the resistance/susceptibly to CBC, moreover rutaceous spedies trade flows will be investigated, as well as the survival mechanisms and fitness on CRR of Xcc and Xfa strains representative of their pathological and genetic diversity. Finally, the genome of Murraya paniculata (sel.2 from Florida Citrus Arboretum, Winter Haven, Florida, USA) will be sequenced as well as transcriptomic analysis performed to highlight the main genes involved in the disease. The project results will contribute to update information on the trade of Rutaceae, acquire new data on the behaviour of ornamental Rutaceae regarding Xcc, expand the knowledge about the epi-endophytic colonization of the pathogen and update the European import-export regulation on plant protection. (Texte intégral)

National audience; Diversity in the South-West Indian Ocean. Surveys, phylotype analysis and phylogenetic tree by egl sequencing.

A team at the 3P Center is developing diagnostic molecular tools for quarantine bacterial and viral plant diseases. The work relies on the abundant and various data obtained from taxonomy, diversity, epidemiology, and genomic studies of the different pathogens, generated by the 3P Center team and collaborators. Several sensitive and specific PCR-based protocols have been developed to detect bacterial and viral pathogens (e.g detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium tissues by nested PCR, (Robène-Soustrade et al., 2006. AEM 72: 1072–1078), detection of X. axonopodis pv. allii in onion seeds by duplex-nested PCR (Robène-Soustrade et al., 2010. AEM 76: 2697– 2703), detection and quantification of a wide range of begomoviruses by five duplex real-time quantitative PCRs (Péréfarres et al., 2011. Virol J 8: 389). These methods are useful diagnostic tools for indexing propagative plant material and for international sanitary surveillance of plant material exchanges. These protocols are intended to be referenced as French official methods and EPPO standards (e.g. X. axonopodis pv. dieffenbachiae). The team has an expertise in comparing and validating different molecular tools (Delcourt et al., 2013. Plant Dis 97: 373–378) and collaborates with the French agency for food, environmental and occupational health safety (ANSES) to validate the different protocols through both intra laboratories and ring tests involving different European laboratories (Chabirand et al., 2014. Plant Pathol 63: 20–30). We are also innovating into new DNAbased diagnostic technologies by developing an efficient and portable microarray technology to detect and identify different pathogenic and/or genetic groups of Ralstonia solanacearum. The team's competencies span development, assessment, and validation of diagnostic tools as well as management of collaborative studies. We can rapidly adapt to new challenges such as the evaluation and improvement of the sanitary situation of cassava in Africa. We propose to assess different existing diagnostic tests and to innovate into new DNA-based diagnostic technologies if necessary, in order to optimize the diagnostic of the main viral and bacterial pathogens of cassava in Africa.

The ancient soil borne plant vascular pathogen Ralstonia solanacearum evolved and adapted to cause severe damage on an unusually wide range of plants. Based on the phylotype classification, it was questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum may be, in an attempt to produce adapted diagnostic tools. A pangenomic microarray was first used to better describe and understand the phylogeny of R. solanacearum, especially three particular ecotypes in the phylotype II: (i) Brown rot strains from sequevars IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from sequevar IIB-4NPB that lack pathogenicity to Cavendish banana but can infect many other plant species; and (iii) Moko disease-causing strains from sequevars IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia. Results revealed a phylogeographic structure within Brown rot strains, allowing European outbreak strains of Andean and African origins to be distinguished, and also showed a close but distinct relationship between Moko ecotype IIB-4 and the emerging IIB-4NPB strains. Based on those results, it is proposed to develop new DNA-based diagnostic tools, including array and routine PCR, to ensure better detection of this complex plant pathogen.

Beauveria brongniartii (Saccardo) Petch epizootics were recorded at two sites in the Dalton area of the Midlands in South Africa on the melolonthid species, Hypopholis sommeri Burmeister (Coleoptera: Scarabaeidae). To identify the disease-causing fungus, 17 different fluorescently-labelled microsatellite PCR primers were used to target 78 isolates of Beauveria spp. DNA. Microsatellite data resolved two distinct clusters of Beauveria isolates which represented the B. bassiana s.s. (Balsamo) Vuillemin and B. brongniartii species groups. Groupings were supported by two gene regions, the nuclear ribosomal ITS and Bloc, of which 38 exemplar Beauveria isolates were represented and sequenced. Microsatellite analyses also showed that B. brongniartii conidia were being cycled from epigeal to subterranean habitats and vice versa in the environment by H. sommeri beetles. This is the first record of this species of fungus causing epizootics on melolonthid larvae and adults of H. sommeri in South Africa.

Special Issue "Integrating Science on Xanthomonas and Xylella for Integrated Plant Disease Management", Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.