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Knowledge of the host range of a biocontrol agent (BCA) is fundamental. Host range determines the BCA's economic potential, as well as the possible risk for non-target organisms. Entomopathogenic fungal strains belonging to the genus Beauveria are widely used as BCA, but our knowledge of their physiological host range is only partial. The aim of this study was to improve our understanding of the physiological host range of three Beauveria strains belonging to two species, B. hoplocheli and B. bassiana. We performed laboratory mortality bioassays to assess their pathogenicity and virulence against nine insect pests, belonging to three orders: Lepidoptera, Coleoptera and Diptera. Mortality rate, mean survival time and mycosis rate were used to estimate virulence. Pathogenicity was assessed as the capacity to cause a disease and induce mortality. Virulence was assessed as the severity of the disease based on mortality rate, mean survival time and mycosis rate. The results of this study revealed significant differences in the physiological host range of the three Beauveria strains tested. The three strains were pathogenic to all Diptera and Lepidoptera species tested. In the case of the Coleoptera, only the B. hoplocheli strain was pathogenic to the white grub Hoplochelus marginalis and only the B. bassiana strains were pathogenic to Alphitobius diaperinus. The B. hoplocheli strain was less virulent on Lepidoptera and Diptera than the two B. bassiana strains. The latter both exhibited very similar virulence patterns. The fact that B. hoplocheli and B. bassiana strains have different host ranges means that they can be used as BCA to target different pests. Impacts on non-target insects across multiple orders cannot be ruled out in the absence of ecological host range studies.

Xanthomonas citri pv. citri IXcc) and X. citri pv. aurantifolii (Xca), are causal agents of Citrus Bacterial Canker ICBC) a severe disease that requires costly control measures and primarily causes defoliation and fruit blemish and drop. Xcc and Xca are quarantine pathogens for UE, net known to occur in the Mediterranean region. The probability of their entry, via import of ornamental rutaceous plants, through bath the commercial trade and passenger pathways, is rated as likely by EFSA (2014). To provide useful information for the pest risk assessment, 25 ornamental rutaceous plants in the genera Atalantia, Balsamocitrus, Clausena, Eremocitrus, Glycosmis, Melicope, Microcitrus, Murraya, Vespris, not covered by Directive 2000/ 29EC, as well as Citrus and Fortune/la, were tested for resistance to Xcc (pathotype A, A*, Aw) and Xca (pathotype Band C) strains in controlled environment by a detached leaf assay. Nine plant species were presumptively classified as non-host, among them Murraya paniculata. Only M. ovatifoliolata and Eremocitrus glauca were susceptible to all pathotypes. The remaining species were susceptible at least to one of the pathotype A strain. Bacterial population densities ranged from 103 to 106 cfu mL-1 and 107 to 109 cfu mL-1 in plants showing HR or no response and typical CBC lesions, respectively. Crystal violet staining showed aggregation of citrus canker strains on M. paniculata leaf similar to that on citrus species but different to that found for a non-citrus Xanthomonas. Moreover, a de novo sequencing of M. paniculata genome, already completed, will serve for RNAseq studies on bath Murraya species.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most destructive plant diseases worldwide. RSSC affects a wide range of host species and includes several ecotypes that represent major constraints and are under strict regulation (i.e brown rot or Moka strains). The reliable characterization of epidemiological strains at the ecotype level is a challenge considering such a complexity and is generally achieved by combining several diagnostic protocols. Therefore, we used microarray technology (ArrayTube) to develop a standard protocol that performs in a single reaction a multiplex characterization of RSSC strains, from the phylotype to the ecotype level (17 targeted groups of interest). Based on 27 sequenced genomes of RSSC, probes were designed with a 50-mer length constraint and thoroughly evaluated for any flaws or secondary structures. Validation data performed on target (n=75) and non-target strains (n=12) showed strong intra-repeatability, inter-repeatability, and reproducibility, as well as good specificity, which allowed for the proper detection of the 17 groups of interest (Cellier et al, 2017). This custom microarray represents a significant improvement in the epidemiological monitoring of Rssc strains worldwide, and it has the potential to provide insights for phylogenetic incongruence of Rssc strains based on the host of isolation and may be used to indicate potentially emergent strains.

International audience; The Rssc encompasses strains that are highly destructive worldwide causing bacterial wilt (BW) on a wide plant host range. BW was alread y described in Madagascar (phylotype I and III) but potato production was hampered by severe outbreaks. Controlling this disease is crucial for Malagasy potato producers. Knowledge of the genetic diversity and population structure of the pathogen was inves tigated, in fine to evaluate resistance of different potato cultivars. We characterized a large collection of strains (n=1224; 74 sites) surveyed from potato areas in 2013, by using different molecular tools. Phylotypes I, II and III were identified and most potato outbreaks were shown to be associated with brown rot strains IIB - 1, whereas members of phylotype I and III caused few. Along with reference strains, epidemic IIB - 1 strains (n=290) were genotyped through a MLVA scheme ( RS2 - MLVA10), which showed a single clonal complex distributed in wide agro - ecological areas, and genetically related to worldwide genotypes. This suggests this IIB - 1 lineage is busting potato production in Madagascar after being introduced (not cryptic) and spread through release of infected potato - seeds. To assess the population structure of long time recognized phylotype III strains, 251 strains were analyzed through the newly developed and highly discriminatory typing scheme RS3 - MLVA16. Malagasy population of phylotype III strains showed a broad genetic basis made of 132 haplotypes in at least 7 clonal complexes; and furthermore distinct from African strains, suggesting its endemic character. At an agro - ecological scale, two major clonal complexes were unified by haplotypes shared between individual areas and the remainder made of haplotypes derived from a unique genotype. This large - scale epidemiological study and the generated sets of data are reference models to further studies of Rssc population in Madagascar and the SWIO* , including evaluation of pathogen aggressiveness and host resistance. (Résumé d'auteur)

Ralstonia solanocearum is a species complex (Rssc), distributed worldwide and economically destructive. This soilborne bacterial plant pathogen encompasses an unusually wide host range. The pathogen invades the roots and coloniz.es the xylem vessels causing bacterial wilt (BW) diseases. Four phylogenetic groups called phylotypes structured the Rssc that relates to their geographic origin of evolution: phylotype I (Asia), phylotype II (America), phylotype III (Africa), and phylotype IV (Indonesia-Japan). In the South-West Indian Ocean (SWIO) islands, although R solanocearum is regularly reported, no recent data is available about its genetic diversity and structure. Our main objectives are then to get a significant collection of R.solanacearum strains to thoroughly analyze the genetic diversity of the strains; characterize the evolutionary forces that shape populations; and evaluate genetic resources for resistance to BW. Presently, 2971 R.solanocearum strains have been collected mainly from Solanaceae: Madagscar* (n=1224), Reunion (n=F789), Mauritius (n=693), Mayotte (n=166), Seychelles (n=92), and Comoros (n=5, to be completed). A preliminary broad molecular typing was done using the multiplex PCR (Fegan and Prior 2005)**, showing 46.6% of phylotype I, 42.3% of phylotype II, 11% of phylotype III, and 0.1% of phylotype IV. A selection of 126 strains that covered the geographic and host diversity was organized to assess phylogenetic relationships. Maximum Likelihood tree (PhyML) was performed on the partial endoglucanase (egl) amplification, along with a set of 1 OO international reference strains. Further characterizations are ongoing through a MLSA scheme based on partial amplification of 6 genes; gdhA, mutS, adK, leuS, rplB and gyrB. This is the first report of a large scale survey in the SWIO. Surprisingly we showed that phylotype I-31 is mainly represented and found in every prospected territory in the SWIO. Also Phylotype IV was first described in Mauritius.

Ralstonia solanacearum is a species complex (Rssc), distributed worldwide and economically destructive. This soilborne bacterial plant pathogen encompasses an unusually wide hast range. The pathogen invades the roots and colonizes the xylem vessels causing bacterial wilt (BW) diseases. Strains distributed into four phylogenetic groups, based on ITS sequences called phylotypes that relates to geographical origin: phylotype 1 (Asia), phylotype Il (America), phylotype Ill (Africa), and phylotype IV (lndonesia-Japan). ln the South-West lndian Ocean (SWIO) islands, although R. solanacearum is regularly reported, no recent data is available about its genetic diversity and structure. Our main objectives are then to ge a significant collection of R. solanacearum strains to thoroughly analyze this genetic diversity; characterize the evolutionary forces that shape populations; and evaluate genetic resources for resistance to BW. 2971 R. solanacearum strains have been collected mainly from Solanaceae: Madagascar* (n=1224), Reunion (n=789), Mauritius (n=785), Mayotte (n=166), Seychelles (n=92), Rodrigues (n=Sl) and Comoros (n=S, to be completed). A preliminary broad molecular typing was done using the multiplex PCR (Fegan and Prier, 2005) showing 49.5% of phylotype I, 40 of phylotype Il, 10.4% of phylotype Ill, and 2 strains of phylotype IV. A selection of 126 strains that covered the geographic and host diversity was organized to assess phylogenetic relationships. Maximum Likelihood tree (PhyML) was performed on th partial endoglucanase (egl) amplification, along with a set of 100 international reference strains. Further characterizations are ongoing through a MLSA/MLST** scheme based on partial amplification of 7 genes; gdhA, mutS, adK, leuS, rplB, gyrB and egl. This is the first report of a large scale survey in the SWIO. lnterestingly we reported here the predominance of one sequevar (1-31) among the SWIO strains whereas the phylotype I is known to encompass a broad genetic basis (16 sequevars) Also sequevar IV-10 was described in Mauritius for the first time.

The Ralstonia solanacearum species complex (Rssc) is a highly diverse cluster of strains, many of which proved destructive worldwide causing bacterial wilt (BW) on a wide plant host range. Potato BW has been described for nearly 30 years in Madagascar highlands (phylotype I and Ill) without major impact on production. Recently, potato production was dramatically devastated by severe outbreaks. Controlling this disease is crucial for Malagasy potato producers and a first important step to achieve this goal is the identification of the strains involved and of their putative origin. Consequently, the genetic diversity and population structure of the Rssc was investigated in the major potato production areas in the highlands. By using different molecular tools we characterized a large collection of strains (n=1224; 75 sites) collected from potato production areas in 2013, April (n=763; 41 sites) and December (n=461; 33 sites). Present for a long time in Madagascar, Phylotypes I and Ill strains were little associated with potato BW outbreaks, while surprisingly, most outbreaks were associated with brown rot strains IIB-1. This constitutes the first report of phylotype IIB-1 strains in Madagascar. Along with reference strains, epidemic IIB-1 strains (n=285) were genotyped through the MLVA RS2-MLVA9 implemented from existing schemes (1, 2]. Malagasy phylotype Il strains separated into two clonal complexes (CC) including one major CCl grouping also most of the worldwide genotypes that distributed in wide agro-ecological areas. Fine scale genetic investigation of the outbreak suggested that the clonal expansion of this IIB- lineage affected potato production in Madagascar after being introduced and massively spread through propagation of latently infected potato-seeds. The survey also allowed the first study about the population structure of phylotype Ill strains. Two hundreds and seventeen phylotype Ill strains were genotyped with the highly discriminatory MLVA scheme RS3-MLVA16 that we newly developed [3]. MLVA distinguished a high genetic diversity in Malagasy population of phylotype Ill strains producing 117 haplotypes grouped in 11 clonal complexes. Malagasy strains were distinct from continental African strains, suggesting little recent immigration from the continent. Many CC a long with the large genetic diversity supported the endemic character of the phylotype Ill population in Madagascar. At an agro-ecological scale, phylotype Ill mostly structured in small clonal complexes associated with a single geographic origin each. But two major CCs grouped some haplotypes shared between individuals from different highland areas, and the remainder made of haplotypes derived from a unique origin supporting long distance dispersal most probably through human activities. Although the most popular Malagasy potato cultivars developed by CIP and FIFAMANOR were apparently developing resistance to phylotype Ill strains in the field, we reported no bacterial wilt resistance properties under artificial environment (growth chambers). As expected, the same potato cultivars were highly susceptible to expanding phylotype IIB-1 lineage.

Xanthomonas axonopodis pv. allii (Xaa) is the causal agent of bacterial blight of onion (BBO), an emerging disease threatening the world onion production, and causing damage to other Alliaceae, including garlic, welsh onion, shallot, chive and leek. Xaa is an EPPO quarantine organism (A1 list i.e. absent from the EPPO region). The international spread of Xaa can probably be explained by its seedborne status (Roumagnac et al., 2000). Consequently it is of crucial importance to validate efficient detection methods which allow the detection of seedborne pathogens for which the level of inoculum can be very low in very large seed lots. We evaluate a detection scheme based on molecular tests (a triplex quantitative real-time PCR assay (Robène et al., 2015) and a duplex nested end-point PCR assay (Robène-Soustrade et al., 2010)) and isolation. The method assessment was performed following the European standard EN ISO 16140 and the EPPO standard PM7/98 (2). An intra-laboratory study was first conducted, where we characterized the analytical specificity (inclusivity and exclusivity), the analytical sensitivity and the repeatability. We finally tested the detection scheme on naturally contaminated onion seed lots showing different contamination rates. In addition to the intralaboratory study, an inter-laboratory reproducibility trial (including five different laboratories) was performed. This detection scheme was shown to be very efficient, combining a maximal inclusivity (100%), a good exclusivity (83%), a high analytical sensitivity (approximately 103 CFU.mL-1), an excellent repeatability (100% for contamination rates higher than 103 CFU.mL-1) and reproducibility (100% for contamination rates higher than 103 CFU.mL-1). These results allowed to validate the detection scheme as official method of analysis for the French officially approved laboratories (MA 038), and to propose it as an EPPO diagnostic protocol for the detection of this emerging pathogen. (Texte integral)

La présente invention se rapporte à de nouveaux outils de détection de Xanthomonas axonopodis pv. allii, en particulier la détection moléculaire de séquences polynucléotidiques spécifiques de cette souche.

14 Pág. (This article belongs to the Special Issue Integrating Science on Xanthomonas and Xylella for Integrated Plant Disease Management), Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host–pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen., This research was funded ORPRAMed Project on “Risk assessment of introduction of Xanthomonas citri subsp. citri through commercial trade of ornamental rutaceous plants in the Mediterranean basin”, ERA-NET ARIMNET 2-Call 2015.C.B., V.C. acknowledge the University of Catania for support under the projects “Investigation of phytopathological problems of the main Sicilian productive contexts and eco-sustainable defense strategies” (PIA.CE.RI. 2020–2022 Linea 2-Research Project MEDIT-ECO.); “Emergent Pests and Pathogens and Relative Sustainable Strategies” (Progetto di ricerca di Ateneo 2018-2020). O.P. and I.R. acknowledge the European Union (ERDF, EAFRD) and Réunion regional council for support