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Bacterial blight of carrot (Daucus carota) is caused by Xanthomonas hortorum pv. carotae (4). The pathogen is seed transmitted and carrot seeds can be an important source of primary inoculum (2). A 2008–2009 outbreak of a disease resembling bacterial blight was observed in Mauritius in 10 ha of carrot crops, primarily in humid areas of the island, at an estimated incidence of 10%. Carrot leaves with angular, water-soaked leaf spots that turned necrotic were collected at Plaine Sophie, Mauritius in December 2008. Yellow, Xanthomonas-like colonies were isolated onto KC agar medium (3). MultiLocus sequence analysis (MLSA) with four genes (atpD, dnaK, efp, and gyrB) was performed as described previously (1) on five carrot strains together with two reference strains of X. hortorum pv. carotae (LMG 8643 and LMG 8644). The reference strains were identical. Of the five Mauritius strains, two (LG1-1 and LG1-4) were identical, and most closely related to, but distinct from, the reference strains (genetic distance of 0.02). The other three strains represented two sequence types identified as Xanthomonas sp. based on a phylogenetic tree derived from concatenated sequences, but were not related to any type strain. PCR assays with a 3S primer pair specific for X. hortorum pv. carotae (2) produced an amplicon of approximately 350 bp from isolates LG1-1, LG1-4, and each of the reference strains. A PCR assay with a 9B primer pair (2) yielded an amplicon of 0.9 kb for strains LG1-1, LG1-4, and LMG 8644, whereas LMG 8643 yielded an amplicon of approximately 2.0 kb (2). Foliage of 4-week-old plants (36 plants per strain) of the carrot cv. Senator F1 were spray inoculated with a suspension of each strain using an 18-h culture in sterile 0.01 M tris buffer (pH 7.2) with approximately 1 × 108 CFU/ml. Plants sprayed with tris buffer were used as a negative control treatment. Plants were incubated in a growth chamber at 26 ± 1°C at a relative humidity of 95 ± 5% and a photoperiod of 16 h. Water-soaked lesions that developed into necrotic areas were observed 12 to 15 days after inoculation of LG1-1, LG1-4, and the two reference strains. Bacteria were recovered from lesions onto KC medium (3) 3 weeks after inoculation with mean Xanthomonas populations of at least 1 × 107 CFU/lesion. Colonies with morphology typical of Xanthomonas were recovered and typed using atpD sequencing to fulfill Koch's postulates. Although Xanthomonas-like bacteria were isolated from symptomatic carrot leaves in Mauritius in 1989, the results of that study were not published. To our knowledge, this is the first report of molecular and pathological characterization of this pathogen in carrot crops in Mauritius. References: (1) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (2) X. Q. Meng et al. Plant Dis. 88:1226, 2004. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

Cassava brown streak disease (CBSD) has emerged as the most important viral disease of cassava (Manihot esculenta) in Africa and is a major threat to food security. The expanding CBSD epidemic in East and Central Africa is caused by two…

Pseudocercospora fijiensis, the causal agent of black Sigatoka disease of banana (Musa spp.) (BLSD), is considered to be the major economic threat for banana cultivated for export (de Lapeyre de Bellaire et al., 2010). The disease has a …

Aims: Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin-Cephalexin-Trimethoprime-pivMecillinam 4 (NCTM4) medium for its isolation. Methods and Results: A total of 104 bacterial strains were inoculated onto NCTM4 and on the previously published Cellobiose-Starch (CS) and Esculin-Trehalose (ET) media. The strain collection included: the anthurium blight pathogen, Xanthomonas strains, for which false positive results are known to occur using serological identification-tests; other bacterial pathogens of anthurium; and representatives of bacteria that are commonly present in the anthurium phyllosphere. Media were evaluated following the ISO 16140 protocol for the validation of alternative methods. Conclusion: Growth of the anthurium blight pathogen was better on NCTM4 and ET media than on CS. NCTM4 provided a better repeatability. It also displayed a lower rate of false positive and false negative results when the pathogen was isolated from plant extracts. Significance and Impact of the Study: This study will lead to improved isolation protocols of the anthurium blight in official procedures. NCTM4 medium could also favourably be used in studies, which aim to further understanding of the biology and epidemiology of this pathogen.

International audience; In November 2013, symptoms of yellowing on leaves and brown necrotic rot on tuberous roots were observed on different cassava landraces (Manihot esculenta) in Dembeni on Mayotte Island, a French Overseas Department in the south-west Indian Ocean. The symptoms, similar to the cassava brown streak disease described in East Africa, suggested the possible involvement of cassava brown streak viruses (Mbanzibwa et al., JGV 2011). The expanding cassava brown streak disease epidemic in East Africa is caused by two ipomoviruses, Cassava brown streak virus (CBSV; Monger et al., Arch. Virol.2010) and Ugandan cassava brown streak virus(UCBSV; Winter et al., JGV 2010). To verify the identity of the causal agent, symptom-bearing leaf samples of 17 affected plants were collected in March and June 2014.For the detection of cassava brown streak viruses, a two-step RT-PCR using CBSDDF2 and CBSDDR primers was used (Mbanzibwa et al., JGV 2011). These primers were designed for simultaneous virus-specific detection of CBSV and UCBSV (amplicons of 344 bp and 440 bp, respectively). RT-PCR products of the expected size for UCBSV were obtained for 11 samples. To confirm the diagnosis, the RT-PCR products were directly and bi-directionally sequenced and contigs assembled. Sequences of 376 bp from eight of the 11 samples were obtained which had 99-100% nucleotide identity (EMBL Aaccession Nos. LN611671 to LN611678). The sequences showed the highest nucleotide identity (86%) with isolates of UCBSV from Uganda and Malawi (FN434109; FN433932) and 85% with an isolate of CBSV from Mozambique (FN434436). A phylogenetic reconstruction with publicly available complete genome sequences of cassava brown streak viruses confirmed that the Mayotte isolates are genetically more related to UCBSV isolates than to CBSV isolates from East Africa, and represent a separate lineage that appears to be almost intermediate between current isolates of CBSV and UCBSV. This is the first report of cassava brown streak disease in Mayotte. Given that cassava is a staple food in the Comoros archipelago and Madagascar, this report is of great significance for managing the health of cassava in this region, and warrants urgent attention from regulatory institutions. (Texte intégral)

International audience; Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.

Huanglongbing is an unculturable vascular citrus pathogen transmitted from infected to healthy plants through grafting or by citrus psyllids, Diaphorina citri mainly in Asia and America and Trioza erytreae in Africa. This phloem limited gram-negative bacterium causes dramatic yield losses and is classified into three species based on 16S rDNA sequence analysis (2): (i) ‘Candidatus Liberibacter asiaticus’ (Las), the most epidemiologically active, widespread and heat tolerant species; (ii) ‘Ca. L. africanus’ (Laf), only found in Africa; and (iii) the newly described ‘Ca. L. americanus’ (Lam), which appeared in 2005 in Brazil (5). Considered as a quarantine organism in America and Europe, Las is actively affecting North America and Asia, and research is leading toward psyllid management and resistance breeding. Despite the fact that Reunion Island has successfully controlled Las by introducing a psyllid parasitoid, Tamarixia radiata (1), this strategy was less effective or reproducible within other territories. D. citri was first detected in Guadeloupe in 1998, where the control of the the psyllid population has been effective with T. radiata (3); and was first detected in Martinique in 2012. Following the outbreak in the United States and the Caribbean, and also supported by reports of symptoms in citrus orchards, local National Plant Protection Organizations (NPPO) organized a detection survey across both islands to verify the occurrence of Huanglongbing. Since 2012, 450 sites were prospected each year in Martinique and Guadeloupe, where 20 leaves from 10 to 30 trees were analyzed. DNA extraction was performed (DNeasy Plant Mini Kit, Qiagen) on fresh or dried leaf midribs, along with negative control midribs (Citrus paradisi ‘Star Rubis’) and PCR amplification was done with the species-specific primers A2/J5 (4) and GB1/GB3 (5). Only Las-specific 703-bp amplicons were obtained (n = 43) and 20 were sequenced (Beckman Coulter Genomics, United Kingdom; sequences available through GenBank Accession Nos. KF699074 to KF699093) and blasted against the National Center for Biotechnology Information non-redondant database (NCBI-nr). BLAST analysis revealed 100% identity with the 50S ribosomal protein subunit L1 (rplA) and L10 (rplJ) of ‘Ca. L. asiaticus’ (all strains), and no significant homology to other organisms. Additionally, sequence assembly on a reference genome (NC_012985) showed 100% homology. Huanglongbing was detected in Guadeloupe on March 2012 at Le Moule (East coast) in a Tahiti lime orchard (C. latifolia) and crossed the island in 6 months. Las was detected in Martinique on May 2013 on Tahiti lime (C. latifolia) at Bellefontaine (Northwest) in a private garden and at Le Lorrain (Northeast) in an orchard. Other species from the Rutaceae family were affected by HLB (C. reticulat and C. sinensis) on both islands; however, few of the positive samples showed HLB symptoms (blotchy mottle patterns and green islands on leaves), but presented symptoms similar to nutrient deficiencies. Despite the former presence of T. radiata in Guadeloupe and its detection in Martinique a few weeks after the detection of D. citri, where it had a mean parasitism rate of 70%, an outbreak of HLB spread across both islands. These analyses confirm the presence of HLB in Martinique and Guadeloupe and to our knowledge represent the first report of Las in the French West Indies. Introduction events remain unclear, but this report raises the importance of plant certification, psyllid population control, and surveillance of territories close to the French West Indies, with regards to the risk that HLB presents to citrus production worldwide. References: (1) B. Aubert et al. Fruits. 38, 1983. (2) J. M. Bové. J. Plant Pathol. 88:1, 2006. (3) J. Etienne et al. Fruits. 56:05, 2001. (4) A. Hocquellet et al. Mol. Cell. Probes 13:5, 1999. (5) D. C. Teixeira et al. Mol. Cell. Probes 19:3, 2005.

International audience; In November 2013, symptoms of yellowing on leaves and brown necrotic rot on tuberous roots (Fig. 1) were observed on different cassava landraces (Manihot esculenta) in Dembeni on Mayotte Island, a French Overseas Department in the southwest Indian Ocean. The symptoms, similar to the cassava brown streak disease described in East Africa, suggested the possible involvement of cassava brown streak viruses (Mbanzibwa et al., 2011). The expanding cassava brown streak disease epidemic in East Africa is caused by two ipomoviruses, Cassava brown streak virus (CBSV; Monger et al., 2010) and Ugandan cassava brown streak virus (UCBSV; Winter et al., 2010). To verify the identity of the causal agent, symptom-bearing leaf samples of 17 affected plants were collected in March and June 2014. Total RNA was extracted from these samples using the RNeasy Plant Mini Kit (Qiagen, France). For the detection of cassava brown streak viruses, a two-step RT-PCR using CBSDDF2 and CBSDDR primers was used (Mbanzibwa et al., 2011). These primers were designed for simultaneous virus-specific detection of CBSV and UCBSV (amplicons of 344 bp and 440 bp, respectively). RT-PCR products of the expected size for UCBSV were obtained for 11 samples. To confirm the diagnosis, the RT-PCR products were directly and bi-directionally sequenced and contigs assembled using DNABaser (Heracle BioSoft, Romania). Sequences of 376 bp from eight of the 11 samples were obtained which had 99-100% nucleotide identity (MEGA6; Tamura et al., 2013) (EMBL Accession Nos. LN611671 to LN611678; Table 1). The sequences showed the highest nucleotide identity (86%) with isolates of UCBSV from Uganda and Malawi (FN434109; FN433932) and 85% with an isolate of CBSV from Mozambique (FN434436). A phylogenetic reconstruction with publicly available complete genome sequences of cassava brown streak viruses confirmed that the Mayotte isolates are genetically more related to UCBSV isolates than to CBSV isolates from East Africa, and represent a separate lineage that appears to be almost intermediate between current isolates of CBSV and UCBSV (MEGA6; Fig. 2). This is the first report of cassava brown streak disease in Mayotte. Given that cassava is a staple food in the Comoros archipelago and Madagascar, this report is of great significance for managing the health of cassava in this region, and warrants urgent attention from regulatory institutions.

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N-PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N-PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90*6, 88*7 and 47*2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92*5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N-PCR assay. The N-PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N-PCR had undeniable advantages compared to the reference method (less labour-intensive and less time-consuming). In addition, post-test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N-PCR assay has since been included in a revised version of the EPPO detection protocol. © 2013 British Society for Plant Pathology.

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

Asiatic citrus canker disease, caused by Xanthomonas citri pv. citri, seriously impacts citrus production worldwide. Two pathogenic variants, A and A*/Aw, have been described within this pathovar. Two additional pathovars of X. citri with a limited geographic distribution and reduced pathogenicity, namely X. citri pvs. aurantifolii and bilvae, are also pathogenic to citrus and some rutaceous species. Rapid and reliable identification is required for these citrus pathogens, which are classified as a quarantine organism in citrus-producing countries. The specificity of nine polymerase chain reaction primers previously designed for the identification of X. citri pv. citri or citrus bacterial canker strains (both pvs. citri and aurantifolii) was assayed on a large strain collection (n = 87), including the two pathotypes of X. citri pv. citri, other genetic related or unrelated pathogenic xanthomonads, and saprophytic xanthomonads. This study gave congruent results with the original articles when testing the same strains or pathovars but the use of a broad inclusivity and exclusivity panel of strains highlighted new findings. Particularly, primers 2/3, 4/7, and KingF/R failed to provide amplification for three strains from the pathotype A*/Aw. Moreover, all pairs of primers detected at least one non-target strain. These data were supported by in silico analysis of the DNA sequences available from National Center for Biotechnology Information databases.

La Direction Générale de l’Alimentation du ministère en charge de l’agriculture a saisi l’Anses en vue de la réalisation d’une évaluation du risque simplifiée (ERS) des LYTS. Ce travail a pour but notamment d’évaluer le risque d’introduction et l’impact économique des LYTS dans la zone ARP. La zone ARP à considérer comprend la Martinique, la Guadeloupe, la Guyane, la Réunion et Mayotte.

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of aroids (BBA), has been reported in many regions and has been isolated on several host genera (1). During February 2004, in a nursery (Mont Dore) in New Caledonia, suspect symptoms have been observed on anthurium and dieffenbachia plants. A survey carried out on the entire island revealed that X. axonopodis pv. dieffenbachiae was present in 41 of the 89 nurseries inspected. During hot and humid weather, marginal or interveinal water-soaked spots surrounded by chlorotic or necrotic areas were observed, usually followed by a systemic phase (stem rotting and death of the plant). During the cold and dry season, only water-soaked spots were observed. Seventy pure cultures isolated from anthurium and dieffenbachia were gram negative, yellow pigmented, and had a mucoid aspect when grown on rich media. All strains responded positively to the Xcd108 monoclonal antibody (Agdia Inc., Elkhart, IN) raised against X. axonopodis pv. dieffenbachiae using indirect ELISA. A set of 18 strains (isolated from 15 anthurium and 3 dieffenbachia plants located in different sites) were further characterized by molecular and pathogenicity tests. All strains reacted positively using a specific nested PCR assay (1). Pathogenicity tests were performed on 8-month-old plants of Anthurium andreanum ‘Carré’, Dieffenbachia maculata ‘Tropic Marianne’, and Syngonium podophyllum ‘Robusta’ by syringue infiltration of a suspension containing approximately 105 CFU mL-1. Each strain was inoculated onto three young leaves (four inoculation sites per leaf) on two plants. Control plants received sterile Tris buffer solution (10 mM, pH 7.2). Plants were maintained in a growth chamber with day and night temperatures of 30 ± 1°C and 26 ± 1°C, respectively, 95 ± 5% relative humidity, 30 μmol m-2·s-1 light intensity and a photoperiod of 12 h (1). On all plants, all strains caused typical water-soaked symptoms within 10 days, evolving into chlorotic then necrotic areas after 20 to 24 days. Amplified fragment length polymorphism (AFLP) markers revealed three haplotypes among these strains, which suggests that several introduction events may have occurred. These AFLP fingerprints were compared with other Xanthomonas spp. pathovars, including most of X. axonopodis pv. dieffenbachiae strains obtained from international culture collections, and were found to belong to the same genomic group as all the X. axonopodis pv. dieffenbachiae strains pathogenic on anthurium. Importation in New Caledonia of aroids from countries in which X. axonopodis pv. dieffenbachiae is present (Hawaii, French Polynesia, the Netherlands, and Australia) occurred before 2004. The wide distribution of BBA is very likely due to the plant material movements occurring in New Caledonia and suggests that the pathogen may have been present on the territory some years before the first official case. Reference: (1) I. Robene-Soustrade et al. Appl. Environ. Microbiol. 72:1072, 2006.

Pronounced yellowing symptoms on the lower and middle leaves of tomato plants, similar to those caused by magnesium deficiencies, were observed in 2004 and 2005 in farmers’ greenhouses in Reunion Island, situated east of Madagascar in the Indian Ocean. Yellow-leaf symptoms included irregular chlorotic mottling, and the interveinal areas of leaves developed red or brown necrotic flecks. Fruit size was reduced on plants with symptoms and yields were indirectly affected. The flamelike pattern of the discoloured leaves, and the abundance of whiteflies on affected plants, suggested the possible involvement of Tomato chlorosis virus (ToCV) or Tomato infectious chlorosis virus (TICV) ( Closteroviridae, Crinivirus ) (Wisler et al ., 1998). Twenty leaf samples with symptoms were collected from tomato plants in March 2005, and total RNA was extracted from these samples using the Qiagen RNeasy Plant Mini kit. For the detection of a potential crinivirus, a nested PCR was performed (Dovas et al ., 2002). The method consisted of a one-step RT–PCR using primers HS-11 and HS-12, followed by nested PCR with primers TIC-3/TIC-4 and ToC-5/ToC-6, for detecting TICV or ToCV, respectively. These primers were designed to amplify the highly conserved region of the heat-shock protein 70 gene. A PCR product of the expected size was observed with ToCV primers for 14 of the 20 leaf samples. No PCR product was observed for the PCR performed with TICV primers. Three PCR products were cloned using pGEM-T easy vector system II (Promega), and sequenced (Genome Express) (EMBL accession nos AJ968394, AJ968395 and AJ968396). Sequences obtained from the three samples had 99·5% nucleotide identity when aligned ( dnaman , Lynnon BioSoft, Quebec, Canada). The most significant sequence alignments (NCBI, blast n) were 98% with ToCV isolates from the USA (GenBank accession no. AF024630), Spain (GenBank accession nos AF233435, AF215818 and AF215817) and Italy (GenBank accession nos AF234029 and AY048854). This is the first report of the presence of ToCV in Reunion Island.

Small, black, angular leaf lesions, which sometimes coalesced, were collected from avocado (Persea americana Miller) leaves in a government nursery located at Grand Anse, Mahé, Seychelles archipelago in 2003. Patterns of diseased plants were highly clustered, suggesting local dispersal in the nursery. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on KC semiselective medium (3). Amplified fragment length polymorphism (AFLP) analysis was performed on two avocado strains together with reference strains of the genetic clusters of Xanthomonas axonopodis (4) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5′-labeled – SacI + C primer for the selective amplification step) (1). The two avocado strains showed identical fingerprints and were closely related to X. axonopodis genetic cluster 9.2 (4). One strain, JZ103-1, was further analyzed by MultiLocus Sequence Analysis (MLSA) using portions of three housekeeping genes (atpD, dnaK, and gyrB) as described previously (1). MLSA data confirmed that the xanthomonad associated with avocado was most closely related to X. axonopodis genetic cluster 9.2. No other strain in this genetic cluster shared an identical sequence type. Avocado cv. Grand collet leaves from the youngest growth flush were infiltrated with a needleless syringe (10 inoculation sites per leaf and three replicates) with bacterial suspensions. Typical, water-soaked lesions that developed into black necrotic spots appeared 6 to 8 days after infiltration on all inoculated leaves when suspensions containing ~1 × 106 CFU ml–1 were used (i.e., ~7 × 102 CFU per inoculation site), while no lesions developed on leaves inoculated with Tris buffer or with suspensions containing ~1 × 104 CFU ml–1. One month after inoculation, mean Xanthomonas population sizes determined on KC semiselective medium (3) from ~1 cm2 leaf fragments showing black lesions ranged from 2 × 106 to 4 × 106 CFU per lesion, typical of a compatible interaction. A few colonies that recovered from lesions obtained after inoculation were typed by AFLP and were identical to the inoculated strain. An extensive branch and trunk canker of avocado caused by a Xanthomonas sp. has been reported in California (2). This bacterium did not cause lesions of avocado leaves or fruit after inoculation (2). This appears to be the sole previous report of a xanthomonad being pathogenic to avocado and the symptoms observed in the Seychelles appear therefore very different from the ones reported in California. No major outbreak of bacterial leaf spot of avocado has been reported in the Seychelles archipelago since 2003. References: (1) N. Ah-You et al. Int. J. Syst. Evol. Microbiol. 59:306, 2009. (2) D. A. Cooksey et al. Plant Dis. 77:95, 1993. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) J. Rademaker et al. Phytopathology 95:1098, 2005.

Bacterial leaf spot of cucurbits caused by Xanthomonas cucurbitae (4) can be a harmful disease of several cucurbit species in tropical environments, mainly within the Cucumis, Cucurbita, and Citrullus genera. The bacterium induces angular, water-soaked leaf spots, which sometimes become necrotic and have a chlorotic halo. Scab-like lesions on fruit can also be observed (2). Water-soaked, angular leaf lesions were collected from approximately 15 watermelon plants (Citrullus lanatus) in a production field located in Mahé, Seychelles in 2003. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on KC semiselective medium (yeast extract 7 g, peptone 7 g, glucose 7 g, agar 18 g, distilled water 1,000 ml, propiconazole 20 μg ml–1, cephalexin 40 mg liter–1, and kasugamycin 20 mg liter–1) from all isolation attempts (3). Amplified fragment length polymorphism (AFLP) analysis was performed on four watermelon strains together with reference strains of Xanthomonas cucurbitae (LMG 690 [type strain] and LMG 8663) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5′-labeled – SacI + C primer for the selective amplification step) (1). The four strains from watermelon showed identical fingerprints and were most closely related to X. cucurbitae. One strain from diseased watermelon (JZ88-1) was further analyzed by MultiLocus Sequence Analysis (MLSA) using portions of three housekeeping genes (atpD, dnaK, and gyrB) as described previously (1). This strain displayed a very high relatedness (99.8 and 98.9% with strain LMG 690 and LMG 8663, respectively) to the two reference strains of X. cucurbitae. AFLP and MLSA were useful for identifying strains at the species level that were consistent with previous results (1). Bottle-gourd (Lagenaria siceraria), pumpkin (Cucurbita maxima), squash cv. Aurore (Cucurbita pepo), cucumber cv. L-04 (Cucumis sativus), cantaloupe melon cv. Cezanne (Cucumis melo), and watermelon cv. Fou-nan (C. lanatus) leaves were infiltrated (10 inoculation sites per leaf and three replicates) with bacterial suspensions (JZ88-1, LMG 690 and LMG 8663) containing approximately 1 × 105 CFU ml–1 (approximately 1 × 102 CFU per inoculation site). Typical water-soaked lesions that developed into necrotic spots were observed 6 to 8 days after inoculation for all inoculated strains on all inoculated plant species. One month after inoculation, Xanthomonas was recovered from lesions and population sizes determined on KC semiselective medium (3) ranging from 1 × 106 to 9 × 106 CFU per lesion were typical of a compatible interaction. Bacterial leaf spot has appeared sporadically in Mahé, Seychelles since 2003, most often with limited incidence. However, growers need to be aware of the potential negative effect of this disease in tropical environments. References: (1) N. Ah-You et al. Int. J. Syst. Evol. Microbiol. 59:306, 2009. (2) J. F. Bradbury. Page 309 in: Guide to Plant Pathogenic Bacteria. CAB International, Slough, UK, 1986. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

In February of 2007, a virus disease survey on tomato plants (Solanum lycopersicum) in greenhouses and open fields was conducted on the island of Mauritius at the request of the Agricultural Research and Extension Unit (AREU), sponsored by the European Union, and funded by the Programme Régional de Protection des Végétaux (PRPV). Yellowing symptoms on the lower and middle leaves of tomato plants and whiteflies (Bemisia tabaci) were observed in greenhouses in Pailles, located in the north region of the island. The interveinal chlorosis pattern of the discolored leaves was similar to symptoms described for Tomato chlorosis virus (ToCV; genus Crinivirus) detected on tomato in 2004 on Reunion Island (1), suggesting the possible involvement of the same virus. Six symptomatic tomato leaf samples were collected from separate plants in the Pailles greenhouses. Total RNA was extracted from these samples with the Qiagen (Courtaboeuf, France) RNeasy Plant Mini Kit. Reverse transcription-PCR was used for molecular diagnosis, independently using two sets of specific ToCV primers. The first set of primers, ToCV-172 and ToCV-610, was designed to amplify the highly conserved region of the heat shock protein 70 (HSP70) gene (2). The second set of primers was designed to amplify the coat protein (CP) gene (forward-CP-ToCV-4384: 5′-ATCCTCTGGTTAGACCGTTAG-3′ and reverse as in Segev et al. [3]). PCR products of the expected size (439 and 725 bp, respectively) were observed for the six samples from the greenhouse from Pailles. For each set of primers, two PCR products obtained from two different samples were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI) and sequenced (Macrogen, Seoul, Korea). The two HSP70 sequences (GenBank-EMBL-DDBJ Accession Nos. AM884013 and AM884014) and the two CP sequences (FM206381 and FM206382) had 100% nucleotide identities (DNAMAN; Lynnon BioSoft, Quebec City, Canada). The highest nucleotide identities of the 439-bp fragment of HSP70 gene (NCBI, BLASTn) were 97% with ToCV isolates from France (DQ355214, DQ355215, and DQ355216), Florida (AY903448), Italy (AM231038 and AY048854), Mayotte Island (AM748818), Portugal (AF234029), and Reunion Island (AM748816). Similarly, the highest nucleotide identities (98%) were obtained with ToCV isolates from France (EU625350) and Spain (DQ136146), with the 725-bp fragments of CP gene. Interestingly, ToCV isolates from Mauritius and Reunion are as divergent as isolates from the rest of the world, which suggests the possibility of different introductions. In conclusion, observed symptoms and laboratory results based on two different regions of the genome confirm the presence of ToCV in symptomatic tomatoes on the island of Mauritius, for the first time to our knowledge. The visual survey carried out in June of 2008 confirmed the presence of typical interveinal chlorosis symptoms in other greenhouses, requiring further studies to assess the incidence of ToCV on tomato crops. References: (1) H. Delatte et al. Plant Pathol. 55:289, 2006. (2) D. Louro et al. Eur. J. Plant Pathol. 106:589, 2000. (3) L. Segev et al. Plant Dis. 88:1160, 2004.

In : November 2005, a survey of greenhouse tomato ( Lycopersicon esculentum ) plants was conducted on Mayotte Island, an island located in the northern part of the Mozambique Channel. Yellow leaf symptoms were observed on the lower and middle leaves of tomato plants from the northern (Koungou), western (Chiconi and Combani) and southern (Chirongui) regions. A flame-like pattern of the discoloured leaves and the similarity of the symptoms to the first description of Tomato chlorosis virus (ToCV, Crinivirus ) on tomato in 2004 in Reunion Island (Delatte et al. , 2006), suggested the possible involvement of the same virus in Mayotte Island. Leaf samples with symptoms were collected from tomato plants from one greenhouse from each of the four affected regions. Total RNA was extracted from four samples using the Qiagen RNeasy Plant Mini Kit. For the detection of a potential crinivirus, a one-step RT-PCR was used with specific primers ToCV-172 and ToCV-610 primers (Louro et al ., 2000). These primers amplify the highly conserved region of the heat shock protein 70 (HSP70) gene of ToCV. PCR products of the expected size (419 bp) were observed for all samples. Three PCR products were cloned using pGEM-T easy vector system II (Promega,) and sequenced (Genome Express) (EMBL Accessions AM748817, AM748818 and AM748819). Sequences obtained from the three amplicons had a 99% nucleotide identity (DNAMAN, Lynnon BioSoft). The highest nucleotide identities of the 412 bp fragment of the HSP70 gene were between 98 and 99% for ToCV isolates from France (EMBL Accessions DQ355214, DQ355215 and DQ355216), from Spain (EMBL Accessions AF233435, AF215818 and DQ136146), from Italy (EMBL Accessions AF234029 and AY048854) and from Reunion (EMBL Accession AM748816). This is the first report of ToCV from Mayotte Island. (Resume d'auteur)

The use of methods of analysis capable of producing reliable analytical results is a prerequisite to the effective control of quarantine plant pathogens. Proficiency testing is considered to be one of the most reliable ways to verify and coordinate analytical results. As a French national reference laboratory in plant pathology, the Anses Plant Health Laboratory organizes proficiency tests in order to ensure that officially approved laboratories (certified by government services) are capable of producing reliable analytical results for the detection of plant pathogens. Proficiency tests in plant pathology have a number of notable features including the processing of qualitative results. This paper presents the experience of the Anses Plant Health Laboratory’s Unit for Tropical Pests and Diseases (LSV-RAPT) as an organizer of proficiency tests in plant pathology. The LSV-RAPT has gained recognition for the methodology it has developed in the form of accreditation as a proficiency testing provider according to the ISO/IEC 17043. The methodology can be applied to many other disciplines that use qualitative detection methods.

National audience; Après avoir rappelé l'histoire des épidémies de TYLCV en culture de tomates à l'ile de la Réunion depuis la détection en 1997 de ce virus et de son vecteur l'aleurode Bemisia tabaci (biotype B), cet article rend compte d'une enquête agronomique menée par le CIRAD et l'ARMEFLHOR en 2010. Elle a établi ou confirmé que : du fait du TYLCV , la filière tomate a évolué vers la production sous abris (la moitié du tonnage produit) ; la culture de plein champ, compromise à moins de 500 m d'altitude (dans les bas), reste possible dans les Hauts ; sous abris la PB (auxiliaires +insecticides compatible) s'est développée ; toujours sous abris, les mesures prophylactiques sont indispensables ; partout, l'usage de plants sains (pépinieristes locaux agréés SOC) se développe ; les variétés résistantes ou tolérantes actuelles sont inadaptées au contexte local. Le maintien de la filière tomate à la Réunion demande d'améliorer les techniques de type PBI sous abris, proposer des variétés resistantes ou tolérantes adaptées, tester de nouvelles techniques et mieux connaitre le virus et son vecteur. Les organismes de la filière et les actions liées à Ecophyto peuvent y aider. La maîtrise du TYLCV et son vecteur implique des itinéraires techniques associant plusieurs méthodes.(résumé d'auteur)