http://ibws.nexenservices.com 20 Cucumis melo, Cucumis sativus, Cucurbita moschata, and Anthurium spp, New Hosts of Ralstonia solanacearum in Martinique (French West Indies) Emmanuel WICKER 1 , Laurence GRASSART 2 , Danielle MIAN 3 , Régine CORANSON-BEAUDU 3 , Denise DUFEAL 3 , Caroline GUILBAUD 4 and Philippe PRIOR 4 1 CIRAD-FLHOR, BP 153, 97202, Fort de France Cedex, Martinique, FWI, 2 Service de la Protection des Végétaux, BP 438, La Pointe des Sables, 97205, Fort de France Cedex, Martinique, FWI, 3 Fédération régionale de défense contre les organismes nuisibles (FREDON), BP 550 – La Pointe des Sables, 97205 Fort de France Cedex, Martinique, FWI, 4 INRA, Unité de Pathologie Végétale, Domaine St Maurice, BP 94, 84140, Montfavet, France. Corresponding author : wicker@cirad.fr In the French West Indies (FWI), endemic bacterial wilt caused by lowland tropical strains of Ralstonia solanacearum is known to be a devastating disease to major solanaceous food and cash-crops like potato, tomato, eggplant and pepper (Prior & Steva, 1990). Since 1999, hybrid and local anthurium (Anthurium sp.) productions which have developed in the wetter and elevated areas of Martinique (Centre-North Atlantic) were dramatically affected by an unknown bacterial disease. The disease was not bacterial spot (Acidovorax anthurii) nor bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae), but bacterial wilt due to novel and unexpected strains of R. solanacearum. Late in 2001 R. solanacearum was isolated for the first time from wilted native heliconia (Heliconia caribaea, Strelitziaceae) established in the same area. Simultaneously, massive wilt damage due to bacterial wilt was also noted for the first time on cantaloupe (Cucumis melo), zucchini (Cucurbita pepo), pumpkin (Cucurbita moschata) and cucumber (Cucumis sativus). These outbreaks were caused by the same new pathotypes of R. solanacearum. Symptoms (colour plate, back-cover). Bacterial wilt on anthurium resulted in a definitive decline of many plantation and the disease extended rapidly in Martinique. Characteristic symptoms consist of greasy, water-soaked lesions (lower face) which turn necrotic with greasy margins (upper face). When the disease becomes systemic, these lesions, generally originating from the insertion point of the leaf on the petiole, develop following the main and secondary veins in a full or partial glove-shape. External infection (disseminated by water) may developed from any natural opening such as hydatodes. Regardless of the way the infection occurs, R. solanacearum establishes itself by flooding into parenchyma tissue, resulting in colonization of the vascular system until most of the leaves or the whole plant collapse. Leaves may turn yellow or not depending on the severity of the systemic invasion, and the stem, a rhizome structure (formally named ‘caudex’) may rot with abundant bacterial ooze. Major characteristic bacterial wilt symptoms on native heliconia, surrounding heavily infested and abandoned anthurium plantations, as browning and rotting of the cigar, which is the youngest rolled-up emerging leaf. Fully expanded leaves may also wilt. A section of the pseudo-stem showed brown-reddish discoloration within and along vascular elements. Bacterial wilt on cucurbit plot began in foci. At that stage, symptoms developed quite late (fruit setting) on cantaloupe and pumpkin. When established in the plot, bacterial wilt may destroy the entire crop only 2-3 weeks after planting. Disease is typically soilborne and develops rapidly from older to youngest leaves which may wilt or not. Leaves turn yellow with necrotic lesions between or along major veins (same as for anthurium). The whole plant collapses and dies, while there are no apparent symptoms on mature fruits. Such bacterial wilt development is very fast on zucchini compared to cantaloupe and pumpkin. Characterization. Bacteria were isolated from oozing stems of rotted rhizomes and surface-disinfected leaves of anthurium, and from stems of wilted cucurbits. The culture medium used was Kelman’s triphenyltetrazolium chloride medium (TZC). Individual bacterial colonies were selected and assessed for purity after 48 h incubation at 28±1°C. Representative isolates were mucoid, with pink-reddish swirling egg- shaped pigmentation pattern characteristic of R. solanacearum (Kelman, 1954). All strains tested were oxidase positive, strictly aerobic, nitrate and citrate positive, and induced a hypersensitive reaction on tobacco leaves (cv. Xanthi) within 24 h. Strains from anthurium, pumpkin, zucchini and cantaloupe were tested for biovar determination using a modified version of the procedures of Hayward (1964), and were metabolically classified as biovar 1. PCR amplification, using the R. solanacearum species-specific primers set 759 and 760 (Opina et al. 1997) generated the expected 280bp amplicon (internal control for the following multiplex-PCR) with all isolates tested. In terms of genetic diversity, typing was assayed using multiplex PCRs for subspecies: phylotype- and Musa-specific primers (Fegan and Prior 2002, Prior and Fegan 2002). Our results clearly demonstrate that these anthurium and cucurbit strains are not distinguishable from reference strains ANT307 and 11212 (Prior and Fegan 2002), falling in the phylotype II, sequevar 4 (or MLG25) cluster, unifying strains (ecotype SFR/A) that induce the insect-transmitted form of the Moko disease (banana bacterial wilt). Pathogenicity tests were performed on cantaloupe (Cucumis melo, cv. Charentais), cucumber (C. sativus, cv. Gemini), pumpkin (Cucurbita moschata, cv. Phoenix), Anthurium andreanum (cv. Amigo), and Cavendish banana (Musa acuminata) cv. Grande naine. Inoculation was by infiltrating a bacterial suspension (10 7 cfu.ml -1 ) into the stem or pseudo-stem, or by pouring 10 ml of inoculum (10 8 cfu.ml -1 ) onto the pot surface after wounding of roots. One or two weeks after inoculation, these tests reproduced symptoms observed in natural conditions : wilting and necrosis to cucurbit leaves, water-soaking and blackening to anthurium leaves and R. solanacearum was successfully recovered from these symptoms. All strains were avirulent on banana (encoded NPB) as no symptom were observed 45 days after inoculations. However, R. solanacearum was consistently recovered from these asymptomatic plants. Discussion From our knowledge, this is the first record of cantaloupe and pumpkin as hosts of R. solanacearum. In the literature, bacterial wilt caused by biovar 1 strains was reported on summer squash, zucchini summer squash and cucumber from Brazil (Oliveira and Moura, 1994; Sinigaglia et al., 2001). Natural occurrence of bacterial wilt on anthurium was reported to be caused by biovar 3 strains in Singapore (Yik et al., 1994), Reunion Island (Poussier et al., 2000) and Mauritius Island (Dookun et al., 2001), or by biovar 4 strains in Taïwan (Su and Leu, 1995). Unlike regular biovar 3 (phylotype I) strains pathogenic to anthurium which developed as focus under the shade-house, behaviour of strains falling in phylotype II seems to be dramatically active with regard to major epidemiological traits like fast spreading, rapid disease onset and acquisition of new hosts. This is the first time that biovar 1 strains are found to cause so much damage in anthurium fields. These novel strains had also the unique feature of being genotypically indistinguishable from Moko strains, while being not pathogenic to banana. Recently, biovar 1 strains infecting anthurium pots were reported from Florida, but these strains were reported pathogenic to banana (Norman and Yuen, 1999). At present we don’t know if they also belong to phylotype II, sequevar 4. Using the hierarchical classification from Fegan and Prior (2002) and relevant molecular tests, these cucurbitaceous and anthurium NPB strains fall in phylotype II, sequevar 4 and