Bacterial wilt of Solanaceous crops, described in Martinique in the 1960's, was mainly caused by biovar 3 and biovar 1 strains of Ralstonia solanacearum. From.
Emerging Strains of Ralstonia solanacearum in Martinique (French West Indies): A Case Study for Epidemiology of Bacterial Wilt Emmanuel Wicker1, Laurence Grassart2, Régine Coranson-Beaudu1, Danielle Mian3, Caroline Guilbaud4 and Philippe Prior4 1 PRAM CIRAD, BP214, 97285 Le Lamentin Cédex 02, Martinique (F.W.I.) 2 DAF/SPV, BP 438, La Pointe des Sables, 97205 Fort de France Cédex, Martinique (F.W.I.) 3 FREDON, BP 550, La Pointe des Sables, 97205 Fort de France Cédex Martinique (F.W.I.) 4 INRA Unité de Pathologie Végétale, Domaine St Maurice, BP 94, F-84140 Montfavet, France Keywords: Solanaceae, tomato, genotyping, rotations, molecular diversity, phytobacteria Abstract Bacterial wilt of Solanaceous crops, described in Martinique in the 1960’s, was mainly caused by biovar 3 and biovar 1 strains of Ralstonia solanacearum. From 1999 anthurium fields were strongly affected by biovar 1 strains, genotypically identical to insect-transmitted “Moko” (bacterial wilt of banana) strains (MLG25, sequevar 4, ecotype SFR/A), but not pathogenic (NP) on Cavendish Banana. The disease has now spread over the island on a widened range of species and crops (Heliconia caribea, Cucurbitaceous crops, several weeds). Fegan and Prior (2004) revised the intraspecific classification within R. solanacearum and proposed molecular tools to distinguish four phylotypes (I to IV, each of which are subdivided in sequevars) and to discriminate between banana-pathogenic strains and nonpathogenic strains (NP) within the R. solanacaearum strains belonging to phylotype II, sequevar 4. A collection of 215 isolates collected from 1989 to 2003 during several surveys, were characterized according to this classification. Isolates from Cucurbitaceae, Heliconia, weeds, as well as most of the anthurium isolates were typed in this group of new strains belonging to phylotype II sequevar 4NP, which appeared in Martinique in 1998. The isolates from Solanaceae were distributed among new (phylotype II sequevar 4NP) and “historical” strains (phylotypes I and II, biovar 3 and 1 respectively), but the ratio of new strains on tomato has increased dramatically since 2002. These results suggest the emergence of a new population of R. solanacaearum in Martinique, with a broad host range, which is most probably endemic (since it is well established on weeds), and is spreading rapidly. This new population may overcome the “historical” population, particularly on tomato. The emergence of these new strains on vegetable crops may be induced by rotations with cucurbitaceous and musaceous plants. INTRODUCTION Bacterial wilt caused by Ralstonia solanacearum Yaabuchi et al. (Smith) is a major threat for agriculture worldwide. This soilborne bacterium is prevalent throughout all tropical and subtropical regions, as well as some temperate zones, with a host range comprising 53 botanical families (Hayward, 1964). In Martinique (French West Indies), Ralstonia (formerly Pseudomonas) solanacearum has been described since the 1960’s as the causal agent of bacterial wilt on solanaceous crops (Digat and Escudié, 1967). Severe outbreaks were observed on eggplant in 1983 (Messiaen, 1983), and the tomato, eggplant, pepper crops were regularly affected by this wilt, whereas no Moko (bacterial wilt of banana) has ever been reported. The pathogen populations were mainly composed of biovar III strains, with a minority of biovar I strains (Prior and Steva, 1990). The phytosanitary situation dramatically changed in 1999. In April of 1999, R. solanacearum was first isolated from diseased Anthurium andreanum plants showing leaf yellowing and necrosis, as well as rotting of the rhizome Proc. 1st IS on Tomato Diseases Eds. M.T. Momol, P. Ji and J.B. Jones Acta Hort 695, ISHS 2005
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with abundant bacterial ooze. This new bacterial disease caused severe damages in the main anthurium production areas. Bacterial strains were surprisingly genotyped as phylotype II sequevar 4, thus clustered in the same group as strains inducing insecttransmitted Moko disease (Prior and Fegan, 2002). Inoculation tests showed however, that these strains were not pathogenic on banana Cavendish (Prior, Dufeal, unpublished data, 1999), leading to their identification as phylotype II/sequevar 4NP (for “nonpathogenic”). In 2001, the same bacterium was isolated from native Heliconia (Heliconia caribaea). In December 2001, bacterial wilt caused by R. solanacearum was observed on cantaloupe (Cucumis melo) fields in the southern part of the island. Since, new cases have been regularly reported on cucumber (Cucumis sativus), pumpkin (Cucurbita moschata) and zucchini (Cucurbita pepo). Symptoms including wilt, leaf yellowing and development of necrotic lesions on the leaves have been described elsewhere (Wicker et al., 2002). This is the first report of strains of R. solanacearum being naturally pathogenic to cantaloupe and pumpkin, and genotyped as phylotype II sequevar 4NP. This unusual epidemic situation in Martinique leads to several questions : (i) what is the origin of these strains of “anthruium-curcurbit” pathotype, were they introduced or endemic; (ii) should these new outbreaks be considered as sporadic events or as the expression of an new emerging population of R. solanacearum; (iii) what is the actual prevalence of these new strains in Martinique; (iv) do factors exist that may explain this emergence. All these questions led us to set up a new, extensive characterization of the R. solanacearum populations in Martinique, thanks to a collaboration of FREDON (Fédération Régionale de Défense contre les Organismes Nuisibles, or Regional Federation for the Control of Pests and Diseases), SPV (Service de Protection des Végétaux, Plant Protection Service), CIRAD (International Center for Agronomic Research for Development) and INRA (National Institute for Agronomic Research). MATERIALS AND METHODS Surveys A first survey was carried out from April to December 1999 and from March to August 2000 by FREDON and SPV, in the main production zones of anthurium, in the upper and humid central areas of the island. In 2001 and 2002, a second survey was conducted in the contaminated anthurium fields, to search for alternative hosts among weeds. A third survey was led by CIRAD from September 2002 to February 2003, throughout the island, in vegetable and horticultural crops. A questionnaire was submitted to the growers cultivating the sampled fields, to know about rotations, previous crops on their fields, irrigation and seed sources. Leaf, stem and rhizome samples were taken from the field, and brought to the laboratory for observations and bacterial isolation and identification. Genetic Diversity and Pathogenicity of R. solanacearum Isolates Isolates were regularly collected from 1989 to 2002 following routine diagnostic of wilted or decaying plants in the SPV laboratory, and also from different bacterial wilt surveys carried out from 1999 to 2002 in anthurium fields. All the isolates collected in 2002-2003 were purified and stored in the CIRAD laboratory (Pole de Recherche Agronomique de Martinique, PRAM). Isolations were made from oozing stems of rotted rhizomes and surface-disinfected leaves of anthurium, stems of wilted cucurbits and solanaceous plants and roots and stems of symptomless weeds. 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 exhibiting a mucoid colony form, with pink-reddish swirling pigmentation pattern characteristic of R. solanacearum (Kelman, 1954) were chosen. Recently, Fegan and Prior (2002) proposed a new intraspecific classification for R. solanacearum, reporting four “sub-species” or phylotypes (I to IV) within the R. solanacearum species complex. From this work, they developed multiplex-PCR 146
procedures combining species-specific primers 759/760 (Opina et al., 1997) and phylotype- and Musa-specific primers (Fegan and Prior, 2002; Prior and Fegan, 2002). These multiplex-PCRs were used to type strains in this study. Multiplex-PCR reactions were run using pure bacterial cultures as DNA template, i.e., without any prior DNA extraction. Bacterial cultures were first tested with the “phylotype” multiplex-PCR to verify that they belonged to the species R. solanacearum and to determine which phylotype (I to IV) they belonged to; strains characterized as phylotype II were then submitted to the “Musa” multiplex-PCR, to determine whether they were non-Musa strains (most probably sequevar 5) or if they belonged to “Musa” strains (sequevars 3, 4, 4NP or 6) (Fegan and Prior, 2002; Prior and Fegan, 2002). To test for differences in geographical distributions of phylotypes, samples were clustered into four groups, corresponding to the four soil and climatic areas defined in Martinique: North-Windward (North-East), North-Leeward (North-West), Center, South. RESULTS Phylotyping of the SPV and CIRAD collections, collected respectively from 1989 to 2002 and in 2002-2003, revealed three groups of strains (Fig. 1): phylotype I strains (ex-biovars 3), phylotype II strains (ex-biovar 1) and phylotype II/sequevar 4 NP strains (ex-biovar 1, named II/4NP in the following). Phylotype I and II strains are strains well-known for decades and were thus called “historical” or “regular” strains. They represented 37% (collection SPV) to 55% (collection CIRAD) of the total strains collected. Whereas, the “new” II/4NP strains constituted the most important group of sampled strains. The emergence of the II/4NP strains is graphically represented in Fig. 2. Strains sampled before 1999 were isolated from Solanaceae only, and were genotyped as phylotype I and phylotype II. Only one II/4NP strain was isolated during this period, in 1998 from a diseased tomato plant. From 1999, a large proportion of strains was isolated from anthurium and Cucurbits (corresponding to new unexpected outbreaks), and all were genotyped as II/4NP. Strains baited on Solanaceae were solely genotyped as “regular” strains, with the exception of one strain which belonged to the II/4NP genotype, again isolated from tomato. The survey conducted in 2002-2003 revealed that the situation was changing. The II/4NP strains, or “new” strains, were no longer restricted to anthurium and Cucurbitaceae, but were also consistently isolated on Solanaceae, and more particularly on tomato. Tomato may thus be an epidemiological “bridge” between “regular” (Phylotype I and II) and “emerging” (II/4NP) strains. On the other hand, the strains isolated from anthurium consisted of both “emergent” (II/4NP) and “regular” phylotype II genotypes. Geographical Distribution of the Strains Surveys indicated that bacterial wilt still was a major limiting factor for Solanaceous crops in all production zones of Martinique (North-West and North-East, Centre, South). Diseased spots on anthurium were first observed in the elevated and humid zones in the Centre of Martinique, and more recently in the South-East, and on the North-Eastern coast. On Cucurbitaceae, the first cases were observed in 2001 in regions of the South-Eastern part of the island. In 2002 and 2003, new cases, especially on watermelon (Citrullus lunatus), were detected on the Northern and North-Eastern coastal zones. Finally, R. solanacearum was isolated from the following weeds: Solanum americanum, Cleome viscosa, Ageratum conyzoides, Commelina diffusa, Portulaca oleracea and New-Guinea Impatiens (Impatiens hawkerii). The data from the survey 2002-2003 showed that the “emerging” strains were prevalent throughout Martinique (Fig. 3), and were found both on vegetable crops and on floriculture (anthurium and Heliconia plantations), except in the South where only phylotype II strains were found on anthurium. The population distributions were not significantly different between areas (Khi-two test, P=0.05). However, considering only the strains isolated from tomato, the proportion of II/4NP strains was significantly more 147
important in North-Windward area (Khi-two test, P
