First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Plectranthus amboinicus in Martinique.

Abstract

Plectranthus amboinicus, commonly known as Gwo ten in the French West Indies (Martinique), is a semi-succulent perennial plant of the Lamiaceae family. This aromatic plant wich is widespread naturally throughout the tropics is of economic importance because of the therapeutic and nutritional properties attributed to its natural phytochemical compounds wich are highly valued in the pharmaceutical industry. In March 2019, wilted P. amboinicus plants intercropped with tomato plants (cv. Heatmaster) in order to reduce the insect-pest damages on tomato, were observed in a field located at the CIRAD experimental station in Lamentin, Martinique (14.663194 N, -60.999167 W). Average disease incidence of 65.74% was recorded on P. amboinicus, in 3 plots with an area of 22.04 m2. The initial symptoms observed were irregular, black, necrotic lesions on leaves. After 10 days, plants wilted and black stripes were observed on stems. Within 4 weeks, more than 50% of plants were fully wilted. Longitudinal stem sections of the wilted plants showed brown vascular discoloration. The cut stems of the wilted plants released a whitish bacterial ooze in water. In all, 108 stem sections were collected, surface disinfected with 70% ethanol and each was crushed in 2 mL of Tris-buffer, then processed for bacterial isolation by plating on modified Semi-Selective Medium from South Africa SMSA (Engelbrecht 1994). Typical Ralstonia solanacearum colonies grew on SMSA medium for 100 of the 108 samples after incubation for 48h at 28°C and were identified as Ralstonia solanacearum using diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all the strains in R. solanacearum Phylotype IIA. A subset of 11 strains was selected for sequevar identification. All the strains were identified as sequevar I-39 (100% nucleotide identity with strain ANT92 - Genbank accession EF371828), by partial egl sequencing (Fegan and Prior 2005) (GenBank Accession Nos. MT314067 to MT314077). This sequevar has been reported to be widespread in the Caribbean and tropical America on vegetable crops (particularly on tomato), but not on P. amboinicus (Deberdt et al. 2014; Ramsubhag et al. 2012; Wicker et al. 2007). To fulfil Koch's postulates, a reference strain, isolated from diseased P. amboinicus (CFBP 8733, Phylotype IIA/sequevar 39), was inoculated on 30 healthy P. amboinicus plants. A common tomato cultivar grown in Martinique (cv. Heatmaster) was also inoculated on 30 plants with the same bacterial suspension. Three-weeks-old plants of both crops grown in sterilized field soil were inoculated by soil drenching with 20 ml of a calibrated suspension (108 CFU/mL). P. amboinicus and tomato plants drenched with sterile water served as a negative controls. Plants were grown in a fully controlled environment at day/night temperatures of 30-26°C ± 2°C under high relative humidity (80%). The P. amboinicus plants started wilting 9 days after inoculation, and within four weeks 60% of the P. amboinicus plants had wilted. The tomato plants started wilting 5 days after inoculation with 62% of wilted plants within four weeks. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. No symptoms were observed and no R. solanacearum strains were isolated from negative controls plants. To our knowledge, this is the first report of R. solanacearum causing bacterial wilt on Gwo ten (P. amboinicus) in Martinique. The importance of this discovery lies in the reporting of an additional host for R. solanacearum, which can be associated with other crops as tomato crop in order to reduce the abundance of insect-pests. Further studies need to be conducted to assess the precise distribution of bacterial wilt disease on P. amboinicus in Martinique and to develop a plan of action avoiding its association with R. solanacearum host crops as tomato for reducing epidemic risk.