First Report of Bacterial Wilt of Amaranth (Amaranthus cruentus) Caused by Ralstonia solanacearum in Benin

Amaranth (Amaranthus cruentus) is an important leafy vegetable cultivated in Benin. In September 2017, wilted amaranth plants of accessions AC NL and Madiira-1 that did not exhibit any foliar discoloration were observed at a field in Cotonou, Benin. Average disease incidences of 72.4 and 27.08% were recorded on accessions AC NL and Madiira-1, respectively, in two plots with an area of ∼8.5 m². The longitudinal sections of most stems of the infected plants showed vascular discoloration, and all the cut stems released whitish bacterial ooze in water. On semiselective modified medium from South Africa (SMSA) agar plates (Engelbrecht 1994), isolated bacterial colonies were morphologically similar to Ralstonia solanacearum. Koch’s postulates were performed with three bacterial isolates by inoculating the susceptible amaranth accession AC NL grown in sterilized field soil. Amaranth plants (10 per isolate) were inoculated by drenching the soil around the crown region of the plant with 40 ml of bacterial suspension (10⁸ CFU/ml). Susceptible tomato cultivar (cv. Tohounvi; five per isolate) was also inoculated with the same bacterial suspensions. Amaranth and tomato plants drenched with sterile distilled water served as the negative control. Three-week-old plants were inoculated and maintained under shade at about 28 to 31°C. Amaranth plants started wilting 12 days after inoculation, and within 4 weeks 73.3% of plants wilted, and all the inoculated tomato plants wilted within 7 days. The noninoculated amaranth and tomato plants remained healthy during the period of the experiment. R. solanacearum was recovered from all symptomatic plants on SMSA medium. Diagnostic polymerase chain reaction (PCR) was conducted using R. solanacearum species-specific primer pairs 759/760 (Opina et al. 1997). The size of the PCR product (282 bp) of the samples from amaranth suggested that the pathogen was R. solanacearum. Multiplex PCR using specific primer for phylotypes I, II, III, and IV (Fegan and Prior 2005) identified the amaranth strain as R. solanacearum phylotype I. Endoglucanase partial gene amplification and sequencing were conducted using the Endo primer pairs (Gutarra et al. 2017). The BLAST search of DNA sequence of the amplified Endo gene region confirmed that the strain was 99% identical to NCBI accessions AB508612 and HM775371 and 98% identical to MF783890. The nucleotide sequence was submitted to the NCBI GenBank and available with the accession number MH397250. Surveys to evaluate the impact of bacterial wilt disease to amaranth farmers in Benin and a plan of action for crop rotation for reducing risk are planned. This is the first recorded report of R. solanacearum causing bacterial wilt on amaranth in the Republic of Benin.