Résumé
In 2013 and 2014, surveys were carried out in the main rice-growing regions of Togo (Kovié and Kpalimé) to evaluate the prevalence of plant-pathogenic bacteria, such as Xanthomonas oryzae. Symptomatic rice leaves showing characteristics of bacterial leaf blight, as typically caused by X. oryzae pv. oryzae (Niño-Liu et al. 2006), were collected, surface-sterilized, and macerated in sterile water. Upon plating on semiselective peptone-sucrose-agar (PSA) medium (Poulin et al. 2014), yellowish colonies of bacteria were isolated. Similar bacterial colonies were isolated from rice grains. Since all isolates were negative in a diagnostic multiplex PCR assay for X. oryzae (Lang et al. 2010), a portion of the 16S rDNA was amplified using universal primers, F1 and R13 (Dorsch and Stackebrandt 1992). DNA sequence analysis indicated that the bacteria belong to the genus Pantoea. New gyrB-specific PCR primers (PANsp_gyrB-F, 5′-TTCCAGGARAAYATYTACTGCTT; PANsp_gyrB-R, 5′- CGGTCATGATRATRATGCTGTG) were developed based on 26 Pantoea genome sequences. gyrB fragments of four bacterial isolates from regions where the prevalence and severity of the disease was most important were amplified and sequenced for further diagnosis. Sequence comparisons of the obtained 603-bp DNA fragments revealed that the sequences of the leaf isolate ARC60 (GenBank accession no. KX385187) and of the seed isolate ARC651 (KX342014) were 98% identical to the corresponding gyrB gene fragment from the Pantoea ananatis type strain LMG 2665 (JMJJ01000009). Interestingly, the sequences of the leaf isolate ARC229 (KX342015) and of the seed isolate ARC646 (KX342016) were 99% and 100% identical to the corresponding gyrB gene fragment from the P. stewartii type strain LMG 2632 (JPKO01000005), respectively. Using species-specific gyrB primers for Pantoea (Kini et al. 2016), we confirmed 91 isolates from 12 localities as P. ananatis or P. stewartii. Pathogenicity of the four isolates was tested on rice plants. Four-week-old rice seedlings of the cultivars Azucena and Nipponbare were inoculated by leaf infiltration at the central vein using a needleless syringe. Bacterial suspensions containing 108 CFU/ml prepared in sterile water were used and sterile water served as a negative control. Inoculated plants were kept in a greenhouse at 28°C and 80% relative humidity. After 7 days, the infiltrated leaves showed necrotic lesions at the inoculation site, which later expanded and turned from straw yellow to light brown color and ultimately developed into typical blight symptoms at 15 to 20 days post inoculation. Symptoms of inoculated leaves were similar to those that had been initially observed in fields and water-treated controls remained symptomless. Yellow- pigmented colonies were reisolated from the infected rice leaves, which were similar to the original isolates, and the PCR-amplified gyrB fragments were 100% identical to the original sequences, thus fulfilling Koch’s postulates. Based on our analyses, we conclude that the bacteria that were isolated from rice leaves and grains in Togo were P. ananatis and P. stewartii. To our knowledge, this is the first report of leaf blight of rice caused by species of Pantoea in Togo.