Résumé
From 2011 to 2015, surveys were conducted in rice fields of Benin to assess the importance of bacterial leaf blight (BLB) of rice caused by Xanthomonas oryzae pv. oryzae. BLB-like diseased leaf samples were collected showing yellowing symptoms or one to two orange to brown stripes on one or both halves of the leaf blade. Older symptoms enlarged to the entire leaf and showed brown stripes below the leaf tip and along the leaf margins. Severely affected leaves became grayish-brown. To diagnose the disease, symptomatic leaf pieces were surface-sterilized and macerated in sterile water. Upon plating on semiselective peptone-sucrose-agar (PSA) medium (Poulin et al. 2014), straw-colored to yellow colonies were obtained after incubation at 28°C for 1 to 2 days. In parallel, the leaf sap was subjected to a diagnostic multiplex PCR assay for X. oryzae pathovars (Lang et al. 2010). Over 3,000 samples tested were negative. Because leaf blight can also be caused by species of Pantoea (Lee et al. 2010; Mondal et al. 2011), the isolates were tested with PCR primers that amplify a gyrB fragment from Pantoea spp. (Brady et al. 2008), often resulting in PCR amplicons of the expected size. However, since these primers were not specific to the genus Pantoea but also amplified loci from other Enterobacteriaceae, we used genomic information from 26 Pantoea strains to develop species-specific primers targeting the gyrB gene of P. ananatis (PANAN_gyrB-F, 5′-TGACGATGCCCGTGAAGG; PANAN_gyrB-R, 5′-TAATCAACGTGGCRACTTCC) and P. stewartii (PANST_gyrB-F1, 5′-AGGGATACAGCAAGAAGGC; PANST_gyrB-R1, 5′- TAGCCACTTCCTGAGACG). Single colonies were isolated from PCR-positive samples, including strains ARC22 and ARC570. gyrB fragments of both isolates were amplified and sequenced using the above primers. The BLASTN searches of a trimmed 426 and 508 bp DNA fragment revealed that the nucleotide sequences were 99% identical to the gyrB gene from P. ananatis strain 17671 (GenBank accession no. KF554589) and P. stewartii strain 626 (KF554590), respectively. The partial sequences of the gyrB gene were deposited at GenBank under KT729518 for ARC22 and KT729519 for ARC570. Pathogenicity assays were conducted on 35-day-old rice plants. To this purpose, bacteria were grown overnight on PSA and inoculum adjusted to 108 cells/ml. Inoculation was done by either clipping half of the leaves or by infiltrating the leaves at about 5 to 15 cm below the leaf tips. One to two leaves per plant, three plants per accession, and seven cultivars were inoculated. Control plants were inoculated with sterile water. Fifteen to twenty-one days after incubation, inoculated leaves showed typical BLB-like lesions whereas control plants remained symptomless. The reisolated bacteria from diseased leaves yielded colonies identical to those described above and confirmed as P. ananatis and P. stewartii by PCR and sequence analysis of the gyrB gene portion, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of a new bacterial disease caused by species of Pantoea in Benin. Symptoms corresponding to the disease were found at all 14 localities in Benin where field prospections were performed and prevalence of the disease varied between 30 and 100%.