A diagnostic multiplex PCR scheme for identification of plant-associated bacteria of the genus Pantoea.

Unrefereed reprintThe genus Pantoea forms a complex of more than 25 species, among which several cause diseases of several crop plants, including rice. Notably, strains of Pantoea ananatis and Pantoea stewartii have been found to cause bacterial leaf blight of rice in Togo and Benin, while other authors have observed that Pantoea agglomerans can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools

First Report of a New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis and Pantoea stewartii in Togo

International audienceIn 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

First Report of a New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis and Pantoea stewartii in Benin

International audienceFrom 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%