Bacterial Blight on Millet Caused by Burkholderia cepacia in Brazil

Millet (Pennisetum glaucum) is a cereal crop of high importance in some regions of the world, and in Brazil the flour is mainly used for the preparation of cakes, biscuits, and porridge. In March 2015, we observed symptoms of yellowing, chlorosis, and blight on millet leaves of cultivar ADR-300 (about 30% incidence) in a plantation located in the municipality of Baixa Grande do Ribeiro (07°51′32″S, 45°12′56″W), Piauí state, Brazil. Samples were collected from a millet field, and five bacterial strains (CCRMBC01 to CCRMBC05) were isolated in trypan blue tetracycline medium (Hagedorn et al. 1987). Pathogenicity tests were performed on 35-day-old millet plants of cultivar BRS 1501, and inoculations were carried out by foliar spraying of bacterial suspension (10⁸ CFU/ml). The negative control consisted of leaves treated with sterile distilled water (SDW). We used three replicates each with three plants. Inoculated leaves showed symptoms of yellowing, chlorosis, and blight 5 days after inoculation, whereas leaves treated with SDW remained symptomless. Seven days after inoculation we fulfilled Koch’s postulates by reisolating the pathogen. BOX-A1R-based BOX sequence polymerase chain reaction (BOX-PCR) analyses were performed according to the method of Louws et al. (1994) to confirm the identity of the strains that were reisolated from Koch’s postulates. The five strains showed the same BOX-PCR profile and were considered clones. For this reason, only the strain CCRMBC01 was used in the biochemical and phylogenetic analyses. The Biolog Gen III system was used for the biochemical identification, and the strain CCRMBC01 was identified as Burkholderia cepacia at a 67% similarity level. The molecular identification was performed by sequencing a 1,500-bp fragment of the 16S rRNA region (Weisburg et al. 1991), a 376-bp fragment of the recA gene (Ginther et al. 2015), and a 454-bp fragment of the gyrB gene (Spilker et al. 2009). Using the BLASTn tool, we observed that sequences of the 16S rRNA region (GenBank MH622193), recA (GenBank MH631060), and gyrB (GenBank MH631060) genes of the strain CCRMBC01 showed 99, 99, and 98% of identity, respectively, with sequences from the type strain of B. cepacia (LMG 1222^T; GenBank AF416381, FJ670559, and AY996867). In the 16S rRNA region maximum likelihood phylogenetic tree, the strain CCRMBC01 clustered with several strains of the B. cepacia complex (BCC), including the type strain of B. cepacia (LMG 1222^T, AF416381). Phylogenetic analysis of concatenated sequences of the recA and gyrB genes based on maximum likelihood, along with sequences from all the types or representative strains of BCC (https://pubmlst.org/bcc/) showed that the strain CCRMBC01 clustered with the LMG 1222^T strain of B. cepacia, with 99% bootstrap support. From our information, this is the first report of B. cepacia causing bacterial blight in millet in Brazil and in the world. In addition, this report sheds light on the host range for B. cepacia, which is a bacterium that has been neglected as a plant pathogen.