First report of Kosakonia cowanii causing bacterial blight on Coffea arabica

From 2012 to 2019, 6-month to 2-year-old coffee (Coffea arabica) plants showing leaf blight, stem blackening, brownish, necrotic, irregular leaf spots and shoot tip dieback (Figure 1A, B, C, and D) symptoms were observed at the municipalities of Araguari, Indianópolis, Monte Carmelo, Nova Ponte and Romaria, Minas Gerais State, Brazil. Bacterial exudates from infected tissues, mainly from the shoot tip, were observed under microscopy; no bacterial exudates were found in leaf tissues. Bacterial colonies were isolated from symptomatic disinfested tissues (leaves and shoot tip) using medium 523 (Figure 1E). The isolated bacteria were facultative anaerobe, Gram-negative, cream-colored on YDC medium, growth at 37 ^oC, arginine and oxidase negative, catalase and asparagine positive (Schaad et al. 2002), and positive hypersensitivity reaction in tobacco leaves. Pathogenicity was confirmed by spraying coffee plants (at the three-leaf stage) until runoff, with a 1 × 108 CFU/mL bacterial suspension inoculate. The plants were kept in the moist chamber for 24 hours, before and after inoculation under greenhouse conditions. No symptoms were observed under the mock-inoculated plants. Disease symptoms on inoculated plants were observed 13 days after inoculation. The bacteria were then reisolated to satisfy Koch's postulates, the colony phenotypes were identical to the original ones. Bacterial genomic DNA of four isolates (UFU D1, UFU G119, UFU H1, and UFU I87) was extracted, and the 16S rRNA gene region was amplified using the 8F and 1492R universal primers, and then compared with sequences deposited in the GenBank. The results aligned closely with those of Kosakonia cowanii (GenBank NR_025566.1), with 99.4% similarity and 96% query coverage for the sequence. In addition to sequencing the 16S rRNA gene, which is considered the barcode for bacteria, other single-copy genes are necessary for species confirmation. In this study, to confirm the species and generate more data on these new isolates, 150 bp paired-end libraries were constructed and sequenced using DNBSEQ. A minimum of 1 gigabase was obtained for each isolate, and the genomes were assembled using the SPAdes genome assembler v3.15.4. Subsequently, the in-house developed algorithm SpM (Species Matching) was used to identify the species. The phylogenetic tree for the 16S gene was constructed using the maximum likelihood algorithm available in PhyML (v3.1/3.0 aLRT). The Phylogeny.fr platform was used to perform the phylogenetic reconstruction. This analysis revealed that our K. cowanii isolates (H1, D1, G119, and I87) from the coffee tree clustered with other K. cowanii strains isolated from different locations (Figure 2A). The Average Nucleotide Identity (ANI) analysis was performed using the fastANI program on all complete genomes of the Kosakonia genus obtained from the NCBI database. The results indicated that our K. cowanii isolates from the coffee tree clustered closely with other strains of the K. cowanii species (Figure 2B). Genomic analyses confirmed that all isolates belong to the species K. cowanii, corroborating the 16S rRNA gene analyses. The genomes have been deposited in NCBI with the accession numbers CP160410 (K. cowanii strain UFU H1, size 4,749,805 bp and coverage 36.21x), CP160412 (strain UFU G119, size 4,509,735 bp and coverage 31.26x), CP162577 (strain UFU D1, size 4,867,107 bp and coverage 19.0x), and CP162576 (strain UFU I87, size 4,552,151 bp and coverage 18.23x). These results are of great importance for better understanding the role of K. cowanii as a disease-causing pathogen of coffee plants. K. cowanii has already been described infecting Eucalyptus with symptoms of bacterial blight in Uruguay (Brady et al. 2009), causing disease in soybeans (Krawczyk and Borodynko-Filas 2020), also associated as endophytic in plants (Thomas et al. 2007), and as human pathogens (Yang et al. 2018). Thus, to our knowledge, this is the first report of bacterial blight caused by K. cowanii on coffee plants. The isolates are deposited in the phytopathogenic bacteria collection at Instituto de Ciências Agrárias, Universidade Federal de Uberlândia, Brazil, under the codes UFU D1, UFU G119, UFU H1 and UFU I87.