New species of pathogenic Pseudomonas isolated from citrus in Tunisia: proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.. KC_E10

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016-2017 from fruits and leaves of Citrus sinuensis cv 'Valencia late' and C. limon cv 'Eureka' plants with black pit disease symptoms. A gene sequence analysis of the housekeeping gene rpoD was used for strains identification at the species level. Results demonstrated their affiliation to the genus Pseudomonas and revealed the presence of 11 strains representatives of two putative new species. Their similarities with the currently known Pseudomonas species type strains were below the threshold values established for members of the same species in the genus. These 11 strains were deeply analyzed morphologically, genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences and phenotypic characteristics were determined by API20NE and Biolog GENIII. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv ‘Eureka’ while representative strains KC12T and E10BT did not produce an hypersensitive response on tobacco. Main proteins profiles were determined by whole-cell mass spectrometry and compared with their closest relatives. The genomes of both representatives were sequenced and their genome sequences compared with their close related species genomes. Average nucleotide index and Genome to Genome distances of KC12T to the closest type strain P. grimontii were 89.6 and 35.1 respectively; for strain E10BT these values were 86.8 and 34.9% with P. canadensis. The results obtained support the conclusion that strains KC12T, KC17 KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12T (=CECT XXXX and CFBPYYYY). The strains E10BT, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10BT (=CECT XXXX and CCUG YYYY).