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Recent occurrence in Tunisia of Citrus blast and black pit disease, caused by Pseudomonas syringae pv. syringae (Pss), the few available antibacterial chemicals, and the increasing resistance of Pss to copper sulfate highlighted the need for new antibacterial agents. In vitro analysis of the antagonistic activity of fifty-four native epiphytic bacterial isolates from the Citrus phyllosphere towards pathogenic Pss resulted in the selection of eight potential biocontrol strains according to their various antagonistic activities. Based on analysis of the (partial) 16S rRNA gene sequence and the rpoD housekeeping gene, five isolates were presumptively identified as Pseudomonas atacamensis (strains Y28, Y58 and Y128), P. cremoris (strain Y40), and P. lactis (strain Y21). In vivo test of these antagonistic strains showed no pathogenicity towards plant tissues. Three strains (Y28, Y128 and Y21) exhibited considerable inhibitory activities and significantly reduced disease severity in five Citrus cultivars (Cassar, Eurêka, Maltaise, Washington Navel and Pomelo) on shoots, leaves and fruits. Determination of genes coding for antibiotic production showed the presence of gacA gene in the five strains mentioned above. However, the genes prnD, pltC, pltB, phlD and phzC as well as phzD (and their homologs phzF and phzA), described earlier for Pf-5, were not detected among the tested Tunisian antagonistic Pseudomonas strains. This study presents the first description of P. atacamensis, P. cremoris, and P. lactis as potential biocontrol agents. The characterized bacterial strains may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for sustainable control of Citrus. blast and black pit disease. [ABSTRACT FROM AUTHOR]

Citrus blast and black pit caused by Pseudomonas syringae pv. syringae (Pss) is the only bacterial disease reported in Tunisian Citrus orchards. The phylogenetic relationship between Pss strains was studied based on multilocus sequence analysis (MLSA), using partial sequences of housekeeping genes rpoD, rpoB, gyrB, cts, and pfk for 14 representative Pss Citrus strains, including the reference strain LMG5496. The MLSA revealed that the studied Tunisian Citrus strains are closely related to LMG5496 and cluster in phylogroup 02. Based on the cts gene, the majority of Citrus strains clustered in clades "a" and "b". However, five strains were placed in a newly defined clade "g". We describe the presence of six different type III secreted effectors (T3SEs). These were found with frequencies of 100% for the effector hopAN1 and the helper hrpK1, 65% for hopT1‐2, and 14% for hopN1, hopR1, and hopQ1‐2. Investigation of copper resistance showed that 67% of our Pss Citrus strains from Tunisia are resistant to copper sulphate in vitro, and the copper resistance genes copABCDR were detected in 23% of the strains. Our results present new data concerning the genetic diversity and phylogeny, presence of T3SEs, and copper resistance within the Pss populations that affect Citrus in Tunisia. [ABSTRACT FROM AUTHOR]