Several studies have reported effects of the plant phenolic acids cinnamic acid (CA) and salicylic acid (SA) on the virulence of soft rot enterobacteria. However, the mechanisms involved in these processes are not yet fully understood. Here, we investigated whether CA and SA interfere with the quorum sensing (QS) system of two Pectobacterium species, P. aroidearum and P. carotovorum ssp. brasiliense, which are known to produce N-acyl-homoserine lactone (AHL) QS signals. Our results clearly indicate that both phenolic compounds affect the QS machinery of the two species, consequently altering the expression of bacterial virulence factors. Although, in control treatments, the expression of QS-related genes increased over time, the exposure of bacteria to non-lethal concentrations of CA or SA inhibited the expression of QS genes, including expI, expR, PC1_1442 (luxR transcriptional regulator) and luxS (a component of the AI-2 system). Other virulence genes known to be regulated by the QS system, such as pecS, pel, peh and yheO, were also down-regulated relative to the control. In agreement with the low levels of expression of expI and expR, CA and SA also reduced the level of the AHL signal. The effects of CA and SA on AHL signalling were confirmed in compensation assays, in which exogenous application of N-(β-ketocaproyl)-l-homoserine lactone (eAHL) led to the recovery of the reduction in virulence caused by the two phenolic acids. Collectively, the results of gene expression studies, bioluminescence assays, virulence assays and compensation assays with eAHL clearly support a mechanism by which CA and SA interfere with Pectobacterium virulence via the QS machinery.

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

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The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10 µ g mL -1, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5-10 µ g mL -1, also as a drench) reduced the rate of infection by 75-85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.

Aims:  Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results:  Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions:  The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study:  The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.