A multi-actor, participatory approach to identify policy and technical barriers to better farming practices that protect our drinking water sources.

Despite an improvement of water quality across Europe there are many pollution hotspots for both nitrates and PPPs, mainly due to agricultural activities. The BMPs and MMs to reduce pollution from agriculture are well known, and there are policy instruments in place to ensure drinking water standards, but the current approach has not been efficient enough. Within the H2020 Water Protect project the premise was that there is a need for a multi-actor, participatory approach to tackle the issue from a new angle, and to assess why the uptake of known BMPs and MMs was not better among farmers. Seven "Action Labs" were selected that represent major physical, socio-economical, cultural and farming settings across Europe. A methodology of multi-actor engagement was chosen but with different approaches due to the local context. Initially the level of farmers' awareness about water quality issues was matched to the observed uptake rates of BMPs and MMs. In a second survey barriers hindering the uptake of measures were identified. The first survey revealed a low general awareness on the potential pollution to drinking water sources. Despite this, between 24% to 88% of the surveyed farmers per Action Lab were already voluntarily adopting one quarter of the selected BMPs and MMs. The second survey demonstrated the need to address organisational, legislative, sociological and technical barriers. The lack of coordination between different institutional bodies promoting measures and the financial incentives needed to invest and operate these often-costly measures need to be considered. The multi-actor, participatory approach with its improved awareness and collaboration made it possible to identify the crucial factors for improvement - to build a social acceptance among all actors and communicate the issues and solutions from the start.

Characterization of Cichopeptins, New Phytotoxic Cyclic Lipodepsipeptides Produced by Pseudomonas cichorii SF1-54 and Their Role in Bacterial Midrib Rot Disease of Lettuce.

The lettuce midrib rot pathogen Pseudomonas cichorii SF1-54 produces seven bioactive compounds with biosurfactant properties. Two compounds exhibited necrosis-inducing activity on chicory leaves. The structure of the two phytotoxic compounds, named cichopeptin A and B, was tentatively characterized. They are related cyclic lipopeptides composed of an unsaturated C12-fatty acid chain linked to the N-terminus of a 22-amino acid peptide moiety. Cichopeptin B differs from cichopeptin A only in the last C-terminal amino acid residue, which is probably Val instead of Leu/Ile. Based on peptide sequence similarity, cichopeptins are new cyclic lipopeptides related to corpeptin, produced by the tomato pathogen Pseudomonas corrugata. Production of cichopeptin is stimulated by glycine betaine but not by choline, an upstream precursor of glycine betaine. Furthermore, a gene cluster encoding cichopeptin synthethases, cipABCDEF, is responsible for cichopeptin biosynthesis. A cipA-deletion mutant exhibited significantly less virulence and rotten midribs than the parental strain upon spray inoculation on lettuce. However, the parental and mutant strains multiplied in lettuce leaves at a similar rate. These results demonstrate that cichopeptins contribute to virulence of P. cichorii SF1-54 on lettuce.

New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation.

Pseudomonas cichorii is the causal agent of lettuce midrib rot, characterized by a dark-brown to green-black discoloration of the midrib. Formation of necrotic lesions by several plant-pathogenic pseudomonads is associated with production of phytotoxic lipopeptides, which contribute to virulence. Therefore, the ability of P. cichorii SF1-54 to produce lipopeptides was investigated. A cell-free culture filtrate of SF1-54 showed surfactant, antimicrobial, and phytotoxic activities which are typical for lipopeptides. High-performance liquid chromatography analysis of P. cichorii SF1-54 culture filtrate revealed the presence of seven compounds with lipopeptide characteristics. Two related lipopeptides, named cichofactin A and B, were studied in more detail: they are linear lipopeptides with a decanoic and dodecanoic lipid chain, respectively, connected to the N-terminus of an eight-amino-acid peptide moiety. Both cichofactins are new members of the syringafactin lipopeptide family. Furthermore, two nonribosomal peptide synthethase-encoding genes, cifA and cifB, were identified as responsible for cichofactin biosynthesis. A cifAB deletion mutant no longer produced cichofactins and was impaired in swarming motility but showed enhanced biofilm formation. Upon spray inoculation on lettuce, the cichofactin-deficient mutant caused significantly less rotten midribs than the wild type, indicating that cichofactins are involved in pathogenicity of P. cichorii SF1-54. Further analysis revealed that P. cichorii isolates vary greatly in swarming motility and cichofactin production.

Biosurfactants in plant-Pseudomonas interactions and their importance to biocontrol.

Production of biosurfactants is a common feature in bacteria, and in particular in plant-associated species. These bacteria include many plant beneficial and plant pathogenic Pseudomonas spp., which produce primarily cyclic lipopeptide and rhamnolipid type biosurfactants. Pseudomonas-derived biosurfactants are involved in many important bacterial functions. By modifying surface properties, biosurfactants can influence common traits such as surface motility, biofilm formation and colonization. Biosurfactants can alter the bio-availability of exogenous compounds, such as nutrients, to promote their uptake, and of endogenous metabolites, including phenazine antibiotics, resulting in an enhanced biological activity. Antibiotic activity of biosurfactants towards microbes could play a role in intraspecific competition, self-defence and pathogenesis. In addition, bacterial surfactants can affect plants in different ways, either protecting them from disease, or acting as a toxin in a plant-pathogen interaction. Biosurfactants are involved in the biocontrol activity of an increasing number of Pseudomonas strains. Consequently, further insight into the roles and activities of surfactants produced by bacteria could provide means to optimize the use of biological control as an alternative crop protection strategy.

Pseudomonas cichorii as the causal agent of midrib rot, an emerging disease of greenhouse-grown butterhead lettuce in Flanders.

Bacterial midrib rot of greenhouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) is an emerging disease in Flanders (Belgium) and fluorescent pseudomonads are suspected to play an important role in the disease. Isolations from infected lettuces, collected from 14 commercial greenhouses in Flanders, yielded 149 isolates that were characterized polyphasically, which included morphological characteristics, pigmentation, pathogenicity tests by both injection and spraying of lettuce, LOPAT characteristics, FAME analysis, BOX-PCR fingerprinting, 16S rRNA and rpoB gene sequencing, as well as DNA-DNA hybridization. Ninety-eight isolates (66%) exhibited a fluorescent pigmentation and were associated with the genus Pseudomonas. Fifty-five of them induced an HR+ (hypersensitive reaction in tobacco leaves) response. The other 43 fluorescent isolates were most probably saprophytic bacteria and about half of them were able to cause rot on potato tuber slices. BOX-PCR genomic fingerprinting was used to assess the genetic diversity of the Pseudomonas midrib rot isolates. The delineated BOX-PCR patterns matched quite well with Pseudomonas morphotypes defined on the basis of colony appearance and variation in fluorescent pigmentation. 16S rRNA and rpoB gene sequence analyses allowed most of the fluorescent isolates to be allocated to Pseudomonas, and they belonged to either the Pseudomonas fluorescens group, Pseudomonas putida group, or the Pseudomonas cichorii/syringae group. In particular, the isolates allocated to this latter group constituted the vast majority of HR+ isolates and were identified as P. cichorii by DNA-DNA hybridization. They were demonstrated by spray-inoculation tests on greenhouse-grown lettuce to induce the midrib rot disease and could be re-isolated from lesions of inoculated plants. Four HR+ non-fluorescent isolates associated with one sample that showed an atypical midrib rot were identified as Dickeya sp.