Patrizia Bella

N-acyl-homoserine-lactone quorum sensing in tomato phytopathogenic Pseudomonas spp. is involved in the regulation of lipodepsipeptide production

Pseudomonas corrugata and Pseudomonas mediterranea are two closely related phytopathogenic bacteria both causal agents of tomato pith necrosis. P. corrugata produces phytotoxic and antimicrobial cationic lipodepsipeptides (LDPs) which are thought to act as major virulence factors. Previous studies have demonstrated that P. corrugata CFBP 5454 has an N-acyl homoserine lactone (AHL) quorum sensing (QS) system PcoI/PcoR and that LDP production occurs at high population densities. No molecular studies on virulence have thus far been reported for P. mediterranea. In this study, we show that P. mediterranea also produces LDPs as well as possessing an AHL-dependent QS system, designated PmeI/PmeR, which is highly homologous to the PcoI/R system of P. corrugata producing and responding to C(6)-AHL. Downstream of pmeI, a partial DNA sequence revealed the presence of a homolog of the rfiA gene of P. corrugata which encodes a transcriptional regulator involved in bacterial virulence. Pathogenicity tests and MALDI-TOF spectra of wild-type strains of both bacterial species and their respective QSs and rfiA derivative mutants revealed that, in the absence of LDPs, the strains induce very weak symptoms indicating that LDPs may act as major virulence factors. Mutational analysis of both QS systems suggests that their mode of action is in places different.

The Transcriptional Activator rfiA Is Quorum-Sensing Regulated by Cotranscription with the luxI Homolog pcoI and Is Essential for Plant Virulence in Pseudomonas corrugata

The gram-negative phytopathogen Pseudomonas corrugata has an acyl-homoserine lactone (AHL) quorum-sensing (QS) system called PcoI/PcoR that is involved in virulence on tomato. This work identifies, downstream of pcoI, a gene designated rfiA, which we demonstrate is directly linked to QS by cotranscription with pcoI. The deduced RfiA protein contains a DNA-binding domain characteristic of the LuxR family but lacks the autoinducer-binding terminus characteristic of the QS LuxR-family proteins. We also identified, downstream of rfiA, an operon designated pcoABC, encoding for the three components of a tripartite resistance nodulation-cell-division (RND) transporter system. The expression of pcoABC is regulated by RfiA. We found that lipodepsipeptide (LDP) production is cell density dependent and mutants of pcoI, pcoR, and rfiA are unable to inhibit the growth of the LDP-sensitive microorganisms Rhodotorula pilimanae and Bacillus megaterium. P. corrugata rfiA mutants were significantly reduced in their ability to cause necrosis development in tomato pith. In addition, it was established that PcoR in the absence of AHL also played a role in virulence on tomato. A model for the role of PcoI, PcoR, and RfiA in tomato pith necrosis is presented.

Pseudomonas corrugata crpCDE is part of the cyclic lipopeptide corpeptin biosynthetic gene cluster and is involved in bacterial virulence in tomato and in hypersensitive response in Nicotiana benthamiana

Pseudomonas corrugata CFBP 5454 produces two kinds of cyclic lipopeptides (CLPs), cormycin A and corpeptins, both of which possess surfactant, antimicrobial and phytotoxic activities. In this study, we identified genes coding for a putative non-ribosomal peptide synthetase and an ABC-type transport system involved in corpeptin production. These genes belong to the same transcriptional unit, designated crpCDE. The genetic organization of this locus is highly similar to other Pseudomonas CLP biosynthetic clusters. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis revealed that transporter and synthetase genomic knock-out mutants were unable to produce corpeptins, but continued to produce cormycin A. This suggests that CrpCDE is the only system involved in corpeptin production in P. corrugata CFBP 5454. In addition, phylogenetic analysis revealed that the CrpE ABC transporter clustered with the transporters of CLPs with a long peptide chain. Strains depleted in corpeptin production were significantly less virulent than the wild-type strain when inoculated in tomato plants and induced only chlorosis when infiltrated into Nicotiana benthamiana leaves. Thus, corpeptins are important effectors of P. corrugata interaction with plants. Expression analysis revealed that crpC transcription occurs at high cell density. Two LuxR transcriptional regulators, PcoR and RfiA, have a pivotal role in crpC expression and thus in corpeptin production.

Grape and environmental mycoflora monitoring in old, traditionally cultivated vineyards on Mount Etna, southern Italy

BACKGROUND Grape contamination by several fungal species occurs during a vineyards preharvest and harvest. Agronomic management and microclimatic conditions can affect fungi occurrence and epidemiology, thus explaining qualitative differences in mycoflora composition, including the presence of phytopathogenic or mycotoxigenic fungi. In this study a two-year grape, air and soil mycoflora monitoring programme was undertaken in vineyards on Mount Etna (eastern Sicily, Italy). The mycoflora composition was investigated at pea berry and veraison phenological phases from air and soil and at ripening from sample grapes. RESULTS Mycoflora in air and soil varied according to the phenological stage. In the air samples, penicillia were dominant over aspergilli at the pea berry phase, but their ratio was inverted at early veraison. Black aspergilli (BA) were isolated from the vine environment and grape samples, where BA were represented mainly by Aspergillus niger aggregate, which showed no or low ochratoxin A (OTA) production. Aspergillus carbonarius was either not identified or identified at low frequency, although most of the isolates produced OTA. CONCLUSION Monitoring focused on the environmental mycoflora composition and highlighted the good health profile of various Sicilian autochthonous grape cultivars. In addition, data suggest that the lower relative humidity occurring at the highest altitudes reduces BA incidence.

Draft Genome Sequence of Pseudomonas mediterranea Strain CFBP 5447T, a Producer of Filmable Medium-Chain-Length Polyhydroxyalkanoates

ABSTRACT Pseudomonas mediterranea strain CFBP 5447T is a phytopathogenic bacterium isolated from tomato plants affected by pith necrosis disease. Moreover, its ability to produce medium-chain-length polyhydroxyalkanoates (mcl-PHAs) in culture from different carbon sources and valuable microbial products, such as cyclic lipopeptides, has been well documented. Here, we report the first draft genome sequence of this species.

Over-expression of CsGSTU promotes tolerance to the herbicide alachlor and resistance to Pseudomonas syringae pv. tabaci in transgenic tobacco

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with enzyme properties, recombinant CsGSTs were in vitro expressed and tested for GST activity using alachlor as substrate. The recombinant GSTU2 enzyme was twice more active than GSTU1 in conjugating alachlor to GSH thus indicating that CsGSTU2 might play a crucial role in the plant defense against the herbicide. Moreover, as a consequence of the infiltration with a bacterial suspension of the P. syringae pv. tabaci, transgenic tobacco plants but not wild type plants bestowed the capability to limit toxic metabolite diffusion through plant tissues as indicated by the absence of chlorotic halos formation. Consequently, the transgenic tobacco plants described in the present study might be utilized for phytoremediation of residual xenobiotics in the environment and might represent a model for engineering plants that resist to pathogen attack.

Antimicrobial Activity of the Extracts of Terfezia Claveryi and Tirmania Pinoyi Against Gram-positive and Gram-negative Bacteria Causal Agent of Diseases in Tomato

Antimicrobial Activity of the Extracts of Terfezia claveryi and Tirmania pinoyi Against Gram-positive and Gram-negative Bacteria Causal Agent of Diseases in Tomato Maria Letizia Gargano , Patrizia Bella , Stefano Panno, Vincenzo Arizza, Luigi Inguglia, Vittoria Catara, Giuseppe Venturella, Salvatore Davino* Department of Agricultural and Forest Science (SAF), University of Palermo, Viale delle Scienze Bld. 5, 90123 Palermo (Italy) Department of Biological, Biochemistry and Pharmacological Science and Technology (STEBICEF), Via Archirafi 18-28, 90122 Palermo, (Italy) Department of Agricultural, Food and Environment (Di3A), Via Santa Sofia 100, 91123 Catania, (Italy) salvatore.davino@unipa.it # These authors contributed equally to the study

Survival of two biocontrol Pseudomonas strains in tomato fruits after inoculation at flowering through fruit ripening.

Pseudomonas syringae strain 1.1S and P. fluorescens strain A506 were applied to tomato plants to investigate their ability to survive during plant growth and ripening of fruits. Both strains were used to spray tomato flowers and green tomato fruits. Ripe tomato fruits were harvested and subjected to microbiological analysis. Peptone wash water and homogenates of fruit pulp were plated on selective media before and after enrichment. All tomatoes produced from inoculated flowers contained P. syringae on the surface of fruits (100% in enriched samples) as well as in the pulp homogenates (100% before and after enrichment). All tomatoes produced from inoculated green fruits contained P. syringae on the surface (38% and 100% in wash water and in enriched samples, respectively) as well as in the pulp homogenates (90% and 100% before and after enrichment, respectively). Of the P. fluorescens surface-positive tomatoes, 37% were from fruits receiving flower inoculation and 77% were from fruits receiving green fruit inoculation. P. fluorescens was harboured in higher percentages in pulp homogenates of tomatoes produced from treated flowers and green fruits (62% and 100%, respectively). Results suggest that antagonistic P. syringae and P. fluorescens strains survive in and on tomato fruits from the time of inoculation at flowering or at early stage of fruit development through fruit ripening. Tomato flower and green surfaces of fruits are possible sites at which Pseudomonas may attach and remain viable during fruit development.