Vittoria Catara

Pseudomonas corrugata: plant pathogen and/or biological resource?

UNLABELLED SUMMARY Pseudomonas corrugata is the causal agent of tomato pith necrosis yet it is also used in the biological control of plant pathogenic bacteria and fungi. Potentially it could be used in other fields, such as the production of commercial biomolecules with a wide range of application and including bioremediation. This review reports the multiple characteristics of the bacterium, highlights its known molecular features and speculates on the possible underlying mechanisms of action. TAXONOMY Bacteria; Proteobacteria; gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas. Microbiological properties: Gram-negative, oxidase-positive, non-spore forming rods; non-fluorescent on Kings B medium; produces wrinkled and rarely smooth colonies on yeast peptone glucose agar or nutrient dextrose agar; yellow to brown diffusible pigments are frequently produced. Disease symptoms: The typical symptom on tomato is necrosis and/or hollowing of the pith of the stem; the syndrome determines loss of turgidity of the plant, hydropic/necrotic areas and long conspicuous adventitious roots on the stem. Biological control agent: In vitro assessed against plant pathogenic fungi and bacteria, as well as the phytotoxin indicator microorganims Rhodotorula pilimanae and Bacillus megaterium; in vivo used against pre- and post-harvest plant pathogens.

Specific oligonucleotide primers for the rapid identification and detection of the agent of tomato pith necrosis, Pseudomonas corrugata, by PCR amplification : evidence for two distinct genomic groups

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata, as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPD-PCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugata strains. All strains produced one of the two specific bands, 1100 bp (from the IPVCT10.3-based primers) and 600 bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonas species, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.

Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, whereas the linear quantification range was within 100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfully detected the fungus in woody samples of naturally infected lemon and artificially inoculated sour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold more sensitive than conventional PCR, and preliminary results indicate that the former technique achieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtain suitable DNA samples from fungal cultures and citrus woody samples for PCR assays enable diagnosis to be completed in a short time.

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.

Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes.

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.

CHARACTERIZATION OF ATYPICAL CLAVIBACTER MICHIGANENSIS subsp. MICHIGANENSIS POPULATIONS IN GREENHOUSE TOMATOES IN ITALY

SUMMARY The quarantine bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is the agent of tomato bacterial canker (TBC), one of the most destructive bacterial diseases of this crop, that causes severe economic losses worldwide. During a recent outbreak of TBC in Sicily (insular Italy), it was impossible to amplify the pat-1 gene by PCR with CMM5/CMM6 primers from affected greenhouse-grown plants from three farms, although Cmm-like colonies were consistently isolated. Microlog metabolic profile, 16S rDNA sequencing and the positive amplification of other Cmm DNA targets supported the identification of the strains as Cmm, suggesting the occurrence of a population lacking the virulence gene pat-1. A detailed phenotypic and molecular characterization of these Cmm strains and their virulence to tomatoes, as compared with Cmm reference strains is reported in this study. Pathogenicity tests revealed the strains under study were able to induce TBC, although delayed wilting symptoms were observed. Strains with or without pat-1 were undistinguishable, based on disease indexes.

Transcriptome analysis of Pseudomonas mediterranea and P. corrugata plant pathogens during accumulation of medium-chain-length PHAs by glycerol bioconversion

Pseudomonas corrugata and P. mediterranea are soil inhabitant bacteria, generally living as endophytes on symptomless plants and bare soil, but also capable of causing plant diseases. They share a similar genome size and a high proteome similarity. P. corrugata produces many biomolecules which play an important role in bacterial cell survival and fitness. Both species produce different medium-chain-length PHAs (mcl-PHAs) from the bioconversion of glycerol to a transparent film in P. mediterranea and a sticky elastomer in P. corrugata. In this work, using RNA-seq we investigated the transcriptional profiles of both bacteria at the early stationary growth phase with glycerol as the carbon source. Quantitative analysis of P. mediterranea transcripts versus P. corrugata revealed that 1756 genes were differentially expressed. A total of 175 genes were significantly upregulated in P. mediterranea, while 217 were downregulated. The largest group of upregulated genes was related to transport systems and stress response, energy and central metabolism, and carbon metabolism. Expression levels of most genes coding for enzymes related to PHA biosynthesis and central metabolic pathways showed no differences or only slight variations in pyruvate metabolism. The most relevant result was the significantly increased expression in P. mediterranea of genes involved in alginate production, an important exopolysaccharide, which in other Pseudomonas spp. plays a key role as a virulence factor or in stress tolerance and shows many industrial applications. In conclusion, the results provide useful information on the co-production of mcl-PHAs and alginate from glycerol as carbon source by P. mediterranea in the design of new strategies of genetic regulation to improve the yield of bioproducts or bacterial fitness.

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.