NMR Metabolomics and Chemometrics of Lettuce, Lactuca sativa L., under Different Foliar Organic Fertilization Treatments.

Lettuce plants were grown in a greenhouse affected by the fungal pathogen Fusarium oxysporum to test the effects on plant metabolomics by different organic treatments. Three foliar application treatments were applied: a commercial compost tea made of aerobically fermented plant organic matter, a pure lyophilized microalga Artrospira platensis, commonly named spirulina, and the same microalga previously exposed during its culture to a natural uptake from medium enriched with F. oxysporum fragmented DNA (NAT). The experiment is the first attempt to observe in field conditions, the use and effects of a natural microbial library as a carrier of pathogenic fungal DNA for disease control. Untargeted NMR metabolomics and chemometrics showed that foliar organic application significantly reduced fumaric and formic acids, aromatic amino acids, and nucleosides, while increasing ethanolamine. A strong decrease in phenolic acids and an increase in citric acid and glutamine were specifically observed in the NAT treatment. It is noteworthy that the exposure of a known biostimulant microalga to fungal DNA in its culture medium was sufficient to induce detectable changes in the metabolomic profiles of the fertilized plants. These findings deserve further investigation to assess the potential relevance of the presented approach in the field of crop biostimulation and biocontrol of plant pathogens.

Plant Dynamic Metabolic Response to Bacteriophage Treatment After Xanthomonas campestris pv. campestris Infection.

Periodic epidemics of black rot disease occur worldwide causing substantial yield losses. Xanthomonas campestris pv. campestris (Xcc) represents one of the most common bacteria able to cause the above disease in cruciferous plants such as broccoli, cabbage, cauliflower, and Arabidopsis thaliana. In agriculture, several strategies are being developed to contain the Xanthomonas infection. The use of bacteriophages could represent a valid and efficient approach to overcome this widespread phenomenon. Several studies have highlighted the potential usefulness of implementing phage therapy to control plant diseases as well as Xcc infection. In the present study, we characterized the effect of a lytic phage on the plant Brassica oleracea var. gongylodes infected with Xcc and, for the first time, the correlated plant metabolic response. The results highlighted the potential benefits of bacteriophages: reduction of bacterium proliferation, alteration of the biofilm structure and/or modulation of the plant metabolism and defense response.

Antibiofilm Activity of a Trichoderma Metabolite against Xanthomonas campestris pv. campestris, Alone and in Association with a Phage.

Biofilm protects bacteria against the host's immune system and adverse environmental conditions. Several studies highlight the efficacy of lytic phages in the prevention and eradication of bacterial biofilms. In this study, the lytic activity of Xccφ1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-α-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a φHA6PP complex, was investigated using confocal laser microscopy under static and dynamic conditions. Additionally, the mechanism used by the complex to modulate the genes (rpf, gumB, clp and manA) involved in the biofilm formation and stability was also studied. Our results demonstrated that Xccφ1, alone or in combination with 6PP and HA, interfered with the gene pathways involved in the formation of biofilm. This approach can be used as a model for other biofilm-producing bacteria.

WRKY genes family study reveals tissue-specific and stress-responsive TFs in wild potato species.

Wild potatoes, as dynamic resource adapted to various environmental conditions, represent a powerful and informative reservoir of genes useful for breeding efforts. WRKY transcription factors (TFs) are encoded by one of the largest families in plants and are involved in several biological processes such as growth and development, signal transduction, and plant defence against stress. In this study, 79 and 84 genes encoding putative WRKY TFs have been identified in two wild potato relatives, Solanum commersonii and S. chacoense. Phylogenetic analysis of WRKY proteins divided ScWRKYs and SchWRKYs into three Groups and seven subGroups. Structural and phylogenetic comparative analyses suggested an interspecific variability of WRKYs. Analysis of gene expression profiles in different tissues and under various stresses allowed to select ScWRKY045 as a good candidate in wounding-response, ScWRKY055 as a bacterial infection triggered WRKY and ScWRKY023 as a multiple stress-responsive WRKY gene. Those WRKYs were further studied through interactome analysis allowing the identification of potential co-expression relationships between ScWRKYs/SchWRKYs and genes of various pathways. Overall, this study enabled the discrimination of WRKY genes that could be considered as potential candidates in both breeding programs and functional studies.

Fusarium oxysporum f.sp. radicis-lycopersici induces distinct transcriptome reprogramming in resistant and susceptible isogenic tomato lines.

BACKGROUND: Fusarium oxysporum f.sp. radicis-lycopersici (FORL) is one of the most destructive necrotrophic pathogens affecting tomato crops, causing considerable field and greenhouse yield losses. Despite such major economic impact, little is known about the molecular mechanisms regulating Fusarium oxysporum f.sp. radicis-lycopersici resistance in tomato. RESULTS: A transcriptomic experiment was carried out in order to investigate the main mechanisms of FORL response in resistant and susceptible isogenic tomato lines. Microarray analysis at 15 DPI (days post inoculum) revealed a distinct gene expression pattern between the two genotypes in the inoculated vs non-inoculated conditions. A model of plant response both for compatible and incompatible reactions was proposed. In particular, in the incompatible interaction an activation of defense genes related to secondary metabolite production and tryptophan metabolism was observed. Moreover, maintenance of the cell osmotic potential after the FORL challenging was mediated by a dehydration-induced protein. As for the compatible interaction, activation of an oxidative burst mediated by peroxidases and a cytochrome monooxygenase induced cell degeneration and necrosis. CONCLUSIONS: Our work allowed comprehensive understanding of the molecular basis of the tomato-FORL interaction. The result obtained emphasizes a different transcriptional reaction between the resistant and the susceptible genotype to the FORL challenge. Our findings could lead to the improvement in disease control strategies.

Proteomic investigation of response to FORL infection in tomato roots.

Fusarium oxysporum f. sp. radicis-lycopersici (FORL) leading to fusarium crown and root rot is considered one of the most destructive tomato soilborne diseases occurring in greenhouse and field crops. In this study, response to FORL infection in tomato roots was investigated by differential proteomics in susceptible (Monalbo) and resistant (Momor) isogenic tomato lines, thus leading to identify 33 proteins whose amount changed depending on the pathogen infection, and/or on the two genotypes. FORL infection induced accumulation of pathogen-related proteins (PR proteins) displaying glucanase and endochitinases activity or involved in redox processes in the Monalbo genotype. Interestingly, the level of the above mentioned PR proteins was not influenced by FORL infection in the resistant tomato line, while other proteins involved in general response mechanisms to biotic and/or abiotic stresses showed significant quantitative differences. In particular, the increased level of proteins participating to arginine metabolism and glutathione S-transferase (GST; EC 2.5.1.18) as well as that of protein LOC544002 and phosphoprotein ECPP44-like, suggested their key role in pathogen defence.

Insights on the susceptibility of plant pathogenic fungi to phenazine-1-carboxylic acid and its chemical derivatives.

Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phenazine compounds as main secondary metabolites. These metabolites were identified as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this study, the spectrum of the activity of PCA and 2-OH P was evaluated against a group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA was active against most of the tested plant pathogens, while 2-OH P slightly inhibited a few fungal species. Furthermore, four semisynthesised derivatives of PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their antifungal activity against 11 phytopathogenic species. Results showed that the carboxyl group is a structural feature important for the antifungal activity of PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1-carboxamide, the two more lipophilic and reversible PCA derivatives remained substantially unaltered compared with PCA.

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71.

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide. A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.

Host and non-host plant response to bacterial wilt in potato: role of the lipopolysaccharide isolated from Ralstonia solanacearum and molecular analysis of plant-pathogen interaction.

Ralstonia solanacearum is one of the most devastating phytopathogenic bacteria, in particular its race 3. This microorganism is the causal agent of destructive diseases of different crops including tomato and potato. An important aspect of the interaction between this pathogen, and the host and non-host plants was its biochemical and molecular basis. Thus, the lipopolysaccharides (LPS) were extracted from the R. solanacearum cell wall, purified, and the O-specific polysaccharide (OPS) was isolated and chemically characterized by compositional analyses and NMR spectroscopy. The OPS was constituted of two linear polymers of an approximate ratio of 3 : 1, both of which were built up from three rhamnose and one N-acetylglucosamine residues and differed only in the substitution of one rhamnose residue. The LPS inhibited the hypersensitivity reaction (HR) in non-host tobacco plants and induced localized resistance in host potato plants, both of which were pre-treated with the LPS before being inoculated with the pathogen. A cDNA-AFLP approach was used to study transcriptome variation during the resistant and susceptible interactions. This revealed the presence of metabolites specifically expressed in the S. commersonii-resistant genotypes, which could be involved in the plant-pathogen incompatible reaction. Furthermore, a specific EST collection of the Ralstonia-potato interaction has been built up.

Polymorphisms in nuclear rDNA and mtDNA reveal the polyphyletic nature of isolates of Phomopsis pathogenic to sunflower and a tight monophyletic clade of defined geographic origin.

The molecular diversity of Diaporthe helianthi (anamorph Phomopsis helianthi), the causal agent of sunflower stem canker, was studied in 16 isolates of different geographic origin using nuclear and mitochondrial markers. PCR products corresponding to the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA gene, and to the mitochondrial atp6 gene were sequenced. The ITS1 and ITS2 sequences were compared with those of Phomopsis spp. and Diaporthe spp. obtained from databases. The diversity in the region surrounding the atp6 gene was also studied by restriction analysis using four enzymes. The analyses revealed a marked diversity within the sunflower-isolated strains, which appear to belong to phylogenetically unrelated groups. Noticeably, all the isolates collected in France and in the former Yugoslavia, where severe epiphytotics of sunflower stem canker are frequently reported, showed high similarity to each other forming a clade which clearly differentiated from all other ones within the genus Phomopsis. Conversely, all the isolates collected in Italy, where, despite favourable environmental conditions, the incidence of the disease is low, were only distantly related to the former group and showed sequence similarity with other previously established phylogenetic clades within the Phomopsis/Diaporthe complex.

Structural elucidation of a novel core oligosaccharide backbone of the lipopolysaccharide from the new bacterial species Agrobacterium larrymoorei.

Agrobacterium larrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically. The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria. The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS. The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A. larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: [carbohydrate structure: see text] All sugars are alpha-D-pyranoses if not stated otherwise, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA.