Exploring tomato Solanum pennellii introgression lines for residual biomass and enzymatic digestibility traits.

BACKGROUND: Residual biomass production for fuel conversion represents a unique opportunity to avoid concerns about compromising food supply by using dedicated feedstock crops. Developing tomato varieties suitable for both food consumption and fuel conversion requires the establishment of new selection methods. RESULTS: A tomato Solanum pennellii introgression population was assessed for fruit yield, biomass phenotypic diversity, and for saccharification potential. Introgression lines 2-5, 2-6, 6-3, 7-2, 10-2 and 12-4 showed the best combination of fruit and residual biomass production. Lignin, cellulose, hemicellulose content and saccharification rate showed a wide variation in the tested lines. Within hemicellulose, xylose value was high in IL 6-3, IL 7-2 and IL 6-2, whereas arabinose showed a low content in IL 10-2, IL 6-3 and IL 2-6. The latter line showed also the highest ethanol potential production. Alkali pre-treatment resulted in the highest values of saccharification in most of lines tested, suggesting that chemical pretreatment is an important factor for improving biomass processability. Interestingly, extreme genotypes for more than one single trait were found, allowing the identification of better genotypes. Cell wall related genes mapping in genomic regions involved into tomato biomass production and digestibility variation highlighted potential candidate genes. Molecular expression profile of few of them provided useful information about challenged pathways. CONCLUSIONS: The screening of S. pennellii introgression population resulted very useful for delving into complex traits such as biomass production and digestibility. The extreme genotypes identified could be fruitfully employed for both genetic studies and breeding.

Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr), a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola.

AIMS: To investigate low molecular weight compounds produced in vitro by Lysobacter capsici AZ78 and their toxic activity against sporangia of plant pathogenic oomycetes. METHODS AND RESULTS: Assays carried out in vitro showed that L. capsici AZ78 drastically inhibits the growth of plant pathogenic oomycetes. Accordingly, the preventive application of culture filtrates of L. capsici AZ78 on grapevine and tomato plants reduced the infections, respectively, caused by Plasmopara (Pl.) viticola and Phytophthora infestans. The subsequent chemical analysis of the culture filtrates of L. capsici AZ78 by spectroscopic (essentially 1D and 2D (1)H NMR and (13)C NMR and ESI MS spectra) and optical methods led to the identification of the 2,5-diketopiperazine cyclo(L-Pro-L-Tyr) that inhibited the development of P. infestans sporangia in vitro and on tomato leaves. Furthermore, a genomic region with high sequence identity with genes coding for a hybrid polyketide synthase and nonribosomal peptide synthetase was detected in L. capsici AZ78. CONCLUSIONS: Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr) in vitro that was effective in killing the sporangia of P. infestans and Pl. viticola in vitro. Moreover, this low molecular weight compound prevents the occurrence of late blight lesions when applied on tomato leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of L. capsici AZ78 cells or its own culture filtrates effectively controls both P. infestans and Pl. viticola. Cyclo(L-Pro-L-Tyr) produced by L. capsici AZ78 is toxic against sporangia of both these oomycetes. These data enforce the potential in the use of Lysobacter members for the control of plant pathogenic oomycetes and provide the basis for the development of new low-impact fungicides based on cyclo(L-Pro-L-Tyr).

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity.

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca(2+)-activated K(+) channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.

Phenazines and cancer.

Phenazines are a large group of natural and synthesised nitrogen-containing heterocycles, including more than 100 different compounds of natural origin and over 6000 synthetic compounds. Many of these compounds have been investigated as potential anti-cancer agents. Despite a large number of research publications, no recent attempt to summarise and critically evaluate the experimental findings relating to the anti-cancer activity of this class of compounds has been made. The present review fills this gap in the literature and discusses both natural and synthetic phenazines with a critical focus on in vitro, in vivo and available clinical anti-cancer activities of these compounds.

Lycorine and its derivatives for anticancer drug design.

Amaryllidaceae alkaloids are extensively studied for their biological activities in several pharmaceutical areas, including, for example, Alzheimer's disease for which galanthamine has already reached the market. Among this chemical family, lycorine displays very promising anti-tumor properties. This review first focuses on the chemical diversity of natural and synthetic analogues of lycorine and their metabolites, and then on mechanisms of action and biological targets through which lycorine and its derivatives display their anti-tumor activity. Our analysis of the structure-activity relationships of this family of compounds highlights the existence of various potential leads for the development of novel anticancer agents.

A New Flow Cytometry Technique to Identify Phaeomoniella chlamydospora Exopolysaccharides and Study Mechanisms of Esca Grapevine Foliar Symptoms.

A flow cytometry technique that unequivocally identifies some of the toxic metabolites produced by Phaeomoniella chlamydospora, one of the main fungal pathogens causing esca disease of grapevine (Vitis vinifera), was developed. Antibodies raised against exopolysaccharides (EPS)-metabolites produced by Pa. chlamydospora that have been reported to be phytotoxic-were used as antigen to immunize rats. The specificity of these antibodies was assayed by flow cytometry against Pa. chlamydospora polysaccharides and against EPS with a different structure isolated from other phytopathogenic fungi, including Phaeoacremonium aleophilum and the Botryosphaeriaceae species Neofusicoccum luteum and N. parvum. Using this method, Pa. chlamydospora polysaccharides were detected in the symptomatic leaves of esca-affected grapevines, while healthy and asymptomatic leaves from both healthy and diseased vines did not produce a binding reaction. This method potentially could be used to develop a simple kit to study the mechanisms underlying the development of esca foliar symptoms and to indirectly assess the presence of Pa. chlamydospora in grapevine material.

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes.

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.

Structural determination of the phytotoxic mannan exopolysaccharide from Pseudomonas syringae pv. ciccaronei.

The structural determination was performed of a mannan exopolysaccharide from the gram negative bacterium Pseudomonas syringae pv. ciccaronei, which is the pathogenic agent responsible for the leaf spots of carob plants. The structure, obtained by chemical, enzymatic and spectroscopic methods, consisted of a backbone of alpha-(1-->6)-linked mannopyranose units with 80% substituted at C-2 by mono-, di- and trisaccharide side chains. In addition, terminal glucose units and phosphate groups were found to be present. This is, to the best of our knowledge, the first report of a mannan exopolysaccharide structure from a phytopathogenic bacterium. The pure polysaccharide showed phytotoxic effects, i.e., chlorosis and necrosis on tobacco leaves.

Determination of Ascochyta caulina phytotoxins by high-performance anion exchange chromatography and pulsed amperometric detection.

A simple and sensitive method has been developed for the rapid qualitative and quantitative analysis of the phytotoxins produced by Ascochyta caulina, a potential mycoherbicide for the biocontrol of Chenopodium album. Considering that the two main toxins produced by this fungus, namely ascaulitoxin and trans-4-amino-D-proline, and the third toxin, identified as 2,4,7-triamino-5-hydroxyoctandioic acid, have an amino acid nature, high-performance anion exchange chromatography with pulsed amperometric detection was the best method for their detection. The method was used to measure the toxin content in the culture filtrates of different strains of A. caulina. The developed method could be employed as a tool to select more virulent strains by determining the higher toxin producers, if in vitro toxin accumulation was related to pathogen virulence. The chemical characterisation of the third toxin purified from A. caulina culture filtrates is also reported.

Immunostimulant (1 --> 3)-D-glucans from the cell wall of Cryphonectria parasitica (Murr.) Barr strain 263.

A (1 --> 3)-beta-D-glucan with approximately 30% of the residues having a beta-D-Glc-(1 --> 6) branch is the main water-soluble component of the cell wall polysaccharide of Cryphonectria parasitica (Murr.) Barr strain 263. A (1 --> 3)-glucan with both alpha and beta anomeric linkages was found in the water-insoluble polysaccharide fraction. Both fractions possess immunological activity, being able to induce the production of either tumour necrosis factor alpha (TNF-alpha) or nitrite (NO2-).

O-specific polysaccharide structure of the aqueous lipopolysaccharide fraction from Xanthomonas campestris pv. vitians strain 1839.

The structure of Xanthomonas campestris pv. vitians O-specific polysaccharide of the lipopolysaccharide fraction, extracted from the aqueous phase, was defined, on the basis of chemical and spectroscopical methods, as constituted by the following repeating unit: [--> 3)-alpha-L-Rhap-(1 -->]n 3)-beta-L-Rhap-(1 --> where n is more frequently equal to 2, but it also assumes values equal to 1 and to 3.

Biological characterization of fusapyrone and deoxyfusapyrone, two bioactive secondary metabolites of Fusarium semitectum.

Fusapyrone (1) and deoxyfusapyrone (2), two alpha-pyrones originally isolated from rice cultures of Fusarium semitectum, were tested in several biological assays. Compounds 1 and 2 showed considerable antifungal activity against several plant pathogenic and/or mycotoxigenic filamentous fungi, although they were inactive toward yeasts isolated from plants and the Gram-positive bacterium Bacillus megaterium in disk diffusion assays. Compound 1 was consistently more active than 2. Among the tested fungi, Fusarium species were the least sensitive to the two pyrones, while Alternaria alternata, Ascochyta rabiei, Aspergillusflavus, Botrytis cinerea, Cladosporium cucumerinum, Phoma tracheiphila, and Penicillium verrucosum were the most sensitive. Compounds 1 and 2 also showed good inhibitory activity toward agents of human mycoses. Aspergilli were the most sensitive, while some species-specific variability was found among the Candida spp. In an Artemia salina larvae bioassay, 1 was not toxic at the highest concentration tested (500 microM), whereas the LC(50) of 2 was 37.1 microM (21.8 microg/mL). Neither 1 nor 2 was phytotoxic in a panel of assays that monitored plant-cell toxicity, as well as wilt-, chlorosis-, and necrosis-inducing activity. Moreover, 2 stimulated the root elongation of tomato seedlings at doses of 10 and 100 microM. In consideration of the biological activities evidenced in this study, 1 and 2 appear to be potential candidates for biotechnological applications, as well as good models for studies on mechanism(s) of action and structure-activity relationships.

Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B.

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.

Studies toward the synthesis of pinolidoxin, a phytotoxic nonenolide from the fungus Ascochyta pinodes. Determination of the configuration at the C-7, C-8, and C-9 chiral centers and stereoselective synthesis of the C(6)-C(18) fragment.

The absolute stereochemistry at the C-7, C-8, and C-9 chiral centers of pinolidoxin (1) has been determined by chemical and spectral methods. First, the synthesis of four stereoisomeric fully benzoylated 2,3-erythro-1,2,3,4-heptanetetrols, corresponding to the C(6)-C(18) portion of the natural substance, has been accomplished starting from meso-tartaric acid. As next step, the selection of the synthetic tetrabenzoate possessing "natural" stereochemistry (10a'), suitable for absolute configuration determination, has been carried out by correlation with its "natural" homologue derived from degradation of pinolidoxin. Determination of the stereochemistry at the title chiral centers has been carried out by application of the Mosher's method both to 7a', a compound stereochemically related to 10a', and to pinolidoxin itself. The stereoselective synthesis of a protected form of the C(6)-C(18) portion of pinolidoxin, to be used in its total synthesis, has also been accomplished starting from commercially available D-erythronolactone.

Structure elucidation of the O-chain from the major lipopolysaccharide of the Xanthomonas campestris strain 642.

A novel O-polysaccharide consisting of D-Xylp and L-Rhap in the molar ratio of 1:2.5 was identified as the major component in the lipopolysaccharide fraction of Xanthomonas campestris strain 642, which is responsible for a new bacterial disease of the strawberry plant. Its structure was mainly determined using chemical analysis, Smith degradation and 1D and 2D NMR spectroscopy experiments as: carbohydrate sequence [see text].

Trans-4-aminoproline, a phytotoxic metabolite with herbicidal activity produced by Ascochyta caulina.

A phytotoxic metabolite, characterized through NMR techniques and synthetic methods as trans-4-aminoproline, was isolated from the culture filtrates of Ascochyta caulina, a promising mycoherbicide for biological control of Chenopodium album. The metabolite, which shows interesting phytotoxic properties, together with ascaulitoxin (recently characterized as N.2-beta-D-glucoside of the unusual bis-amino acid 2,4,7-triamino-5-hydroxyoctandioc acid) and another unidentified compound, compose an active fraction of A. caulina culture filtrates with promising herbicidal properties. When assayed on leaves of host and non host dicots, including wild and cultivated plants, the trans-4-aminoproline showed a wide range of toxicity, with leaves of C. album being the most sensitive. Other interesting aspects were its inefficacy on several monocots, both cultivated and wild, and its lack of antifungal, antibiotic and zootoxic activities. This is the first report on trans-4-aminoproline as naturally occurring compound and phytotoxic metabolite produced by A. caulina.

Isolation and characterisation of the lipopolysaccharide from Xanthomonas hortorum pv. vitians.

Xanthomonas hortorum pv. vitians is a Gram-negative bacterium that acts as the causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide (LPS) of this bacterium is suspected to be an important molecule for adhesion to the plants. We have isolated the LPS, prepared the lipid A and the polysaccharide moieties thereof, and characterised all preparations by compositional analysis. Main sugar components are rhamnose and 3-acetamido-3,6-dideoxy-galactose which presumably furnish the O-specific polysaccharide. Other sugars are mannose, glucose, 6-deoxygalactose (fucose), and galacturonic acid, which should be core region constituents, and glucosamine, which builds up the carbohydrate backbone of lipid A. The LPS contains several phosphate groups, most of which are present in the core region. The main fatty acids in the lipid A are C10:0, 3-OH-C10:0 and 3-OH-C12:0. The latter is the only amide-linked fatty acid. Two fatty acids present in small amounts were identified, C8:0 and C11:0.

A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste waters.

Hydroxytyrosol, a polyphenol with very interesting antioxidant properties, which naturally occurs in virgin olive oil and mainly in olive oil mill waste waters, was synthesized by reducing 3, 4-dihydroxyphenylacetic acid with LiAlH(4) in tetrahydrofuran under refluxing for 2 h. The yield of reaction was 82.8%. The spectroscopic and HPLC data of the synthesized compound proved to coincide fully with those of a pure sample obtained by the chromatographic recovery from olive oil mill waste waters (yield = 91 mg/L). This synthetic method appears to be the most convenient compared with those reported in the literature and is more convenient than the chromatographic recovery. The tri- and diacetyl derivatives of the synthetic compound were also prepared for structure-bioactivity relationship studies. A brief discussion is given on the economical and ecological aspects regarding the production of hydroxytyrosol.

Elicitin 172 from an isolate of Phytophthora nicotianae pathogenic to tomato.

Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae, the causal agent of crown and root rot of tomato (Lycopersicon esculentum). The M(r) (10,349 +/- 1) of the purified protein, determined by ES-MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI-MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae, as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum (Capsicum annuum) and vegetable marrow (Cucurbita pepo) from P. capsici.

Structural analysis of a novel putative capsular polysaccharide from Pseudomonas (Burkholderia) caryophylli strain 2151.

A novel putative capsular polysaccharide consisting of D-Glcp and D-Fruf in the molar ratio of 1:1 was isolated as minor constituent from the lipopolysaccharide (LPS) fraction of Pseudomonas (Burkholderia) caryophylli. Its structure was determined, using mainly one- and two-dimensional NMR spectroscopy, as: -->6)-alpha-D-Glcp-(1-->1)-beta-D-Fruf-(2-->.