Echinacea purpurea microbiota: bacterial-fungal interactions and the interplay with host and non-host plant species in vitro dual culture.

Important evidence is reported on the antimicrobial and antagonistic properties of bacterial endophytes in Echinacea purpurea and their role in the modulation of plant synthesis of bioactive compounds. Here, endophytic fungi were isolated from E. purpurea, and the dual culture approach was applied to deepen insights into the complex plant-microbiome interaction network. In vitro experiments were carried out to evaluate the species specificity of the interaction between host (E. purpurea) and non-host (E. angustifolia and Nicotiana tabacum) plant tissues and bacterial or fungal endophytes isolated from living E. purpurea plants to test interactions between fungal and bacterial endophytes. A higher tropism towards plant tissue and growth was observed for both fungal and bacterial isolates compared to controls without plant tissue. The growth of all fungi was significantly inhibited by several bacterial strains that, in turn, were scarcely affected by the presence of fungi. Finally, E. purpurea endophytic bacteria were able to inhibit mycelial growth of the phytopathogen Botrytis cinerea. Bacteria and fungi living in symbiosis with wild Echinacea plants interact with each other and could represent a potential source of bioactive compounds and a biocontrol tool.

Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry.

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

Lepidium meyenii (Maca) does not exert direct androgenic activities.

Maca is the edible root of the Peruvian plant Lepidum meyenii, traditionally employed for its purported aphrodisiac and fertility-enhancing properties. This study aimed at testing the hypothesis that Maca contains testosterone-like compounds, able to bind the human androgen receptor and promote transcription pathways regulated by steroid hormone signaling. Maca extracts (obtained with different solvents: methanol, ethanol, hexane and chloroform) are not able to regulate GRE (glucocorticoid response element) activation. Further experiments are needed to assess which compound, of the several Maca's components, is responsible of the observed in vivo effects.

Mediterranean food and health: building human evidence.

Adherence to a Mediterranean style diet affords protection from degenerative diseases such as cardiovascular disorders and cancer. Identification of the active constituents of the Mediterranean diet is crucial to the formulation of appropriate dietary guidelines. Also, research on the pharmacological properties of the "minor components" of this diet, eg vitamins and polyphenols, is very active and might lead to the formulation of functional foods and nutraceuticals. Even though in vitro data are plentiful, human studies are difficult to perform due to ethical and practical reasons. Yet, intervention trials represent the best approach to validate claims of healthful activities. This article reviews human evidence of the biological properties of olive oil and tomato constituents and illustrates a research approach by which the bioactive elements of a wild plant (Cynara cardunculus) are first studied in vitro to build biochemical evidence, then in vivo to obtain proof of their vasomodularoty activity.

The role of antioxidants in the mediterranean diets: focus on cancer.

The incidence of certain cancers in the Mediterranean area is lower than in other areas of the world (e.g. in northern Europe and the USA). As nutrition and dietary factors comprise one of the three major factors for human carcinogenesis, the hypothesis was formulated that the dietary profile of the Mediterranean diet, rich in antioxidants, might exert preventive actions. Alas, the vast majority of experiments to prove this hypothesis have been obtained in vitro, and most of the necessary information on the absorption, distribution and metabolism of oligonutrients is currently lacking. Yet, even though the exact role of antioxidants in the Mediterranean diet is yet to be fully established, data from observational studies are strong enough to reinforce the notion that a diet low in saturated fat and alcohol and rich in plant food and whole grain, such as the traditional Mediterranean diet, is associated with lower risk of cancer and should be actively promoted.

Molecular variation in plant cell populations evolving in vitro in different physiological contexts.

Previous work has shown the fixation of context-specific random amplified polymorphic DNA (RAPD) patterns in tomato cell cultures grown for 2 years in different hormonal contexts. In this work, RAPD sequences were characterised and RAPD-derived molecular markers used for a further study of variation between and within auto- and auxo-trophic tomato cultures grown in different hormonal equilibria. Results were then compared with those obtained using microsatellite markers located in noncoding regions of differentiation- and hormone-related genes and with those obtained with the external transcribed spacer (ETS) from tomato rDNA. Hybridisation of RAPDs on a tomato genomic DNA bank, or on total DNA after enzymatic digestion, suggested that the markers were repetitive in nature. Sequence analysis. however, showed that the homology between different fragments was due mainly to the presence of homo-AT nucleotide stretches. Moreover, a series of computational methods, such as an information-theory algorithm coupled with AG estimates, suggested that the RAPD fragments isolated in our experiments are noncoding. The amplification of SSR-containing RAPD-derived markers, and of other SSRs located in noncoding regions of tomato functional genes, consistently showed polymorphism between auxo- and auto-trophic somaclones (the latter being either habituated or transgenic for Agrobacterium tumefaciens oncogenes) but not within these same clones. Differences were also found between auxotrophic clones and the differentiated tissue. These findings were confirmed by restriction fragment length polymorphism (RFLP) analysis with the REII repetitive element of the ETS from tomato rDNA, which was isolated during this study. The results obtained suggest a possible role for physiological context in the selection of RAPD patterns during the evolution of tomato cells with different endogenous hormonal equilibria. The results are discussed in terms of a possible role for variation in noncoding regions of hormone-related genes in the adaptation to different physiological contexts.

Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants.

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.

A physiological and molecular analysis of the genus Nicotiana.

An analysis of the evolution of the genus Nicotiana was carried out with physiological and molecular tools. The capacity of explants from seedlings of several species of Nicotiana to differentiate roots or shoots or to habituate was used to ascertain whether the in vitro behavior of species has a nonrandom distribution in the genus. The results obtained allowed us to identify two groups of species, one root-forming prone composed of Paniculatae (subgenus Rustica) and the other composed of Alatae, Repandae, and Noctiflorae (subgenus Petunioides), with a major tendency toward the production of shoots. Habituation capacity was characteristic of species randomly distributed throughout the phylogenetic tree. These data suggest fixation throughout the evolution of coadapted gene complexes (hormone-related genes) involved in the control of developmental processes. RAPDs, on the other hand, used as molecular markers for the clustering of related species, seem entirely coherent both with classical morphological and karyological studies and with in vitro physiological methods, supporting an early subdivision of the whole genus into two diverging developmental patterns.

Genome flux in tomato cell clones cultured in vitro in different physiological equilibria. II. A RAPD analysis of variability.

An analysis of the effect of changing physiological conditions on genome evolution in tomato cell populations has been carried out on long-term in vitro cultured clones grown on different auxin-cytokinin equilibria or selected for low-high competence for active defense against Fusarium oxysporum f.sp. lycopersici. RAPD analysis, confirmed through pattern rehybridization, was used as a random tool to measure the genetic variability. Through the use of a modified ANOVA, variation was shown to depend on both the initial genotype and the physiological conditions. Pattern correlation analysis through a mutual information algorithm suggested the fixation of RAPD patterns specific to physiological equilibria. The results are discussed in view of the possible relevance for evolution at hierarchical levels higher than cell populations. Key words : tomato clones, somaclonal variation, RAPD, coadaptation.

Genome flux in tomato auto- and auxo-trophic cell clones cultured in different auxin/cytokinin equilibria. I. DNA multiplicity and methylation levels.

An analysis of the effect of changing physiological conditions on genetic stability, in terms of epigenetic changes, such as DNA, methylation patterns, and multiplicity of repetitive DNA, was carried out on tomato cell clones grown on media supplemented with different auxin/cytokinin ratios. The effect of endogenous variation in phytohormone equilibria was also indirectly analysed through a comparison of auxotrophic or habituated (autotrophic) cell clones and the differentiated leaf tissue. The data obtained showed significant variation in methylation and multiplicity levels both between clones and between treatments, clearly suggesting a contemporary influence of exogenous hormonal treatments and of the initial/endogenous physiological state of the treated tissue on both phenomena studied.

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis.

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs 'Davis' and 'Red River', respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv 'Red River' can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv 'Davis' the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in 'Davis' cells.

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. I. Selection from a susceptible cultivar for high and low polysaccharide content.

Plant cell walls play a major role in the outcome of host-parasite interactions. Wall fragments released from the plant, and/or the fungal pathogen, can act respectively as endogenous and exogenous elicitors of the defence response, and other wall components, such as callose, lignin, or hydroxyproline-rich glycoproteins, can inhibit pathogen penetration and/or spreading. We have previously demonstrated that calli from tomato cultivars resistant in vivo to Fusarium oxysporum f.sp. lycopersici show a high amount of polysaccharides in vitro. The aim of the present work was to assess the possible role of polysaccharide content and/or synthetic capacity in determining the competence of plant cells for active defence. For this purpose, tomato cell clones with increased and decreased polysaccharide (FL(+), FL(-)) and callose (A(+), A(-)) content have been selected by means of specific stains as visual markers and tested for the effect of these changes on the extent of response to Fusarium. The analysis of several parameters known to be indicative of active defence (cell browning after elicitor treatment, peroxidase and ß-glucanase induction and inhibition of fungal growth in dual culture) clearly shows that FL(+) and A(+) clones have acquired an increased competence for the activation of defence response. The results are thoroughly discussed in terms of an evaluation of the relative importance of constitutive and/or inducible polysaccharide synthetic capacity for plant response to pathogens, and their possible regulation by plant physiological backgrounds.

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici.

With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible ('Red River'), one tolerant ('UC 105') and three resistant ('Davis UC 82', 'Heinz', 'UC 90') tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo - resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.

The pleiotropic phenotype of tomato cells selected for altered response to Fusarium oxysporium f. sp. lycopersici cell wall components.

With the aim of better understanding in vitro host-parasite interactions, tomato cell lines selected for altered response to Fusarium oxysporum f. sp. lycopersici cell wall components were further characterized. Particularly, their behaviour in dual culture in regard to both fungal inhibition and peroxidase activation was analysed and selected, and control cell clones were screened for esopolysaccharide content and toxin tolerance. Interclonal differences in growth response to 2,4-D and DMSO and the capacity to grow on a medium devoid of hormones (habituation) were taken as parameters representative of physiological variability not directly correlated with the response to pathogens. Significant differences between clones selected for increased (F+) and decreased (F-) response to fungal elicitors were found for pathogen inhibition, peroxidase and esopolysaccharide content, toxin tolerance being reduced in F but not significantly different from the control in F+. As expected, clonal variability for the response to 2,4-D and DMSO, although significant, was not connected with hostparasite interactions. The data reported thus show that selection for a character (response to elicitors), probably critical for the response to pathogens, may lead to the recovery of genotypes showing a set of modifications suggestive of a cascade of events leading to active defense.

Correlations between in vivo resistance to Fusarium and in vitro response to fungal elicitors and toxic substances in carnation.

With the aim of ascertaining the existance of a correlation between in vivo resistance to Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic substances, phenylalanine ammonialyase and phytoalexin accumulation, on one hand, and resistance to culture filtrate, on the other, were assayed in "in vitro" cultures of three susceptible and four resistant Dianthus caryophyllus cultivars. Cultivars showing varying degrees of resistance in vivo either tolerated higher culture filtrate concentrations ('Niki') or showed high PAL activity and phytoalexin production when treated with Fusarium elicitor ('Duca'), or responded positively to both treatments ('Mei-Ling', 'Pulcino'). No such responses were shown in tissue cultures of susceptible cultivars. The differential response to the fungal elicitor seemed to be highly specific as genetic differences between cultivars were not observed in tissue cultures treated with other biotic (Phytophthora infestans) and abiotic (HgCl2) elicitors.