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The aim of this research was to use a polyphasic approach to differentiate Penicillium verrucosum from Penicillium nordicum, to compare different techniques, and to select the most suitable for industrial use. In particular, (1) a cultural technique with two substrates selective for these species; (2) a molecular diagnostic test recently set up and a RAPD procedure derived from this assay; (3) an RP-HPLC analysis to quantify ochratoxin A (OTA) production and (4) an automated system based on fungal carbon source utilisation (Biolog Microstation™) were used. Thirty strains isolated from meat products and originally identified as P. verrucosum by morphological methods were re-examined by newer cultural tests and by PCR methods. All were found to belong to P. nordicum. Their biochemical and chemical characterisation supported the results obtained by cultural and molecular techniques and showed the varied ability in P. verrucosum and P. nordicum to metabolise carbon-based sources and to produce OTA at different concentrations, respectively. [ABSTRACT FROM AUTHOR]

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Cucurbitacins, structurally different triterpenes mainly found in the members of Cucurbitaceae, possess a vast pharmacological potential. Genus Cucurbita , Cucumis, and Citrullus are affluent in these bioactive compounds, and, amongst them, Citrullus colocynthis (L.) Schrad. is widely exploited in folk medicine, since a huge number of diseases are successfully treated with organic and aqueous extracts obtained from different organs and tissues of the plant. The well-known pharmacological activities of such species have been attributed to its peculiar composition, which includes cucurbitacins and other bioactive molecules; thus, owing to its high importance as a valuable natural resource for pharmaceuticals and nutraceuticals, C. colocynthis propagation and multiplication protocols are considered significant, but the exploitation of its phytochemical potential is limited by the restricted cultivation conditions and the low rate of seed germination in the natural environment; in fact, the assessment of accumulation rate of specific phytochemicals under controlled conditions is still missing. Axenically sprouted plantlets obtained without the use of culture media or the addition of hormones have been evaluated here for the production of bioactive compounds and relevant bioactive features. Our results proved that derived organic extracts contain cucurbitacins and other bioactives, show antioxidant potential, and exert activity against some pathogenic fungi ( Candida krusei, C. albicans, C. parapsilosis, C. glabrata , and Aspergillus flavus ), supporting the feasibility of a methodology intended to scale-up cultivation of this species as a source of pharmaceutically interesting compounds, achievable from plantlets cultivated under laboratory conditions.

Crop yield and plant products quality are directly or indirectly affected by climate alterations. Adverse climatic conditions often promote the occurrence of different abiotic stresses, which can reduce or enhance the susceptibility to pests or pathogens. Aflatoxin producing fungi, in particular, whose diffusion and deleterious consequences on cereals commodities have been demonstrated to highly depend on the temperature and humidity conditions that threaten increasingly larger areas. Biological methods using intraspecific competitors to prevent fungal development and/or toxin production at the pre-harvest level are particularly promising, even if their efficacy could be affected by the ecological interaction within the resident microbial population. A previously characterized Aspergillus flavus atoxigenic strain was applied in two maize fields to validate its effectiveness as a biocontrol agent against aflatoxin contamination. At one month post-application, at the harvest stage, its persistence within the A. flavus population colonizing the maize kernels in the treated area was assessed, and its efficacy was compared in vitro with a representation of the isolated atoxigenic population. Results proved that our fungal competitor contained the aflatoxin level on maize grains as successfully as a traditional chemical strategy, even if representing less than 30% of the atoxigenic strains re-isolated, and achieved the best performance (in terms of bio-competitive potential) concerning endogenous atoxigenic isolates.

Beneficial interactions between plants and some bacterial species have been long recognized, as they proved to exert various growth-promoting and health-protective activities on economically relevant crops. In this study, the growth promoting and antifungal activity of six bacterial strains, Paenarthrobacter ureafaciens , Beijerinckia fluminensis , Pseudomonas protegens , Arthrobacter sp., Arthrobacter defluii , and Arthrobacter nicotinovorans , were investigated. The tested strains resulted positive for some plant growth promoting (PGP) traits, such as indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate-deaminase (ACC-deaminase), siderophore production, and solubilization of phosphates. The effect of the selected bacteria on Arabidopsis thaliana seedlings growth was assessed using different morphological parameters. Bacterial activity against the phytopathogenic fungal species Aspergillus flavus , Fusarium proliferatum , and Fusarium verticillioides was also assessed, since these cause major yield losses in cereal crops and are well-known mycotoxin producers. Strains Pvr_9 ( B. fluminensis ) and PHA_1 ( P. protegens ) showed an important growth-promoting effect on A. thaliana coupled with a high antifungal activity on all the three fungal species. The analysis of bacterial broths through ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) confirmed the presence of potential PGP-compounds, among these are desferrioxamine B, aminochelin, asperchrome B, quinolobactin siderophores, and salicylic acid.

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus , represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the "virological" perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus , opening new and intriguing hypotheses about the ecological significance of mycoviruses.

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox ® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.

Usually regarded as less evolved than their more recently diverged vascular sisters, which currently dominate vegetation landscape, bryophytes seem having nothing to envy to the defensive arsenal of other plants, since they had acquired a suite of chemical traits that allowed them to adapt and persist on land. In fact, these closest modern relatives of the ancestors to the earliest terrestrial plants proved to be marvelous chemists, as they traditionally were a popular remedy among tribal people all over the world, that exploit their pharmacological properties to cure the most different diseases. The phytochemistry of bryophytes exhibits a stunning assortment of biologically active compounds such as lipids, proteins, steroids, organic acids, alcohols, aliphatic and aromatic compounds, polyphenols, terpenoids, acetogenins and phenylquinones, thus it is not surprising that substances obtained from various species belonging to such ancestral plants are widely employed as antitumor, antipyretic, insecticidal and antimicrobial. This review explores in particular the antifungal potential of the three Bryophyta divisions-mosses (Musci), hornworts (Anthocerotae) and liverworts (Hepaticae)-to be used as a sources of interesting bioactive constituents for both pharmaceutical and agricultural areas, providing an updated overview of the latest relevant insights.

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum , Neofusicoccum parvum , Phaeomoniella chlamydospora , Fomitiporia mediterranea , Fusarium poae , F. culmorum , F. graminearum , F. oxysporum f. sp. lactucae, F. sporotrichioides , Aspergillus flavus , Rhizoctonia solani, Sclerotinia spp. and Verticillium dahliae . The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides , also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi.

Great are the expectations for a new generation of antimicrobials, and strenuous are the research efforts towards the exploration of diverse molecular scaffolds-possibly of natural origin - aimed at the synthesis of new compounds against the spread of hazardous fungi. Also high but winding are the paths leading to the definition of biological targets specifically fitting the drug's structural characteristics. The present study is addressed to inspect differential biological behaviours of cinnamaldehyde and benzaldehyde thiosemicarbazone scaffolds, exploiting the secondary metabolism of the mycotoxigenic phytopathogen Aspergillus flavus. Interestingly, owing to modifications on the parent chemical scaffold, some thiosemicarbazones displayed an increased specificity against one or more developmental processes (conidia germination, aflatoxin biosynthesis, sclerotia production) of A. flavus biology. Through the comparative analysis of results, the ligand-based screening strategy here described has allowed us to delineate which modifications are more promising for distinct purposes: from the control of mycotoxins contamination in food and feed commodities, to the environmental management of microbial pathogens, to the investigation of specific structure-activity features for new generation drug discovery.

Nanoparticles are widely studied for applications in medical science. In recent years, they have been developed for agronomical purposes to target microbial pest such as bacteria, fungi, and viruses. Nanoparticles are also proposed to limit the use of pesticides, whose abuse is causing environmental impact and human health concerns. In this study, nanoparticles were obtained by using poly-(ε-caprolactone), a polyester chosen for its biocompatibility and biodegradability properties. Poly-(ε-caprolactone) nanoparticles were formulated by using poly(vinyl alcohol) or Pluronic® F127 as non-ionic surfactants, and then loaded with benzophenone or valerophenone thiosemicarbazone, two compounds that inhibit aflatoxin production by Aspergillus flavus. The different types of nanoparticles were compared in terms of size, polydispersity index, morphology, and drug loading capacity. Finally, their effects were investigated on growth, development, and aflatoxin production in the aflatoxigenic species Aspergillus flavus, a ubiquitous contaminant of maize, cereal crops, and derived commodities. Aflatoxin production was inhibited to various extents, but the best inhibitory effect was obtained with respect to sclerotia production that was most effectively suppressed by both benzophenone and valerophenone thiosemicarbazone-loaded nanoparticles. These data support the idea that it is possible to use such nanoparticles as an alternate to pesticides for the control of mycotoxigenic sclerotia-forming fungi.

Aflatoxins are secondary fungal metabolites that can contaminate feed and food. They are a cause of growing concern worldwide, because they are potent carcinogenic agents. Thiosemicarbazones are molecules that possess interesting antiaflatoxigenic properties, but in order to use them as crop-protective agents, their cytotoxic and genotoxic profiles must first be assessed. In this paper, a group of thiosemicarbazones and a copper complex are reported as compounds able to antagonize aflatoxin biosynthesis, fungal growth, and sclerotia biogenesis in Aspergillus flavus . The two most interesting thiosemicarbazones found were noncytotoxic on several cell lines (CRL1790, Hs27, HFL1, and U937), and therefore, they were submitted to additional analysis of mutagenicity and genotoxicity on bacteria, plants, and human cells. No mutagenic activity was observed in bacteria, whereas genotoxic activity was revealed by the Alkaline Comet Assay on U937 cells and by the test of chromosomal aberrations in Allium cepa .

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus : compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.

Citrullus colocynthis L. Schrader is an annual plant belonging to the Cucurbitaceae family, widely distributed in the desert areas of the Mediterranean basin. Many pharmacological properties (anti-inflammatory, anti-diabetic, analgesic, anti-epileptic) are ascribed to different organs of this plant; extracts and derivatives of C. colocynthis are used in folk Berber medicine for the treatment of numerous diseases-such as rheumatism arthritis, hypertension bronchitis, mastitis, and even cancer. Clinical studies aimed at confirming the chemical and biological bases of pharmacological activity assigned to many plant/herb extracts used in folk medicine often rely on results obtained from laboratory preliminary tests. We investigated the biological activity of some C. colocynthis stem, leaf, and root extracts on the mycotoxigenic and phytopathogenic fungus Aspergillus flavus , testing a possible correlation between the inhibitory effect on aflatoxin biosynthesis, the phytochemical composition of extracts, and their in vitro antioxidant capacities.

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The issue of food contamination by aflatoxins presently constitutes a social emergency, since they represent a severe risk for human and animal health. On the other hand, the use of pesticides has to be contained, since this generates long term residues in food and in the environment. Here we present the synthesis of a series of chelating ligands based on the thiosemicarbazone scaffold, to be evaluated for their antifungal and antiaflatoxigenic effects. Starting from molecules of natural origin of known antifungal properties, we introduced the thio- group and then the corresponding copper complexes were synthesised. Some molecules highlighted aflatoxin inhibition in the range 67-92% at 100 μM. The most active compounds were evaluated for their cytotoxic effects on human cells. While all the copper complexes showed high cytotoxicity in the micromolar range, one of the ligand has no effect on cell proliferation. This hit was chosen for further analysis of mutagenicity and genotoxicity on bacteria, plants and human cells. Analysis of the data underlined the importance of the safety profile evaluation for hit compounds to be developed as crop-protective agents and at the same time that the thiosemicarbazone scaffold represents a good starting point for the development of aflatoxigenic inhibitors.

Aspergillus flavus is an opportunistic mold that represents a serious threat for human and animal health due to its ability to synthesize and release, on food and feed commodities, different toxic secondary metabolites. Among them, aflatoxin B1 is one of the most dangerous since it is provided with a strong cancerogenic and mutagenic activity. Controlling fungal contamination on the different crops that may host A. flavus is considered a priority by sanitary authorities of an increasing number of countries due also to the fact that, owing to global temperature increase, the geographic areas that are expected to be prone to experience sudden A. flavus outbreaks are widening. Among the different pre- and post-harvest strategies that may be put forward in order to prevent fungal and/or mycotoxin contamination, fungicides are still considered a prominent weapon. We have here analyzed different structural modifications of a natural-derived compound (cuminaldehyde thiosemicarbazone) for their fungistatic and anti-aflatoxigenic activity. In particular, we have focused our attention on one of the compound that presented a prominent anti-aflatoxin specificity, and performed a set of physiological and molecular analyses, taking also advantage of yeast (Saccharomyces cerevisiae) cell as an experimental model.

Aflatoxins represent a serious problem for a food economy based on cereal cultivations used to fodder animal and for human nutrition. The aims of our work are two-fold: first, to perform an evaluation of the activity of newly synthesized thiosemicarbazone compounds as antifungal and anti-mycotoxin agents and, second, to conduct studies on the toxic and genotoxic hazard potentials with a battery of tests with different endpoints. In this paper we report an initial study on two molecules: S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazone and its metal complex, bis(S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazonato)nickel (II). The outcome of the assays on fungi growth and aflatoxin production inhibition show that both molecules possess good antifungal activities, without inducing mutagenic effects on bacteria. From the assays to ascertain that the compounds have no adverse effects on human cells, we have found that they are cytotoxic and, in the case of the nickel compound, they also present genotoxic effects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In Fabaceae, dispersion of forisomes-highly ordered aggregates of sieve element proteins-in response to phytoplasma infection was proposed to limit phloem mass flow and, hence, prevent pathogen spread. In this study, the involvement of filamentous sieve element proteins in the containment of phytoplasmas was investigated in non-Fabaceae plants. Healthy and infected Arabidopsis plants lacking one or two genes related to sieve element filament formation-AtSEOR1 (At3g01680), AtSEOR2 (At3g01670), and AtPP2-A1 (At4g19840)-were analysed. TEM images revealed that phytoplasma infection induces phloem protein filament formation in both the wild-type and mutant lines. This result suggests that, in contrast to previous hypotheses, sieve element filaments can be produced independently of AtSEOR1 and AtSEOR2 genes. Filament presence was accompanied by a compensatory overexpression of sieve element protein genes in infected mutant lines in comparison with wild-type lines. No correlation was found between phloem mass flow limitation and phytoplasma titre, which suggests that sieve element proteins are involved in defence mechanisms other than mechanical limitation of the pathogen., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Callose deposition, phloem-protein conformational changes and cell wall thickening are calcium-mediated occlusions occurring in the plant sieve elements in response to different biotic and abiotic stresses. However, the significance of these structures in plant-phytoplasma interactions requires in-depth investigations. We adopted a novel integrated approach, based on the combined use of microscopic and molecular analyses, to investigate the structural modifications induced in tomato leaf tissues in presence of phytoplasmas, focusing on vascular bundles and on the occlusion structures. Phloem hyperplasia and string-like arrangement of xylem vessels were found in infected vascular tissue. The diverse occlusion structures were differentially modulated in the phloem in response to phytoplasma infection. Callose amount was higher in midribs from infected plants than in healthy ones. Callose was observed at sieve plates but not at pore-plasmodesma units. A putative callose synthase gene encoding a protein with high similarity to Arabidopsis CalS7, responsible for callose deposition at sieve plates, was upregulated in symptomatic leaves, indicating a modulation in the response to stolbur infection. P-proteins showed configuration changes in infected sieve elements, exhibiting condensation of the filaments. The transcripts for a putative P-protein 2 and a sieve element occlusion-related protein were localized in the phloem but only the first one was modulated in the infected tissues., (© 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.)

Main Conclusion: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Phytoplasmas are among the most recently discovered plant pathogenic microorganisms so, many traits of the interactions with host plants and insect vectors are still unclear and need to be investigated. At now, it is impossible to determine the precise sequences leading to the onset of the relationship with the plant host cell. It is still unclear how phytoplasmas, located in the phloem sieve elements, exploit host cell to draw nutrition for their metabolism, growth and multiplication. In this work, basing on microscopical observations, we give insight about the structural interactions established by phytoplasmas and the sieve element plasma membrane, cytoskeleton, sieve endoplasmic reticulum, speculating about a possible functional role.

Background: In the Po Valley aflatoxins play a relevant role: the local food economy is heavily based on cereal cultivations for animal feed and human nutrition. Aims of this project are the identification of new compounds that inhibit Aspergillus proliferation, the development of new inhibitors of aflatoxins production, and the set-up a practical screening procedure to identify the most effective and safe compounds., Design and Methods: New compounds will be synthetized with natural origin molecules as ligands and endogenous metal ions to increase their bioavailability for the fungi as metal complexes. A biotechnological high-throughput screening will be set up to identify efficiently the most powerful substances. The newly synthesized compounds with effective antifungal activities, will be evaluated with battery of tests with different end-points to assess the toxic potential risk for environmental and human health., Expected Impact of the Study for Public Health: The fundamental step in the project will be the synthesis of new compounds and the study of their capability to inhibit aflatoxin biosynthesis. A new, simple, inexpensive and high-throughput method to screen the anti-fungine and anti-mycotoxin activity of the new synthesised compounds will be applied. The evaluation of possible risks for humans due to toxic and genotoxic activities of the molecules will be made with a new approach using different types of cells (bacteria, plants and human cells). Significance for public healthAflatoxins contamination constitutes a health emergency because aflatoxins and mycotoxins, besides being toxic, are among the most carcinogenic substances known. Even if Aspergillus are dominant in tropical regions, recently are becoming a serious problem also in Europe and in Italy, especially in area as the Po Valley in which this problem play a particularly important role, because the local food economy is heavily based not only on cereal cultivations aimed at animal feed but also on the production of derivatives to human nutrition. The aims of this research are the development of new bioactive molecules, obtained by natural molecules and metal ions, that are able to reduce the risk of food contamination by aflatoxin, but are harmless for environmental and health and the evaluation of the newly synthesized compounds using a battery of tests with different end-points to assess the toxic potential risk for environmental and human health.

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by 'Candidatus Phytoplasma solani,' the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes.

Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

With a steadily increasing world population, a more efficient system of food production is of paramount importance. One of the major causes of food spoilage is the presence of fungal pathogens and the production and accumulation of mycotoxins. In the present work we report a study on the activity of a series of functionalized thiosemicarbazones (namely cuminaldehyde, trans-cinnamaldehyde, quinoline-2-carboxyaldehyde, 5-fluoroisatin thiosemicarbazone and 5-fluoroisatin N(4)-methylthiosemicarbazone), as antifungal and anti-mycotoxin agents, against the two major genera of cereal mycotoxigenic fungi, i.e. Fusarium and Aspergillus. These thiosemicarbazones display different patterns of efficacy on fungal growth and on mycotoxin accumulation depending on the fungal species. Some of the molecules display a greater effect on mycotoxin synthesis than on fungal growth., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)