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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.

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.

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.)

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.

NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.)

NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant and ubiquitous enzyme that may exist in different isoenzymic forms. Variation in the composition of the GDH isoenzyme pattern is observed during plant development and specific cell, tissue and organ localization of the different isoforms have been reported. However, the mechanisms involved in the regulation of the isoenzymatic pattern are still obscure. Regulation may be exerted at several levels, i.e. at the level of transcription and translation of the relevant genes, but also when the enzyme is assembled to originate the catalytically active form of the protein. In Arabidopsis thaliana, three genes (GDH1, GDH2 and GDH3) encode three different GDH subunits (β, α and γ) that randomly associate to form a complex array of homo- and hetero-hexamers. In order to asses if the different Arabidopsis GDH isoforms may display different structural properties we have investigated their thermal stability. In particular the stability of GDH1 and GDH3 isoenzymes was studied using site-directed mutagenesis in a heterologous yeast expression system. It was established that the carboxyl terminus of the GDH subunit is involved in the stabilization of the oligomeric structure of the enzyme., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

In higher plants, NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant enzyme that exists in different isoenzymic forms. In Arabidopsis thaliana, three genes (Gdh1, Gdh2 and Gdh3) encode three different GDH subunits (β, α and γ) that randomly associate to form a complex array of homo- and heterohexamers. The modification of the GDH isoenzyme pattern and its regulation was studied during the development of A. thaliana in the gdh1, gdh2 single mutants and the gdh1-2 double mutant, with particular emphasis on GDH3. Investigations showed that the GDH3 isoenzyme could not be detected in closely related Arabidopsis species. The induction and regulation of GDH3 activity in the leaves and roots was investigated following nitrogen deprivation in the presence or absence of sucrose or kinetin. These experiments indicate that GDH3 is likely to play an important role during senescence and nutrient remobilization., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.

Aims: To develop a simple, high-throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia., Methods and Results: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 μl of different formulations of coconut-derived liquid medium. Time-dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV-induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC-based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct 'in situ' readings, the latter method reinforcing the high-throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α-lipoic acid) to the medium., Conclusions: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut-derived culture medium., Significance and Impact of the Study: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.

"First line" defence mechanisms, such as phytochelatin biosynthesis, and "second line" mechanisms, such as stress protein induction, were investigated in cadmium-exposed cells of Trebouxia impressa Ahmadjian, a green microalgal species that is a common photobiont of the lichen Physcia adscendens (Fr.) H. Olivier. When T. impressa cells were exposed to 0, 9 and 18 microM Cd for 6, 18 and 48 h, glutathione and phytochelatins efficiently protected the cells against Cd damage. By contrast, the highest Cd concentration (36 microM) at the longest exposure-time (48 h) caused marked drops in glutathione and phytochelatin content, several types of ultrastructural damage, and decreases in cell density and total chlorophyll concentration. In this case, induction of stress proteins was observed, but only long after the induction of phytochelatins. Thus, stress proteins could represent a "second line" mechanism to counteract Cd stress, activated when there is a decline in the "first line" mechanism of Cd detoxification given by phytochelatins.

Aims: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus., Methods and Results: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression., Conclusions: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium., Significance and Impact of the Study: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Fenarimol, a systemic pyrimidine carbinol fungicide, is considered to be not genotoxic or weakly genotoxic, although the available toxicological data are controversial and incomplete. Our results obtained in vitro with leukocytes of two different rodent species (rat and mouse) show that fenarimol affects DNA, as detected by the single-cell gel electrophoresis (SCGE, Comet) assay. This fungicide is able to induce DNA damage in a dose-related manner, with significant effectiveness at 36 nM, but without significant interspecies differences. Simultaneous exposure of rat leukocytes to fenarimol (36-290 nM) and a model genotoxic compound (50 microg/ml bleomycin) produced a supra-additive cytotoxic and genotoxic effect. This supports previous findings suggesting possible co-toxic, co-mutagenic, cancer-promoting and co-carcinogenic potential of fenarimol, and modification of the effects of other xenobiotics found to be influenced by this agrotoxic chemical, with consequent different toxicological events. The potential for DNA strand breaks to act as a biomarker of genetic toxicity in plants in vivo was also considered, in view of the fact that higher plants represent reliable sensors in an ecosystem. Significant DNA breakage was observed in the nuclei of Impatiens balsamina leaves after in vivo treatment with fenarimol (145 nM, 1h). More than 50% of the cells showed such DNA damage.

Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

Urban airborne particulate is a complex mixture of air pollutants, many of which have not been identified. However, short-term mutagenesis tests together with chemicophysical parameter analysis are able to better assess air quality and genotoxic load. The findings of continuous monitoring (January 1991-August 1998) of urban air genotoxicity of a Po Valley town (Italy) on Salmonella typhimurium and Saccharomyces cerevisiae are reported. During this period, various measures (catalytic devices, unleaded fuels, annual vehicle overhaul, etc.) to improve air-dispersed pollutant control were enforced. However, a continuous presence of genotoxic compounds is shown and more qualitative than quantitative changes are evident. We also demonstrate the ability of the Comet assay to detect DNA-damaging agents in airborne particulate samples. We applied the test to human leukocytes and, with major improvements, to plant cells (Allium cepa roots and epigean tissues of Impatiens balsamina). The first findings on human leukocytes confirm the sensitivity of this assay, its peculiarity and its applicability in assessing genotoxicity in environmental samples. The capability of plants to show the response of multicellular organisms to environmental pollutants largely counterbalances a probable lowering in sensitivity. Moreover, application of the Comet test to epigean tissues could be useful in estimating the bioavailability of and genotoxic damage by air pollutants, including volatile compounds (ozone, benzene, nitrogen oxides, etc.) to higher plants.

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40-80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 1∶1 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.

Extracts from phylloclads of Asparagus officinails were electrophoretically analyzed for isozyme polymorphism. Fourteen enzyme systems were examined using four buffer systems: seven enzymes (acid phosphatase, catalase, glutamate-oxaloacetate transaminase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase) exhibited clear and consistent banding patterns. Isozyme polymorphism was studied in seven pairs of male and female doubled haploids and in their male F1s. Segregation of polymorphic loci was examined in the backcross progenies and was found to be consistent with a simple Mendelian inheritance in all cases, except for three anodical peroxidases, where two factors have been hypothesized. No linkage could be found between isozyme markers that were segregating in the same cross, but association was demonstrated between one malate dehydrogenase locus and the sex determining genes. The availability of isozyme markers may be useful in breeding and, in particular, the localization of one malate dehydrogenase locus on the sex chromosomes may be helpful in mapping the sex genes.

Two additional types of nuclear determinants involved in the control of spontaneous mutability of rho in S. cerevisiae have been identified: mmc and the pet-ts 1, 2, 10, 52 and 53 genes. These genes in their mutated recessive form increase at various extents the number of respiratory deficient cytoplasmic "petite" mutants accumulated. The gene mmc does not affect the respiratory activity and is not temperature-dependent whereas the pet-ts genes determine at the non permissive temperature a respiratory deficient phenotypes even if they affect the mutability of rho at the permissive and at the non permissive temperature. The data here reported suggest that a "replicative complex" exists for the mitochondrial DNA. It is in the purpose of this paper to deal with the relative contribution that mmc and pet-ts gene products have in ensuring the fidelity of this "replicative complex".