Characterization of Dextran Produced by the Food-Related Strain Weissella cibaria C43-11 and of the Relevant Dextransucrase Gene.

A metabolic feature of lactic acid bacteria (LAB) is the production of exopolysaccharides (EPSs), which have technological and functional properties of interest to the food sector. The present study focused on the characterization of the Weissella cibaria strain C43-11, a high EPS producer in the presence of sucrose, in comparison with a low-producing strain (C2-32), and on possible genetic regulatory elements responsible for the modulation of dextransucrase (dsr) genes expression. NMR analysis of the polymeric material produced by the C43-11 strain indicated the presence of dextran consisting mainly of a linear scaffold formed by α-(1-6) glycosidic linkages and a smaller amounts of branches derived from α-(1-2), α-(1-3), and α-(1-4) linkages. Molecular analysis of the dsr genes and the putative transcriptional promoters of the two strains showed differences in their regulatory regions. Such variations may have a role in the modulation of dsr expression levels in the presence of sucrose. The strong upregulation of the dsr gene in the C43-11 strain resulted in a high accumulation of EPS. This is the first report showing differences in the regulatory elements of the dsr gene in W. cibaria and indicates a new perspective of investigation to identify the regulatory mechanism of EPS production.

Identification and Characterisation of pST1023 A Mosaic, Multidrug-Resistant and Mobilisable IncR Plasmid.

We report the identification and characterisation of a mosaic, multidrug-resistant and mobilisable IncR plasmid (pST1023) detected in Salmonella ST1023, a monophasic variant 4,[5],12:i: strain of widespread pandemic lineage, reported as a Southern European clone. pST1023 contains exogenous DNA regions, principally gained from pSLT-derivatives and IncI1 plasmids. Acquisition from IncI1 included oriT and nikAB and these conferred the ability to be mobilisable in the presence of a helper plasmid, as we demonstrated with the conjugative plasmids pST1007-1D (IncFII) or pVC1035 (IncC). A sul3-associated class 1 integron, conferring resistance to aminoglycosides, chloramphenicol and trimethoprim-sulphonamides, was also embedded in the acquired IncI1 DNA segment. pST1023 also harboured an additional site-specific recombination system (rfsF/rsdB) and IS elements of the IS1, IS5 (IS903 group) and IS6 families. Four of the six IS26 elements present constituted two pseudo-compound-transposons, named PCT-sil and PCT-Tn10 (identified here for the first time). The study further highlighted the mosaic genetic architecture and the clinical importance of IncR plasmids. Moreover, it provides the first experimental data on the ability of IncR plasmids to be mobilised and their potential role in the horizontal spread of antimicrobial-resistant genes.

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

Metagenetic Analysis for Microbial Characterization of Focaccia Doughs Obtained by Using Two Different Starters: Traditional Baker's Yeast and a Selected Leuconostoc citreum Strain.

Lactic acid bacteria (LAB) decisively influence the technological, nutritional, organoleptic and preservation properties of bakery products. Therefore, their use has long been considered an excellent strategy to improve the characteristics of those goods. The aim of this study was the evaluation of microbial diversity in different doughs used for the production of a typical Apulian flatbread, named focaccia. Leavening of the analyzed doughs was obtained with baker's yeast or by applying an innovative "yeast-free" protocol based on a liquid sourdough obtained by using Leuconostoc citreum strain C2.27 as a starter. The microbial populations of the doughs were studied by both a culture-dependent approach and metagenetic analyses. The flours used for dough preparation were also subjected to the same analyses. The metagenetic analyses were performed by sequencing the V5-V6 hypervariable regions of the 16S rRNA gene and the V9 hypervariable region of the 18S rRNA gene. The results indicate that these hypervariable regions were suitable for studying the microbiota of doughs, highlighting a significant difference between the microbial community of focaccia dough with baker's yeast and that of the dough inoculated with the bacterial starter. In particular, the dough made with baker's yeast contained a microbiota with a high abundance of Proteobacteria (82% of the bacterial population), known to be negatively correlated with the biochemical properties of the doughs, while the Proteobacteria in dough produced with the L. citreum starter were about 43.5% lower than those in flour and dough prepared using baker's yeast. Moreover, the results show that the L. citreum C2.27 starter was able to dominate the microbial environment and also reveal the absence of the genus Saccharomyces in the dough used for the production of the "yeast-free" focaccia. This result is particularly important because it highlights the suitability of the starter strain for obtaining an innovative "yeast-free" product.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15-28 °C vs 18-34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, "velvet complex") involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

New insight into microbial degradation of mycotoxins during anaerobic digestion.

Anaerobic digestion represents an interesting approach to produce biogas from organic waste materials contaminated by mycotoxins. In this study a shotgun metagenomic analysis of lab-scale bioreactors fed with mycotoxin-contaminated silage has been carried out to characterize the evolution of microbial community under the operating conditions and the key enzymatic activities responsible for mycotoxin degradation. The study was conducted at two different level of contamination for fumonisins and aflatoxin B1. After 15 days biogas production was not influenced by the presence of mycotoxins. Metagenomic analysis revealed that a high contamination rate of mycotoxins interfere with microbial diversity. Degradation of mycotoxins accounted in about 54% for aflatoxin B1 and 60% for fumonisins. The degradation activity of fumonisins resulted in the presence of partially hydrolyzed forms in both tested contamination levels. Accordingly, metagenomic functional analysis revealed the presence of two new carboxylesterase genes belonging to D. bacterium and P. bacterium putatively involved in fumonisin degradation.

Toxigenic Fungi and Mycotoxins in a Climate Change Scenario: Ecology, Genomics, Distribution, Prediction and Prevention of the Risk.

Toxigenic fungi and mycotoxins are very common in food crops, with noticeable differences in their host specificity in terms of pathogenicity and toxin contamination. In addition, such crops may be infected with mixtures of mycotoxigenic fungi, resulting in multi-mycotoxin contamination. Climate represents the key factor in driving the fungal community structure and mycotoxin contamination levels pre- and post-harvest. Thus, there is significant interest in understanding the impact of interacting climate change-related abiotic factors (especially increased temperature, elevated CO2 and extremes in water availability) on the relative risks of mycotoxin contamination and impacts on food safety and security. We have thus examined the available information from the last decade on relative risks of mycotoxin contamination under future climate change scenarios and identified the gaps in knowledge. This has included the available scientific information on the ecology, genomics, distribution of toxigenic fungi and intervention strategies for mycotoxin control worldwide. In addition, some suggestions for prediction and prevention of mycotoxin risks are summarized together with future perspectives and research needs for a better understanding of the impacts of climate change scenarios.

The Role of Grafting in the Resistance of Tomato to Viruses.

Grafting is routinely implemented in modern agriculture to manage soilborne pathogens such as fungi, oomycetes, bacteria, and viruses of solanaceous crops in a sustainable and environmentally friendly approach. Some rootstock/scion combinations use specific genetic resistance mechanisms to impact also some foliar and airborne pathogens, including arthropod or contact-transmitted viruses. These approaches resulted in poor efficiency in the management of plant viruses with superior virulence such as the strains of tomato spotted wilt virus breaking the Sw5 resistance, strains of cucumber mosaic virus carrying necrogenic satellite RNAs, and necrogenic strains of potato virus Y. Three different studies from our lab documented that suitable levels of resistance/tolerance can be obtained by grafting commercial tomato varieties onto the tomato ecotype Manduria (Ma) rescued in the framework of an Apulian (southern Italy) regional program on biodiversity. Here we review the main approaches, methods, and results of the three case studies and propose some mechanisms leading to the tolerance/resistance observed in susceptible tomato varieties grafted onto Ma as well as in self-grafted plants. The proposed mechanisms include virus movement in plants, RNA interference, genes involved in graft wound response, resilience, and tolerance to virus infection.

A loop-mediated isothermal amplification (LAMP) assay for rapid detection of fumonisin producing Aspergillus species.

Fumonisins contamination of food commodities is a worldwide problem, especially for maize. The ability to produce fumonisinsis a trait of several species of Fusarium, mainly F. verticillioides and F. proliferatum on maize, and some Aspergillus species. A. niger and its sister species A. welwitschiae, can contribute to fumonisin B2 (FB2) accumulation in maize kernels, although to a lesser extent than fumonisin-producing Fusarium species. Fumonisins risk monitoring represents an effective strategy in the integrated approach for mycotoxin risk management and reduction. The availability of a user-friendlymolecular assay for the detection oftoxigenic fungal species represents a valuable tool in understanding and managing upcoming mycotoxin contamination. In this study, we developed a LAMP assay, based on the detection of fum10, for a rapid and specific molecular detection of FB2-producing A. niger and A. welwistchiae, potentially useful to perform monitoring directly "on site" in maize chain. Results showed that very low amounts of conidia are suitable to detect the presence of the target gene, thus providing information about the presence of FB2-producing Aspergillus species and the possible upcoming fumonisins contamination in maize. The assay was combined with a suitable protocol for "in field" crude DNA extraction and a colorimetric method for easy naked-eye evaluationof results, offering a reliable and user-friendly tool to support effective reduction strategies of mycotoxin contamination in crop management programs.

Grafting alters tomato transcriptome and enhances tolerance to an airborne virus infection.

Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVYC-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement.

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.

Effects of temperature and water activity change on ecophysiology of ochratoxigenic Aspergillus carbonarius in field-simulating conditions.

Ochratoxin A (OTA) is the primary mycotoxin threat in wine and dried vine fruits. Its presence in grape and wine is strongly related to climatic conditions and the expected climate change could represent a risk of increasing fungal colonization and OTA contamination in grapes. In this regard, the interacting effect of i) different conditions of water availability (0.93 and 0.99aw) and ii) different 10 h/14 h dark/light alternating temperature conditions simulating a nowadays (18/31 °C) and climate change scenario (20/37 °C) in high OTA risk areas of Apulia region, were studied. Lag phases prior to growth, mycelial growth rate, the expression of biosynthesis, transcription factors and regulatory genes of OTA cluster and OTA production were analysed in Aspergillus carbonarius ITEM 5010 under the combined effect of different climatic factors. At 18/31 °C and under water stress conditions (0.93 aw) the growth rate was slower than at 0.99 aw; on the contrary, at 20/37 °C a higher growth rate was observed at 0.93 aw. An over-expression of OTA genes and genes belonging to the global regulator Velvet complex was observed at 18/31 °C and 0.99 aw, with the specific OTA pathway transcription factor bZIP showing the highest expression level. The up-regulated transcription profile of the genes positively correlated with OTA production higher at 18/31 °C than at 20/37 °C and 0.99 aw; while no OTA production was detected at 0.93 aw at each of the temperature conditions tested. These findings provide preliminary evidence that the possible increase of the temperature, likely to happen in some areas of the Apulia region, may results in a reduction of both A. carbonarius spoilage and OTA production in grapes.

A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum.

Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.

Time-dependent effects of Pochonia chlamydosporia endophytism on gene expression profiles of colonized tomato roots.

A transcriptome analysis was produced from tomato roots inoculated with the hyphomycete Pochonia chlamydosporia at three different times. Gene expression data were also yielded from fungus grown in vitro or endophytic. A next-generation sequencing (NGS) and network analysis approach were applied. We identified 3.676 differentially expressed tomato genes (DEG), highlighting a core of 93 transcripts commonly down- or upregulated at every time point, shedding light on endophytism process. Functional categories related to plant information-processing system, which recognizes, percepts, and transmits signals, were associated with gene upregulated early in time, with higher representations in processes such as plant defense regulation later in time. Network analysis of a DEG subset showed dominance of MAP kinase hubs in the uninoculated control samples, replaced by an increased centrality of WRKY transcription factor and ETR-ethylene response factor genes in the colonized roots. Fungus genes expressed during progression of plant colonization, therefore related to the host colonization process or endophytism persistence, were also identified. Data provided a high-resolution insight on tomato transcriptome changes as induced by endophytism, highlighting a specific modulation of stress-responsive transcripts, related to a selective activation of defense pathways, likely required by the fungus to establish a persistent endophytic lifestyle.

Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

UNLABELLED: Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTA cluster of A. niger The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius IMPORTANCE: Ochratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius The encoding gene, AcOTAhal, is contiguous to biosynthetic genes that we have already described (nrps and pks), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius, which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.

Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days.

Rapid prediction of ochratoxin A-producing strains of Penicillium on dry-cured meat by MOS-based electronic nose.

The availability of rapid diagnostic methods for monitoring ochratoxigenic species during the seasoning processes for dry-cured meats is crucial and constitutes a key stage in order to prevent the risk of ochratoxin A (OTA) contamination. A rapid, easy-to-perform and non-invasive method using an electronic nose (e-nose) based on metal oxide semiconductors (MOS) was developed to discriminate dry-cured meat samples in two classes based on the fungal contamination: class P (samples contaminated by OTA-producing Penicillium strains) and class NP (samples contaminated by OTA non-producing Penicillium strains). Two OTA-producing strains of Penicillium nordicum and two OTA non-producing strains of Penicillium nalgiovense and Penicillium salamii, were tested. The feasibility of this approach was initially evaluated by e-nose analysis of 480 samples of both Yeast extract sucrose (YES) and meat-based agar media inoculated with the tested Penicillium strains and incubated up to 14 days. The high recognition percentages (higher than 82%) obtained by Discriminant Function Analysis (DFA), either in calibration and cross-validation (leave-more-out approach), for both YES and meat-based samples demonstrated the validity of the used approach. The e-nose method was subsequently developed and validated for the analysis of dry-cured meat samples. A total of 240 e-nose analyses were carried out using inoculated sausages, seasoned by a laboratory-scale process and sampled at 5, 7, 10 and 14 days. DFA provided calibration models that permitted discrimination of dry-cured meat samples after only 5 days of seasoning with mean recognition percentages in calibration and cross-validation of 98 and 88%, respectively. A further validation of the developed e-nose method was performed using 60 dry-cured meat samples produced by an industrial-scale seasoning process showing a total recognition percentage of 73%. The pattern of volatile compounds of dry-cured meat samples was identified and characterized by a developed HS-SPME/GC-MS method. Seven volatile compounds (2-methyl-1-butanol, octane, 1R-α-pinene, d-limonene, undecane, tetradecanal, 9-(Z)-octadecenoic acid methyl ester) allowed discrimination between dry-cured meat samples of classes P and NP. These results demonstrate that MOS-based electronic nose can be a useful tool for a rapid screening in preventing OTA contamination in the cured meat supply chain.

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Phylogenetic study of polyketide synthases and nonribosomal peptide synthetases involved in the biosynthesis of mycotoxins.

Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: ß-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species.