B. Patiño

Universidad Complutense de Madrid, Microbiology, Faculty Member

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Papers by B. Patiño

Analysis of genes related with fumonisin production in Gibberella fujikuroi

Pectin degrading enzymes secreted by six isolates of Fusarium oxysporum

Mycological Research, 1993

... Modified Czapek-Dox medium (Vazquez et ai., 1986) with 1 % pectin was used to grow the fungus... more

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

International Journal of Food Microbiology, 2011

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and re... more Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.

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Molecular diversity between maize and banana population of Fusarium verticillioides

Effect of preharvest anti-fungal compounds on Aspergillus steynii and A. carbonarius under fluctuating and extreme environmental conditions

International Journal of Food Microbiology, 2012

Ochratoxin A (OTA) has been found in pre-harvest and freshly harvested wheat. Spanish climatic co... more Ochratoxin A (OTA) has been found in pre-harvest and freshly harvested wheat. Spanish climatic conditions point to Aspergillus species as probably responsible for this OTA. In this study the effectiveness of 5 non-specific antifungal chemicals used on wheat fields (25.9% tebuconazole+60.0% N,N-capramide dimethyl; 12.70% tebuconazole+12.7% prothioconazole+59.5% N,N-amide dimethyldecane; 12.5% epoxiconazole; 12.5% tetraconazole; and 70% thiophanate methyl) and an extract from Equisetum arvense were investigated in vitro on wheat by recording growth (colony size, fungal growth and DNA concentration) and OTA production of two ochratoxigenic isolates of Aspergillus carbonarius and three of A. steynii, simulating current and extreme climatic conditions. Inoculated wheat was incubated under two alternating temperature cycles (20/30°C and 25/35°C) with photoperiod (14/10h lightness/darkness), and two moisture levels (40 and 25%). The Aspergillus species tested seemed to be able to persist in predicted future climatic conditions, in particular, A. steynii, a high OTA producer. Azoles were effective in controlling the growth of A. carbonarius and A. steynii, and this effectiveness may not be compromised by the increase in temperature and decrease of humidity. However, azoles are not useful for the prevention of OTA accumulation, which could be only reduced in A. carbonarius under non-extreme conditions. Although some adjustment will probably be required, further studies should be conducted in the field, since the antifungals used in this study are applied at flowering and not directly on the grain. Moreover, timing of antifungal application may need to be optimized. Finally, Equisetum extract showed promising results as an antifungal, however further work to adjust the applied concentrations is required.

The course of pectin degradation by polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici

Microbios, 1997

The production of polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici (FORL) oc... more The production of polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici (FORL) occurred sequentially, and endo- and exo-polygalacturonases (PG) predominated at different times during growth. At first FORL produced a PG which did not release monomers from pectins and was unable to hydrolyse dimers and trimers. Later a complex was produced in which exo-PG seemed to be the predominant activity; the monomer was released rapidly and this activity was able to degrade dimers and trimers.

Fumonisin production by Gibberella fujikuroi strains fromPinus species

International Journal of Food Microbiology, 2003

Fumonisins are important mycotoxins basically produced by strains from the Gibberella fujikuroi s... more Fumonisins are important mycotoxins basically produced by strains from the Gibberella fujikuroi species complex (with anamorphs in Fusarium genus) which contaminate food and feed products representing a risk to human and animal health. In this work, we report for the first time the fumonisin production of Fusarium moniliforme Sheldon strains associated to edible pine nuts of Pinus pinea. P. pinea is an important and widely distributed Pinus species in the Mediterranean area where their pine nuts are consumed raw or slightly processed in diverse food products. In this work, characterization and further identification of those strains were performed by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the intergenic spacer region of the rDNA (IGS) with the aid of the eight mating populations (A-H) described for G. fujikuroi species complex. The method was powerful to detect polymorphism, allowing discrimination between individuals and could be used to study the genetic relationships among them and within the G. fujikuroi species complex. Fusarium strains associated to Pinus radiata were also included in the present study. These strains did not produce fumonisins and showed no close relation with the strains isolated from P. pinea. The approach used in this work was rapid and proved to be efficient to assist identification and to characterize and analyse relatedness of new isolates within the G. fujikuroi species complex.

Characterization and in vitro expression patterns of an exopolygalacturonase encoding gene from Fusarium oxysporum f. sp. radicis lycopersici

Journal of applied …, 2003

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Detection of potentially mycotoxigenic Aspergillus species in Capsicum powder by a highly sensitive PCR-based detection method

Food Control, 2011

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Revision of ochratoxin a production capacity by the main species of Aspergillus section Circumdati. Aspergillus steynii revealed as the main risk of OTA contamination

Food Control, 2011

Ochratoxin A (OTA) is a highly toxigenic mycotoxin commonly present in a number of diverse agropr... more Ochratoxin A (OTA) is a highly toxigenic mycotoxin commonly present in a number of diverse agroproducts. Aspergillus Section Circumdati includes some of the most important OTA-producing species: Aspergillus ochraceus, Aspergillus elegans and the recently described Aspergillus steynii and Aspergillus westerdijkiae. In this work, OTA production by several strains of these four species from diverse origins and food matrices was examined.

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Control of polygalacturonase synthesis in Fusarium oxyspotum f.sp. radicis lycopersici

Canadian Journal of Microbiology, 1997

Genetic control of polygalacturonase (PG) activity from Fusarium oxysporum f.sp. radicis lycopers... more Genetic control of polygalacturonase (PG) activity from Fusarium oxysporum f.sp. radicis lycopersici was analyzed on pectin and glucose cultures. One exopolygalacturonase from F. oxysporum f.sp. radicis lycopersici was strongly induced, in stationary culture, when the fungus was grown on apple pectin, while on glucose no extracellular PG activity could be detected. Although SDS–PAGE detected the presence of a putative PG band (66 kDa) in both conditions, specific antibodies obtained against the purified PG only detected it in PG-inducing conditions, that is to say, when apple pectin was used as the carbon source. Northern blot analysis of RNA of two isolates of F. oxysporum f.sp. radicis lycopersici (r6 and r2) confirmed that this regulation of PG synthesis was exerted at the transcriptional level. Only one single mRNA species of around 1400 nucleotides was detected on the cultures containing pectin and was absent in glucose-grown cultures. Southern blot analysis of genomic DNA indi...

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The effect of different pectic growth substrates on beta-glucosidase in Fusarium oxysporum f. sp. radicis lycopersici: partial purification and characterization

The new microbiologica, 1997

Fusarium oxysporum f. sp. radicis lycopersici produces beta-glucosidase activities when it grows ... more Fusarium oxysporum f. sp. radicis lycopersici produces beta-glucosidase activities when it grows on pectin and glucose. The pectins were better substrates than glucose. In the medium containing galacturonic acid or sucrose the activity was present in low levels and at the end of autolysis. A beta-glucosidase from the pectin medium was purified by ion exchange chromatography followed by gel filtration. The enzyme was a unique band of protein in SDS-PAGE and isoelectric focussing. It had a molecular weight of 86,000 and a pI of 4.8. This beta-glucosidase was a glycoprotein.

Heterologous expresión and enzymatic characterization of exopolygalacturonase PGX1

Regulation of polygalacturonases in two isolates of Fusarium oxysporum f. sp. Radicis lycopersici (FORL)

Pectins and Pectinases, Proceedings of an International Symposium, 1996

... Eva Stratilov~i, Emilia Breierov~i and Ren~ita Vadkertiov~i Institute of Chemistry SAS, Ddbra... more

Heterologous expression and enzymatic characterisation of exopolygalacturonase PGX1

Current Research Topics in Applied Microbiology and Microbial Biotechnology - Proceedings of the II International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2007), 2009

ABSTRACT Fusurium oxysporum f. sp. radicis lycopersi (FORL) is a tomato pathogen which produces s... more ABSTRACT Fusurium oxysporum f. sp. radicis lycopersi (FORL) is a tomato pathogen which produces several cell wall degrading enzymes during the process of colonisation. Among these enzymes, polygalacturonases (PGs) are important because of their role in deconstruction the plant cell wall during host plant infection. Production of PGs in heterologous systems is an alternative approach to characterize individual enzymes, a difficult task, since complex patterns of PG isoforms, arising from different genes and patterns of postraductional modifications are,generally present in in vitro cultures. In this work, we have Successfully performed the heterologous expression of one exopolygalacturonase of FORL for the first time in the methylotrophic yeast Pichia pastoris. The enzyme produced showed an exo mode of action as predicted, and optimal pH and temperature values of 5 and 55 degrees C, respectively. Heterologous PGX1 showed higher affinity for trigalacturonic acid than for digalacturonic or polygalacturonic acid. The enzymatic features analyzed are discussed in relation to the other exopolygalacturonase (PGX2) described in F. oxysporum f.sp. radicis lycopersici.

In silico and in vitro analysis of promoter regions of two exopolygalacturonase coding genes of Fusarium oxysporum f.sp radicis lycopersici and regulation in Saccharomyces cerevisiae

ABSTRACT Fusarium oxysporum f.sp. radicis lycopersici (FORL) causes an important disease in tomat... more ABSTRACT Fusarium oxysporum f.sp. radicis lycopersici (FORL) causes an important disease in tomato characterized by cell wall degradation. The characterization of cell wall-degrading polygalacturonase enzymes and their regulation is crucial to gain knowledge on the role of these enzymes in pathogenesis. The objectives of this work were to perform an in silico and in vitro analyses of the upstream regions of two exopolygalacturonase (EXOPG) coding genes of FORL and to develop an easy and reliable system to perform the functional analysis of those promoters. The analysis of the upstream regions of pgx1 and pgx2 genes revealed differences in the distribution of regulatory motifs. These upstream regions were fused with the beta-galactosidase reporter gene in a Saccharomyces cerevisiae vector. The induction of the reporter gene was analyzed in response to different carbon sources and the results compared to the induction pattern of pgx1 and pgx2 genes in in vitro cultures of FORL.

Conventional and Real-Time PCR Assays for Detection and Quantification of the Expression of Fumonisin Biosynthetic Genefum5 inFusarium verticillioides

Modern Multidisciplinary Applied Microbiology, 2006

... [9] MT González-Jaén, S. Mirete, В. Patino, E. López -Errasquín and С. Vázquez 2004, EJPP ПО,... more

MYCOTOXINS | Toxicology

Encyclopedia of Food Microbiology, 2014

Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244

International Journal of Food Microbiology, 2011

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within... more Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.

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ITS-based detection and quantification of Aspergillus ochraceus and Aspergillus westerdijkiae in grapes and green coffee beans by real-time quantitative PCR

International Journal of Food Microbiology, 2009

Real-time qPCR OTA Grapes Green coffee beans ITS Aspergillus ochraceus and A. westerdijkiae are c... more Real-time qPCR OTA Grapes Green coffee beans ITS Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (OTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR® Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10 6 spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point of the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production.

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Analysis of genes related with fumonisin production in Gibberella fujikuroi