Effect of moisture on wheat grains lipid patterns and infection with Fusarium graminearum.

Suitable conditions of temperature and humidity are required to maintain wheat grains quality, but during processing and storage, the grains can be exposed to adverse environmental conditions and presence of infectious fungi. Fusarium graminearum, the main causal agent of Fusarium head blight on wheat, affects crop yields and grain quality by alteration of their biochemical components and mycotoxin contamination, which reduces the possibilities of wheat end use and compromises food safety. Lipid degradation by hydrolytic, oxidative and microbial deterioration is the predominant cause of the loss of sensory acceptability, nutritional value and baking quality. The aim of this research was to determine the influence of adverse environmental conditions -as the increasing moisture - on lipid patterns of whole wheat flours contaminated with F. graminearum in relation to the infection degree. In vitro cultures of F. graminearum were carried out on wheat grains under different degrees of relative humidity (11, 50, 75 and 100%) throughout 45 days of incubation at 28 °C. The fungal biomass measured by q-PCR increased proportionally with the humidity. A decrease in the signals of saturated (palmitic and estearic) and unsaturated (oleic, linoleic and linolenic) fatty acids, analyzed as fatty acid methyl esters (FAMEs) by GC-MS, was observed in relation with the humidity and infection degree. The degradation rate of the lipids was high during the first 15 days of incubation, reaching the fatty acids content, values around 20-40% of those found in the control. From that moment on, the rate of degradation was slower or even null. It was observed that in all treatments, the linolenic acid reached the highest degradation ratio in comparison with the other fatty acids, which may be caused by the action of lipoxygenases. The lipase activity and the content of deoxynivalenol were also determinate on the flours. The lipase activity increased until day 25 of incubation reaching twice the initial value. The deoxynivalenol content also increased along incubation while fatty acids decreased. Our results demonstrated that the magnitude in the signal of fatty acids in whole wheat flours varied in relation to the degree of humidity and fungal infection of the grains from which they were obtained. Otherwise, lipids and their oxidation products are related with the pathogenesis and production of mycotoxins. These observations highlight the importance of an adequate manipulation of wheat grains on the processing chain to prevent quality changes and mycotoxins contamination.

Characterization of Fusarium graminearum isolates recovered from wheat samples from Argentina by Fourier transform infrared spectroscopy: Phenotypic diversity and detection of specific markers of aggressiveness.

Fusarium graminearum is the primary causal agent of Fusarium head blight of wheat in Argentina. This disease affects crop yields and grain quality also reducing the wheat end-use, and causing mycotoxin contamination. The aim of this work was to analyze the phenotypic characteristics associated with phenotypic diversity and aggressiveness of 34 F. graminearum sensu stricto isolates recovered from Argentinean fields in the 2008 growing season using the Fourier Transform Infrared (FTIR) dried film technology. We applied this technique also to search for spectral specific markers associated with aggressiveness. The combination of FTIR technology with hierarchical cluster analysis allowed us to determine that this population constitutes a highly diverse and heterogeneous group of fungi with significant phenotypic variance. Still, when the spectral features of a set of these isolates were compared against their aggressiveness, as measured by disease severity, thousand grains weight, and relative yield reduction, we found that the more aggressive isolates were richer in lipid content. Therefore, we could define several spectroscopic markers (>CH stretching modes in the 3000-2800 window, >CO and CO vibrational modes of esters at 1765-1707cm-1 and 1474-900cm-1, respectively), mostly assigned to lipid content that could be associated with F. graminearum aggressiveness. All together, by the application of FTIR techniques and simple multivariate analyses, it was possible to gain significant insights into the phenotypic characterization of F. graminearum local isolates, and to establish the existence of a direct relationship between lipid content and fungal aggressiveness. Considering that lipids have a major role as mediators in the interaction between plants and fungi our results could represent an attractive outcome in the study of Fusarium pathogenesis.

Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina.

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers.

Diversidad genética de aislamientos de Fusarium graminearum sensu lato asociados con la fusariosis de la espiga en trigo de diversas localidades argentinas / Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

La fusariosis de la espiga de trigo es una importante enfermedad para la región pampeana Argentina; Fusarium graminearum es el principal patógeno asociado. Se estudió el polimorfismo del ADN de un conjunto de aislamientos utilizando las técnicas de IGS-RFLP e ISSR. La técnica de IGS-RFLP produjo 41 bandas, 30 de ellas fueron polimórficas. El análisis de los ISSR mostró 87 bandas con 47 bandas polimórficas. La primera de estas metodologías fue más eficiente, ya que detectó mayor promedio polimórfico (59,91%) que la segunda (44,11%). Los valores promedio del contenido de información polimórfica (PIC) fueron 0,211 y 0,129, respectivamente. Se identificaron 20 haplotipos por IGS-RFLP, mientras que el análisis de los ISSR reveló 15 haplotipos. La agrupación de genotipos obtenida en ambos dendrogramas fue diferente. Los grupos genéticos obtenidos por la técnica de IGS-RFLP mostraron una asociación parcial con el origen geográfico. Este es el primer reporte que analiza la variabilidad genética en poblaciones de F. graminearum de trigo empleando marcadores IGS-RFLP e ISSR en Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers

Production, characterization, and identification using proteomic tools of a polygalacturonase from Fusarium graminearum.

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease.

Acyl-CoA oxidase activity from Beauveria bassiana, an entomopathogenic fungus.

Beauveria bassiana produces acyl-Co oxidase (ACO) in the P(20000 g) fraction of glucose and alkane-grown cultures that catalyze the oxidation of acyl-CoAs of different chain length. The activity was measured indirectly over the formation of H2O2 via the oxidative-coupled assay system. ACO activity was assessed spectrophotometrically in the P(20000 g) fraction of glucose-grown (FS0) and n-alkane grown cultures (FS(alk)), employing acyl-CoAs of 16 to 24 carbons as substrates. A significant increment in the activity was observed in FS(alk) as compared to that of controls (FS0) in all conditions tested. Tetracosane-grown cultures showed the highest activity with lignoceroyl-CoA. The reaction conditions were optimized employing lignoceroyl-CoA as substrate. A variable lag phase was observed when the activity was measured as a function of time. In the presence of 3-amino-1,2,4-triazole (AT) to prevent H2O2 consumption by endogenous catalase, the lag phase became shorter and disappeared when AT concentrations were raised from 40 to 200 mM, thus enhancing acyl-CoA oxidation. Enzyme activity reached its maximal value in the presence of 240 microg peroxidase, 0.08% Triton X-100 and 36 microM bovine serum albumin. The apparent Km using lignoceroyl as substrate was estimated 2.5 microM. ACO showed high activity and stability between 30 and 40 degrees C, as well as between 7.0 and 9.0 pH, for 120 min, being 7.0 the optimum pH.