Quantification of DNA of Fusarium culmorum and Trichothecene Genotypes 3ADON and NIV in the Grain of Winter Wheat.

Fusarium head blight (FHB) is a wheat disease caused by fungi of the genus Fusarium. The aim of the study was to find relationships between the weather conditions in the experimental years and the locations and the amount of F. culmorum DNA and trichothecene genotypes, as well as the proportions between them. A three-year field experiment (2017, 2018 and 2019) was established in two locations (Poznan, Radzików). The DNA of F. culmorum was detected in all grain samples in an average amount of 20,124 pg per 1 µg of wheat DNA. The average amount of DNA from the 3ADON genotype was 4879 pg/µg and the amount of DNA from the NIV genotype was 3330 pg/µg. Weather conditions strongly affected the amount of DNA of F. culmorum and trichothecene genotypes detected in the grain. In the three experimental years, a high variability was observed in the coefficients of correlation between DNA concentrations and the FHB index, FDK, ergosterol and the corresponding toxins. There were significant correlations between disease incidence, fungal biomass (quantified as the total amount of fungal DNA or DNA trichothecene genotypes) and toxins (DON, 3AcDON and NIV) concentrations. The 3ADON trichothecene genotype dominated over the NIV genotype (ratio 1.5); however, this varied greatly depending on environmental conditions.

Influence of Endosperm Starch Composition on Maize Response to Fusarium temperatum Scaufl. & Munaut.

Fusarium temperatum Scaufl. & Munaut is a newly described taxon belonging to the Fusarium fujikuroi species complex (FFSC) and a frequent causative factor of maize ear rot. The aim of the present study was to determine the responses to the disease in maize populations differing in endosperm features that were classified to flint, dent, and a group of plants with intermediate kernel characteristics. In inoculation studies, substantial variation of host response to the fungus was found among the tested maize types. The dent-type kernels contained significantly less amylose (28.27%) and exhibited significantly higher rates of infection (IFER = 2.10) and contamination by beauvericin (7.40 mg kg-1) than plants of the flint maize subpopulation. The study documents a significant positive correlation between the Fusarium ear rot intensity (IFER) and ergosterol content (the R value ranged from 0.396 in 2015 to 0.735 in 2018) and between IFER and the presence of beauvericin (the R value ranged from 0.364 in 2015 to 0.785 in 2017). The negative correlation between (IFER) and amylose content (ranging from R = -0.303 to R= -0.180) stresses the role of the endosperm starch composition in the kernel resistance to Fusarium ear rot. The conducted study indicated that the risk of kernel infection and contamination with fungal metabolites (beauvericin and ergosterol) was associated with the maize type kernels.

Modelling the Effects of Weather Conditions on Cereal Grain Contamination with Deoxynivalenol in the Baltic Sea Region.

Fusarium head blight (FHB) is one of the most serious diseases of small-grain cereals worldwide, resulting in yield reduction and an accumulation of the mycotoxin deoxynivalenol (DON) in grain. Weather conditions are known to have a significant effect on the ability of fusaria to infect cereals and produce toxins. In the past 10 years, severe outbreaks of FHB, and grain DON contamination exceeding the EU health safety limits, have occurred in countries in the Baltic Sea region. In this study, extensive data from field trials in Sweden, Poland and Lithuania were analysed to identify the most crucial weather variables for the ability of Fusarium to produce DON. Models were developed for the prediction of DON contamination levels in harvested grain exceeding 200 µg kg-1 for oats, spring barley and spring wheat in Sweden and winter wheat in Poland, and 1250 µg kg-1 for spring wheat in Lithuania. These models were able to predict high DON levels with an accuracy of 70-81%. Relative humidity (RH) and precipitation (PREC) were identified as the weather factors with the greatest influence on DON accumulation in grain, with high RH and PREC around flowering and later in grain development and ripening correlated with high DON levels. High temperatures during grain development and senescence reduced the risk of DON accumulation. The performance of the models, based only on weather variables, was relatively accurate. In future studies, it might be of interest to determine whether inclusion of variables such as pre-crop, agronomic factors and crop resistance to FHB could further improve the performance of the models.

Biomarkers of Deoxynivalenol, Citrinin, Ochratoxin A and Zearalenone in Pigs after Exposure to Naturally Contaminated Feed Close to Guidance Values.

This study applied multi-mycotoxin liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methods to determine the biomarkers of exposure in urine and serum samples from a dose-response study with pigs. The 24 studied pigs were divided into three groups: a control and two experimental ones (with different levels of feed contamination). They were exposed to feed prepared from cereals contaminated with deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and citrinin (CIT) for 14 days. After that, both experimental groups received the same feed as the control group for the next 14 days to determine the kinetics of the disappearance of mycotoxin biomarkers. Urine samples were collected daily in the morning and blood samples-eight-times during the experiment. The study reported herein was the first prolonged exposure experiment for multiple mycotoxins like OTA and CIT in pigs. The urinary and serum levels of all biomarkers correlated well with the respective toxin intake; thereby demonstrating that they are suitable biomarkers of exposure in pigs. Urine is a good candidate to monitor DON, ZEN, OTA, CIT exposure while serum may be used to monitor DON, OTA and CIT. Additionally, OTA has even been quantified in both matrices in the experimental groups two weeks after changing the contaminated feed back to the control, this result differed from those produced by the other mycotoxins which were only quantified during the first two weeks. Therefore both matrices are suitable candidates to monitor prolonged OTA exposure in pigs.

Sucrose phosphate synthase (SPS), sucrose synthase (SUS) and their products in the leaves of Miscanthus × giganteus and Zea mays at low temperature.

MAIN CONCLUSION: The changes in the expression of key sugar metabolism enzymes (SPS and SUS), sucrose content and arrangement of chloroplast starch may play a significant role in the cold response in M. giganteus and maize plants. To understand the mechanism of the chilling-response of two closely-related C4 plants, we investigated the changes in the expression of sucrose phosphate synthase (SPS) and sucrose synthase (SUS) as well as changes in their potential products: sucrose, cellulose and starch in the leaves of Miscanthus × giganteus and Zea mays. Low temperature (12-14 °C) increased SPS content in Miscanthus (MG) and chilling-sensitive maize line (Zm-S), but not in chilling-tolerant one (Zm-T). In Zm-S line, chilling also caused the higher intensity of labelling of SPS in the cytoplasm of mesophyll cells, as demonstrated by electron microscopy. SUS labelling was also increased by cold stress only in MG plants what was observed in the secondary wall between mesophyll and bundle sheath cells, as well as in the vacuoles of companion cells. Cold led to a marked increase in total starch grain area in the chloroplasts of Zm-S line. In turn, Fourier transform infrared spectroscopy (FTIR) showed a slight shift in the cellulose band position, which may indicate the formation of more compact cellulose arrangement in Zm-T maize line. In conclusion, this work presents new findings supporting diversified cold-response, not only between two C4 plant species but also within one species of maize.

Identification of Proteomic Components Associated with Resistance to Fusarium Head Blight in Rye.

Rye was used here to dissect molecular mechanisms of resistance to Fusarium head blight (FHB) and to go deeper with our understanding of that process in cereals. F. culmorum-damaged kernels of two lines different in their potential of resistance to FHB were analyzed using two-dimensional gel electrophoresis and mass spectrometry to identify resistance markers. The proteome profiling was accompanied by measurements of α- and ß-amylase activities and mycotoxin content. The proteomic studies indicated a total of 18 spots with clear differences in protein abundance between the more resistant and more susceptible rye lines after infection. Eight proteins were involved in carbohydrate metabolism of which six proteins showed a significantly higher abundance in the resistant line. The other proteins recognized here were involved in stress response and redox homeostasis. Three remaining proteins were associated with protease inhibition/resistance and lignin biosynthesis, revealing higher accumulation levels in the susceptible rye line. After inoculation, the activities of α- and ß-amylases, higher in the susceptible line, were probably responsible for a higher level of starch decomposition after infection and a higher susceptibility to FHB. The presented results could be a good reference for further research to improve crop resistance to FHB.

Relationship between Fusarium Head Blight, Kernel Damage, Concentration of Fusarium Biomass, and Fusarium Toxins in Grain of Winter Wheat Inoculated with Fusarium culmorum.

Winter wheat lines were evaluated for their reaction to Fusarium head blight (FHB) after inoculation with Fusarium culmorum in two field experiments. A mixture of two F. culmorum chemotypes was applied (3ADON-deoxynivalenol producing, NIV-nivalenol producing). Different types of resistance were evaluated, including head infection, kernel damage, Fusarium biomass content and trichothecenes B (deoxynivalenol (DON), and nivalenol (NIV)) accumulation in grain. The aim of the study was to find relationships between different types of resistance. Head infection (FHB index) and Fusarium damaged kernels (FDK) were visually scored. Fusarium biomass was analysed using real-time PCR. Trichothecenes B accumulation was analysed using gas chromatography. Wheat lines differ in their reaction to inoculation for all parameters describing FHB resistance. We found a wide variability of FHB indexes, FDK, and Fusarium biomass content. Both toxins were present. DON content was about 60% higher than NIV and variability of this proportion between lines was observed. Significant correlation was found between head infection symptoms and FDK. Head infection was correlated with F. culmorum biomass and NIV concentration in grain. No correlation was found between the FHB index and DON concentration. Similarly, FDK was not correlated with DON content, but it was with NIV content; however, the coefficients were higher than for the FHB index. Fusarium biomass amount was positively correlated with both toxins as well as with the FHB index and FDK. Environmental conditions significantly influenced the DON/NIV ratio in grain. In locations where less F. culmorum biomass was detected, the DON amount was higher than NIV, while in locations where more F. culmorum biomass was observed, NIV prevailed over DON.

Chilling-induced physiological, anatomical and biochemical responses in the leaves of Miscanthus × giganteus and maize (Zea mays L.).

Miscanthus × giganteus and Zea mays, closely-related C4 grasses, originated from warm climates react differently to low temperature. To investigate the response to cold (12-14 °C) in these species, the photosynthetic and anatomical parameters as well as biochemical properties of the cell wall were studied. The research was performed using M. giganteus (MG) and two Z. mays lines differentiated for chilling-sensitivity: chilling-tolerant (Zm-T) and chilling-sensitive (Zm-S). The chilled plants of Zm-S line demonstrated strong inhibition of net CO2 assimilation and a clear decrease in F'v/F'm, Fv/Fm and ɸPSII, while in MG and Zm-T plants these parameters were almost unchanged. The anatomical studies revealed that MG plants had thinner leaves, epidermis and mesophyll cell layer as well as thicker cell walls in the comparison to both maize lines. Cold led to an increase in leaf thickness and mesophyll cell layer thickness in the Zm-T maize line, while the opposite response was observed in Zm-S. In turn, in chilled plants of MG and Zm-T lines, some anatomical parameters associated with bundle sheath cells were higher. In addition, Zm-S line showed the strong increase in the cell wall thickness at cold for mesophyll and bundle sheath cells. Chilling-treatment induced the changes in the cell wall biochemistry of tested species, mainly in the content of glucuronoarabinoxylan, uronic acid, ß-glucan and phenolic compounds. This work presents a new approach in searching of mechanism(s) of tolerance/sensitivity to low temperature in two thermophilic plants: Miscanthus and maize.

Low temperature caused modifications in the arrangement of cell wall pectins due to changes of osmotic potential of cells of maize leaves (Zea mays L.).

The cell wall emerged as one of the important structures in plant stress responses. To investigate the effect of cold on the cell wall properties, the content and localization of pectins and pectin methylesterase (PME) activity, were studied in two maize inbred lines characterized by different sensitivity to cold. Low temperature (14/12 °C) caused a reduction of pectin content and PME activity in leaves of chilling-sensitive maize line, especially after prolonged treatment (28 h and 7 days). Furthermore, immunocytohistological studies, using JIM5 and JIM7 antibodies, revealed a decrease of labeling of both low- and high-methylesterified pectins in this maize line. The osmotic potential, quantified by means of incipient plasmolysis was lower in several types of cells of chilling-sensitive maize line which was correlated with the accumulation of sucrose. These studies present new finding on the effect of cold stress on the cell wall properties in conjunction with changes in the osmotic potential of maize leaf cells.

Higher Fusarium Toxin Accumulation in Grain of Winter Triticale Lines Inoculated with Fusarium culmorum as Compared with Wheat.

Resistance to Fusarium head blight in 32 winter triticale and 34 winter wheat accessions was evaluated. Triticale and wheat were sown in field experiments in two locations. At the time of flowering, heads were inoculated with three Fusarium culmorum isolates. Fusarium head blight index was scored and after the harvest percentage of Fusarium damaged kernels was assessed. Grain was analysed for type B trichothecenes (deoxynivalenol and derivatives, nivalenol) and zearalenone (ZEN) content. The average Fusarium head blight indexes were 28.0% for wheat and 19.2% for triticale accessions. The percentage of Fusarium damaged kernels was also higher for wheat and came to 55.6%, while for triticale this figure was 40.2%. The average content of deoxynivalenol (DON) for wheat amounted to 11.65 mg/kg and was lower than the result for triticale which was 14.12 mg/kg. The average contents of nivalenol were similar in both cereals: 4.13 mg/kg and 5.19 mg/kg for wheat and triticale respectively. Considerable amounts of DON derivatives in the cereals were also detected. The ZEN content in the grain was 0.60 mg/kg for wheat and 0.66 mg/kg for triticale. Relationships between Fusarium head blight index, Fusarium damaged kernels and mycotoxin contents were statistically significant for wheat and mostly insignificant for triticale. Triticale proved to have less infected heads and kernels than wheat. However, the content of type B trichothecenes was higher in triticale grain than in wheat grain.

Alterations in Kernel Proteome after Infection with Fusarium culmorum in Two Triticale Cultivars with Contrasting Resistance to Fusarium Head Blight.

Highlight: The level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to Fusarium head blight. Triticale was used here as a model to recognize new components of molecular mechanism of resistance to Fusarium head blight (FHB) in cereals. Fusarium-damaged kernels (FDK) of two lines distinct in levels of resistance to FHB were applied into a proteome profiling using two-dimensional gel electrophoresis (2-DE) to create protein maps and mass spectrometry (MS) to identify the proteins differentially accumulated between the analyzed lines. This proteomic research was supported by a measurement of alpha- and beta-amylase activities, mycotoxin content, and fungal biomass in the analyzed kernels. The 2-DE analysis indicated a total of 23 spots with clear differences in a protein content between the more resistant and more susceptible triticale lines after infection with Fusarium culmorum. A majority of the proteins were involved in a cell carbohydrate metabolism, stressing the importance of this protein group in a plant response to Fusarium infection. The increased accumulation levels of different isoforms of plant beta-amylase were observed for a more susceptible triticale line after inoculation but these were not supported by a total level of beta-amylase activity, showing the highest value in the control conditions. The more resistant line was characterized by a higher abundance of alpha-amylase inhibitor CM2 subunit and simultaneously a lower activity of alpha-amylase after inoculation. We suggest that the level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to FHB.

Performance of winter triticale lines under high disease (Fusarium head blight) pressure.

Resistance to Fusarium head blight of 29 winter triticale lines and 3 cultivars was evaluated. Triticale was sown in field experiments in two locations. At flowering, triticale heads were inoculated with three Fusarium culmorum isolates. FHB index was scored and after the harvest percentage of Fusarium damaged kernels was assessed. Grain was analyzed for a content of trichothecenes B (deoxynivalenol and derivatives, nivalenol) and zearalenone. The average FHB indexes were similar in both locations and amounted 19.8% in Radzików, and 19.9%. in Cerekwica. Percentage of Fusarium damaged kernels was higher in Cerekwica (53.7%) than in Radzików (26.8%). An average content of DON in Radzików amounted to 8.690 ppm and was lower than in the second locaiion--19.543 ppm. In Cerekwica there were also large quantities of NIV in grain. The average content was 10.048 ppm, while in Radzików it was very low--0.324 ppm. Considerable amounts of DON derivatives in both locations were detected (1,815 ppm of 3AcDON and 1,913 ppm of 15AcDON). The content of the ZON in the grain from Cerekwica was very high and amounted to 1123 ppb, while in Radzików it was 6 times lower--200 ppb. Relationships between FHB index and mycotoxin contents were statistically insignificant in both locations. In contrast, FDK percentages correlated significantly with concentration of mycotoxins. In both locations the parallel experiments with 36 winter wheat were carried out. Triticale proved to be less infected than wheat. However, the content of trichothecenes, was higher in triticale grain than in wheat grain.