Aggressiveness and Mycotoxin Production by Fusarium meridionale Compared with F. graminearum on Maize Ears and Stalks in the Field.

Fusarium meridionale and F. graminearum both cause Gibberella ear rot (GER) and Gibberella stalk rot (GSR) of maize in Brazil, but the former is much more common. Recent work with two isolates of each from maize suggested this dominance could be caused by greater aggressiveness and competitiveness of F. meridionale on maize. We evaluated pathogenicity and toxigenicity of 16 isolates of F. graminearum and 24 isolates of F. meridionale recovered from both wheat and maize. Strains were individually inoculated into ears of four maize hybrids in field trials. GER severity varied significantly between isolates within each species. Although ranges overlapped, the average GER severity induced by F. meridionale (25.2%) was two times as high overall as that induced by F. graminearum (12.8%) for isolates obtained from maize but was similar for those isolated from wheat (19.9 and 21.4%, respectively). In contrast, severity of GSR was slightly higher for F. graminearum (22.2%) than for F. meridionale (19.8%), with no effect of the host of origin. Deoxynivalenol and its acetylated form 15ADON were the main mycotoxins produced by F. graminearum (7/16 strains), and nivalenol toxin was produced by F. meridionale (17/24 strains). Six isolates of F. graminearum and three of F. meridionale also produced zearalenone. Results confirmed that F. meridionale from maize is, on average, more aggressive on maize but also suggested greater complexity related to diversity among the isolates within each species and their interactions with different hybrids. Further studies involving other components of the disease cycle are needed to more fully explain observed patterns of host dominance.

The Dominance of Fusarium meridionale Over F. graminearum Causing Gibberella Ear Rot in Brazil May Be Due to Increased Aggressiveness and Competitiveness.

In Brazil, Gibberella ear rot (GER) of maize is caused mainly by Fusarium meridionale, whereas F. graminearum is a minor contributor. To test the hypothesis that F. meridionale is more aggressive than F. graminearum on maize, six experiments were conducted in the south (summer) and one in the central-south (winter), totaling seven conditions (year × location × hybrid). Treatments consisted of F. graminearum or F. meridionale (two isolates of each) inoculated once 4 days after silk, inoculated sequentially and alternately (F. graminearum → F. meridionale or F. meridionale → F. graminearum) 6 days apart, or (in the central-south) inoculated sequentially without alternating species (F. meridionale → F. meridionale or F. graminearum → F. graminearum). Overall, severity was two times greater in the south (37.0%), where summer temperatures were warmer (20 to 25°C) than in central-south. In the south, severity was greatest in F. meridionale treatments (67.8%); followed by F. meridionale → F. graminearum (41.1%), then F. graminearum → F. meridionale (19.4%), and lowest in F. graminearum (2.1%), suggesting an antagonistic relationship. In the central-south (15 to 20°C), severity was generally higher in the sequential nonalternating inoculation treatments (F. meridionale → F. meridionale or F. graminearum → F. graminearum) than when either species was inoculated only once. Only nivalenol (NIV) or deoxynivalenol was detected when F. meridionale or F. graminearum, respectively, was inoculated singly, or sequentially with no alternation. Both toxins were found in grains harvested from the F. meridionale → F. graminearum treatment, whereas only NIV was found in kernels from the F. graminearum → F. meridionale treatment, suggesting that F. meridionale was more competitive than F. graminearum in coinoculations. The dominance of F. meridionale as a cause of GER in Brazil may be due in part to its higher aggressiveness and competitiveness compared with F. graminearum.

A mycotoxin transporter (4D) from a library of deoxynivalenol-tolerant microorganisms.

New strategies are needed to mitigate the mycotoxin deoxynivalenol (DON) in feed and food products. Microbial DNA fragments were generated from a library of DON-tolerant microorganisms. These fragments were screened in DON-sensitive yeast strains for their ability to modify or transport DON. Fragments were cloned into a PCR8/TOPO vector, and recombined into the yeast vector, pYES-DEST52. Resulting yeast transformants were screened in the presence of 100 ppm DON. Transformants that were able to grow in the presence of DON were plated on a selective medium, and the cloned microbial DNA fragments were sequenced. BLAST queries of one microbial DNA fragment (4D) showed a high degree of similarity to an ABC transporter. A series of screening and inhibition assays were conducted with a transport inhibitor (propanol), to test the hypothesis that 4D is a mycotoxin transporter. DON concentrations did not change for yeast transformants expressing 4D. The ability of yeast transformants expressing 4D to transport DON was inhibited by the addition of propanol. Moreover, yeast transformants expressing a known efflux pump (PDR5) showed similar trends in propanol transport inhibition compared to 4D. Future work should consider mycotoxin transporters such as 4D to the development of transgenic plants to limit DON accumulation in seeds.

Modification of the Mycotoxin Deoxynivalenol Using Microorganisms Isolated from Environmental Samples.

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral salt media containing 100 µg/mL DON and analysis by gas chromatography mass spectrometry (GC/MS). Two mixed cultures derived from soil samples consistently decreased DON levels in assays using DON as the sole carbon source. Nuclear magnetic resonance (NMR) analysis indicated that 3-keto-4-deoxynivalenol was the major by-product of DON. Via 16S rRNA sequencing, these mixed cultures, including mostly members of the genera Acinetobacter, Leadbetterella, and Gemmata, were revealed. Incubation of one of these mixed cultures with wheat samples naturally contaminated with 7.1 µg/mL DON indicated nearly complete conversion of DON to the less toxic 3-epimer-DON (3-epi-DON). Our work extends previous studies that have demonstrated the potential for bioprospecting for microorganisms from the environment to remediate or modify mycotoxins for commercial applications, such as the reduction of mycotoxins in fuel ethanol co-products.

A comparison of two milling strategies to reduce the mycotoxin deoxynivalenol in barley.

Winter barley (Hordeum vulgare L.), a potential feedstock for fuel ethanol production, may be contaminated with the trichothecene mycotoxin deoxynivalenol (DON). DON is a threat to feed and food safety in the United States and may become concentrated during the production of distillers dried grains with solubles (DDGS). DDGS is a coproduct of fuel ethanol production and is increasingly being used as feed for domestic animals. Therefore, new strategies to reduce the threat of DON in DDGS need to be developed and implemented for grain destined for fuel ethanol production. It is known that large concentrations of DON accumulate in the hulls of wheat and barley. Consequently, improved methods are needed to carefully remove the hull from the grain and preserve the starchy endosperm. Whole kernels from five Virginia winter barley genotypes were used to evaluate the abilities of two different milling strategies (roller milling and precision milling (FitzMill)) for their ability to remove the hull-enriched tissue from the kernel while maintaining starch levels and reducing DON levels in the endosperm-enriched tissue. After whole kernels were milled, DON and starch levels were quantified in the hull-enriched fractions and endosperm-enriched fractions. Initial milling experiments demonstrated that the precision mill system (6 min run time) is able to reduce more DON than the roller mill but with higher starch losses. The average percent DON removed from the kernel with the roller mill was 36.7% ± 5.5 and the average percent DON removed from the dehulled kernel with the precision mill was 85.1% ± 9.0. Endosperm-enriched fractions collected from the roller mill and precision mill contained starch levels ranging from 49.0% ± 12.1 to 59.1% ± 0.5 and 58.5% ± 1.6 to 65.3% ± 3.9, respectively. On average, the precision mill removed a mass of 23.1% ± 6.8 and resulted in starch losses of 9.6% ± 6.3, but produced an endosperm-enriched fraction with relatively very little average DON (5.5 ± 2.7 µg g(-1)). In contrast, on average, the roller mill removed a mass of 12.2% ± 1.6 and resulted in starch losses of 2.1% ± 0.5, but produced an endosperm-enriched fraction with high average DON (20.7 ± 13.5 µg g(-1)). In a time course precision milling experiment, we tested barley genotypes Nomini, Atlantic, and VA96-44-304 and attempted to reduce the starch loss seen in the first experiment while maintaining low DON concentrations. Decreasing the run time of the precision mill from 5 to 2 min, reduced starch loss at the expense of higher DON concentrations. Aspirated fractions revealed that the precision milled hull-enriched fraction contained endosperm-enriched components that were highly contaminated with DON. This work has important implications for the reduction of mycotoxins such as DON in barley fuel ethanol coproducts and barley enriched animal feeds and human foods.

Survey of mycotoxins in corn distillers' dried grains with solubles from seventy-eight ethanol plants in twelve States in the U.S. In 2011.

Fuel ethanol co-products known as distillers' dried grains with solubles (DDGS) are a significant source of energy, protein, and phosphorous in animal feed. Fuel ethanol production may concentrate mycotoxins present in corn into DDGS. One hundred and forty one corn DDGS lots collected in 2011 from 78 ethanol plants located in 12 states were screened for the mycotoxins deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), nivalenol (NIV), and zearalenone (ZON). DON ranged from <0.50 to 14.62 µg g-1, 15-ADON ranged from <0.10 to 7.55 µg g-1, and ZON ranged from <0.10 to 2.12 µg g-1. None of the DDGS lots contained 3-ADON or NIV. Plants in OH had the highest levels of DON overall (mean of 9.51 µg g-1), and plants in NY, MI, IN, NE, and WI had mean DON levels >1 and <4 µg g-1. Twenty six percent (36/141) of the DDGS lots contained 1.0 to 5.0 µg g-1 DON, 2% (3/141) contained >5.0 and <10.0 µg g-1 DON, and 3% (4/141) contained >10.0 µg g-1 DON. All DDGS lots contaminated with unacceptable levels of DON evaded detection prior to their commercial distribution and were likely sold as feed products.

Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modified during long-term (decades) desiccation but is protected from oxidative damage and degradation.

Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5'-GWCWATCGCC-3') based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.