Identification of unique or elevated levels of kernel proteins in aflatoxin-resistant maize genotypes through proteome analysis

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize (Zea mays L.). Resistant maize genotypes have been identified, but the incorporation of resistance into commercial lines has been slow due to the lack of selectable markers. Here we report the identification of potential markers in resistant maize lines using a proteomics approach. Kernel embryo proteins from each of two resistant genotypes have been compared with those from a composite of five susceptible genotypes using large format two-dimensional gel electrophoresis. Through these comparisons, both quantitative and qualitative differences have been identified. Protein spots have been sequenced, and based on peptide sequence homology analysis, are categorized as follows: storage proteins (globulin 1 and globulin 2), late embryogenesis abundant (LEA) proteins related to drought or desiccation (LEA3 and LEA14), water- or osmo-stress related proteins (WSI18 and aldose reductase), and heat-stress related proteins (HSP16.9). Aldose reductase activity measured in resistant and susceptible genotypes before and after infection suggests the importance of constitutive levels of this enzyme to resistance. Results of this study point to a correlation between host resistance and stress tolerance. The putative function of each identified protein is discussed.     

An ecological risk assessment for spinosad use on cotton

Spinosad is a reduced-risk insecticide with a novel mode of action that provides an alternative to older classes of insecticides such as organophosphates, carbamates and pyrethroids. A comprehensive ecological risk assessment for spinosad use in US cotton crops is presented within a framework of tiered levels of refinement following the guidelines of the US EPA for ecological risk assessments. Toxicity information for a variety of species is documented and utilized, environmental concentrations estimated, and risk characterizations in the form of risk quotients are quantified. Results indicate that spinosad use in cotton does not exceed the most conservative Tier I levels of concern (LOC) values for groundwater, mammals and birds or acute risk to aquatic organisms. Use of very conservative Tier I screening methods resulted in exceeding LOC values for chronic exposure for some aquatic organisms, thus prompting further refinement. When the exposure prediction was refined using less conservative, Tier II mechanistic environmental fate transport models to predict offsite transport and environmental concentrations, chronic risk was not predicted for these species. Spinosad is acutely toxic to bees under laboratory conditions, but toxicity of residue studies and field studies indicate that under actual use conditions the impact on bees is minimal.     

Assessment of a bovine viral diarrhea virus antigen capture ELISA and a microtiter virus isolation ELISA using pooled ear notch and serum samples.

To reduce the cost of whole herd screening for bovine viral diarrhea virus persistently infected animals, the sensitivity and specificity of an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a microtiter virus isolation ELISA using saline from ear notch samples or pooled serum was determined. Pooled saline from ear notch samples, assayed by AC-ELISA, gave a sensitivity and specificity of 98% and 94%, respectively, for pools containing 2 samples and 72% and 100%, respectively, for pools of 5. The sensitivity of pooled ear notch or serum samples for bovine viral diarrhea virus detection by microtiter virus isolation (sensitivity < 5%) or serum samples for detection by AC-ELISA (sensitivity < 15%) is too low to be used for whole herd screening. Pooling saline from ear notch samples from 2 animals tested by AC-ELISA, however, could provide a less expensive, reliable method for whole herd screening for bovine viral diarrhea virus.     

Identification of Maize Kernel Endosperm Proteins Associated with Resistance to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize (Zea mays). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics, and resistance-associated proteins were identified. Here, we report the comparison of maize endosperm proteins from five resistant and five susceptible genotypes, and the identification of additional resistance-associated proteins using the same approach. Ten protein spots were upregulated twofold or higher in resistant lines compared with susceptible ones. Peptide sequencing of these proteins identified them as a globulin-2 protein, late embryogenesis abundant proteins (LEA3 and LEA14), a stress-related peroxiredoxin antioxidant (PER1), heat-shock proteins (HSP17.2), a cold-regulated protein (COR), and an antifungal trypsin-inhibitor protein (TI). The gene encoding one such upregulated protein, PER1, was cloned and overexpressed in Escherichia coli. The overexpressed PER1 protein demonstrated peroxidase activity in vitro. In addition, per1 expression was significantly higher in the resistant genotype Mp420 than in the susceptible genotype B73 during the late stage of kernel development, and was significantly induced upon A. flavus infection, suggesting that it may play an important role in enhancing kernel stress tolerance and aflatoxin resistance. The significance of other identified proteins to host resistance and stress tolerance also is discussed.     

Plot-scale manipulations of organic matter inputs to soils correlate with shifts in microbial community composition in a lowland tropical rain forest

Little is known about the organisms responsible for decomposition in terrestrial ecosystems, or how variations in their relative abundance may influence soil carbon (C) cycling. Here, we altered organic matter in situ by manipulating both litter and throughfall inputs to tropical rain forest soils, and then used qPCR and error-corrected bar-coded pyrosequencing to investigate how the resulting changes in soil chemical properties affected microbial community structure. The plot-scale manipulations drove significant changes in microbial community composition: Acidobacteria were present in greater relative abundance in litter removal plots than in double-litter plots, while Alphaproteobacteria were found in higher relative abundance in double-litter and throughfall reduction plots than in control or litter removal plots. In addition, the bacterial:archaeal ratio was higher in double-litter than no-litter plots. The relative abundances of Actinobacteria, Alphaproteobacteria and Gammaproteobacteria were positively correlated with microbial biomass C and nitrogen (N), and soil N and C pools, while acidobacterial relative abundance was negatively correlated with these same factors. Bacterial:archaeal ratios were positively correlated with soil moisture, total soil C and N, extractable ammonium pools, and soil C:N ratios. Additionally, bacterial:archaeal ratios were positively related to the relative abundance of Actinobacteria, Gammaproteobacteria, and Actinobacteria, and negatively correlated to the relative abundance of Nitrospira and Acidobacteria. Together, our results support the copiotrophic/oligotrophic model of soil heterotrophic microbes suggested by Fierer et al. (2007).