Estimation of Total Phenolic Compounds and Non-Targeted Volatile Metabolomics in Leaf Tissues of American Chestnut (<i>Castanea dentata</i>), Chinese Chestnut (<i>Castanea mollissima</i>) and the Backcross Breeding Generations

The American chestnut (Castanea dentata) was once a dominant tree species in the Appalachian Mountains and played a critical role in the ecological system. However, it was nearly eliminated by chestnut blight caused by the Ascomycetous fungus Cryphonectria parasitica. Identification of compounds specific to species and backcross hybrids may help further refine disease resistance breeding and testing. Phenolic compounds produced by plants are significant to their defense mechanisms against fungal pathogens. Therefore, an analytical platform has been developed to estimate the total phenolic content in leaf tissues of the American chestnut, Chinese chestnut (Castanea mollissima), and their backcross breeding generations (B3F2 and B3F3) using the Folin-Ciocalteu reagent assay with UV/Vis spectrophotometry which may be used to predict blight resistance. Adsorption (765 nm) results from leaf tissue extraction in methanol/water (95%:5% v/v) and pH 2, show that the variations among these four tree species are significant (ANOVA p = 2.3 × 10-7). The kinetics of phenolic compound solid-liquid extraction was elaborated using Peleg, second order, Elovich, and power law models. In addition, extensive analysis using headspace solid phase microextraction (SPME) gas chromatography and mass spectrometry was conducted to identify volatile organic compounds (VOCs) from the leaf of American chestnut, Chinese chestnut, and their backcross hybrids B3F2 and B3F3. A total of 67 VOCs were identified among all chestnut types. Many of the metabolites associated with the Chinese chestnut have been reported to have antifungal properties, whereas the native and hybrid American chestnut metabolites have not. Most of the antifungal metabolites showed the strongest efficacy towards the Ascomycota phylum. A partial least squares discriminant analysis (PLS-DA) model (R2X = 0.884, R2Y = 0.917, Q2 = 0.584) differentiated chestnut species and hybrids within the first five principal component (PCs).

Analysis of trace dicyandiamide in stream water using solid phase extraction and liquid chromatography UV spectrometry

An improved method for trace level quantification of dicyandiamide in stream water has been developed. This method includes sample pretreatment using solid phase extraction.The extraction procedure（including loading, washing, and eluting） used a flow rate of1.0 m L/min, and dicyandiamide was eluted with 20 m L of a methanol/acetonitrile mixture（V/V = 2：3）, followed by pre-concentration using nitrogen evaporation and analysis with high performance liquid chromatography–ultraviolet spectroscopy（HPLC–UV）. Sample extraction was carried out using a Waters Sep-Pak AC-2 Cartridge（with activated carbon）.Separation was achieved on a ZIC-Hydrophilic Interaction Liquid Chromatography（ZIC-HILIC）（50 mm × 2.1 mm, 3.5 μm） chromatography column and quantification was accomplished based on UV absorbance. A reliable linear relationship was obtained for the calibration curve using standard solutions（R^2〉 0.999）. Recoveries for dicyandiamide ranged from 84.6% to 96.8%, and the relative standard deviations（RSDs, n = 3） were below 6.1% with a detection limit of 5.0 ng/m L for stream water samples.