Inferring the presence of aflatoxin-producing Aspergillus flavus strains using RNA sequencing and electronic probes as a transcriptomic screening tool.

E-probe Diagnostic for Nucleic acid Analysis (EDNA) is a bioinformatic tool originally developed to detect plant pathogens in metagenomic databases. However, enhancements made to EDNA increased its capacity to conduct hypothesis directed detection of specific gene targets present in transcriptomic databases. To target specific pathogenicity factors used by the pathogen to infect its host or other targets of interest, e-probes need to be developed for transcripts related to that function. In this study, EDNA transcriptomics (EDNAtran) was developed to detect the expression of genes related to aflatoxin production at the transcriptomic level. E-probes were designed from genes up-regulated during A. flavus aflatoxin production. EDNAtran detected gene transcripts related to aflatoxin production in a transcriptomic database from corn, where aflatoxin was produced. The results were significantly different from e-probes being used in the transcriptomic database where aflatoxin was not produced (atoxigenic AF36 strain and toxigenic AF70 in Potato Dextrose Broth).

Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia.

Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264 bp PCR product for S. minor, a 218 bp product for S. homoeocarpa, a 171 bp product for S. sclerotiorum, and a 97 bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001 pg/µL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction.

Capillary electrophoresis of some free fatty acids using partially aqueous electrolyte systems and indirect UV detection. Application to the analysis of oleic and linoleic acids in peanut breeding lines.

This study has shown for the first time the suitability of CE with a partially aqueous electrolyte system for the analysis of free fatty acids (FFAs) in small portions of single peanut seeds. The partially aqueous electrolyte system consisted of 40 mM Tris, 2.5 mM adenosine-5'-monophosphate (AMP) and 7 mM alpha-CD in (N-methylformamide) NMF/dioxane/water (5:3:2 by volume) mixture, pH 8-9. While AMP served as the background UV absorber for indirect UV detection of the FFAs, the alpha-CD functioned as the selectivity modulator by affecting the relative effective electrophoretic mobilities of the various FFAs due to their differential association with alpha-CD. This CE method allowed the screening of peanut seeds for their content of oleic and linoleic acids, which is essential in breeding of peanuts of high-oleic acid content. The extraction method of FFAs from peanut seeds is very reproducible with a high recovery approaching quantitative yield (approximately 97% recovery).