Industrial hemp (Cannabis sativa L.) production is increasing dramatically in the US due to recent changes which lift restrictions on the growth and sale of hemp products; however, due to the decades-long prohibition of hemp, there is a lack of current research with respect to varieties and best agricultural practices for the many uses of this versatile crop. Natural fiber production relies on retting, a microbially-mediated process necessary for the separation of fibers from the plant which can occur unevenly in the field environment and result in inconsistent fiber quality and lower processing efficiency. In this study, the microbiome of hemp stalks is investigated throughout the retting process using 16S rRNA gene amplicon sequencing using the Illumina MiSeq platform. Field retting conditions were simulated in a controlled greenhouse environment in order to determine the effects of different moisture levels and soil contact on the retting process. Samples were taken over six time points, reflecting the community of freshly cut stalks to optimally-retted material, and finally over-retted material showing degraded fibers. The results show a very consistent population throughout retting, dominated primarily by Proteobacteria, but showing an increase in the abundance of the Bacteroidetes, namely Chryseobacterium, in time points corresponding to optimally-retted and over-retted stalks in treatments receiving higher moisture levels, but not in the low-moisture treatment. Soil application did not appear to influence the microbial community throughout retting, indicating a resilient population present in and on the hemp stalks at harvest.

Endophytic microorganisms live in intercellular and vascular spaces of plants and span the continuum from beneficial to pathogenic in both plants and animals. Increasing human consumption of fruits and vegetables has placed an emphasis on identifying and studying those microbes that colonize the internal tissues of plants, with a goal of limiting populations of enteric pathogens in store-bought foods meant for raw consumption, such as leafy greens. Culture-independent (i.e., metagenomic) methods are increasingly used to obtain an accurate snapshot of plant microbial communities, yet technical hurdles limit the accuracy and throughput of these methods. This includes the low-throughput nature of plant tissue maceration, and the prevalence of plant plastid DNA in metagenomic DNA extracts, which is typically coamplified via PCR strategies that target the bacterial 16S rRNA gene. In this study, we use kale (Brassica oleracea) as a model to explore the leafy green endophytic microbiome and to compare how two tissue maceration techniques used in traditional endophyte research compare in culture-independent microbiome studies using the Illumina Miseq platform. Three different brands of store-bought kale were surface sterilized and subjected to two tissue maceration strategies: enzyme digestion and blender processing. Analysis of 16S rRNA gene amplicon libraries revealed two highly abundant operational taxonomic units present in all libraries, one classified to the genus Pseudomonas and one to the family Enterobacteriaceae. Community structure and membership were highly similar among brands and between tissue maceration strategies, suggesting that both enzyme digestion and blender processing are suitable methods. Nonetheless, enzyme digestion may increase sample throughput and minimize steps involved in sample processing.

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A goal of the cooperative agreement between the U.S. Food and Drug Administration and the Center for Tobacco Reference Products (CTRP) at the University of Kentucky was to produce a standard reference cigarette for researchers to further tobacco science. The 1R6F certified reference cigarette for analytical testing was developed to be typical of an American blend cigarette. The cigarettes have been used in proficiency testing (PT) schemes under the supervision of the CTRP since 2016 for a variety of parameters. Data were compared from 8 rounds of PT schemes submitted by participating laboratories, which include various international stakeholders comprised of academic institutions, federal research/regulatory laboratories, tobacco product manufacturers, and independent contract testing laboratories. Evaluation of the datasets for harmful and potentially harmful constituents (HPHCs) within the mainstream smoke of the 1R6F certified reference cigarette included tobacco-specific nitrosamines (TSNA), tar, nicotine, and carbon monoxide (TNCO), and Benzo[α]pyrene (BαP). Of the datasets evaluated, less than 5 % resulted in either a warning or alert, and less than 2 % resulted in an alert. Comparisons across multiple PT rounds revealed no significant differences in the consensus means of the tested parameters. Evaluation of data submitted by participating laboratories for multiple proficiency test rounds shows that the 1R6F certified reference cigarette is an acceptable material as a control reference for the analysis of HPHCs which is imperative since some stakeholders may be required to participate in, and receive satisfactory results, from PT for accreditation purposes.