Isolation and Characterization of Agrobacterium Strains from Soil: A Laboratory Capstone Experience

Abstract

In this investigation, the students’ goal was to isolate and characterize Agrobacterium strains from soil. Following selection and enrichment on 1A-t medium, putative Agrobacterium isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the Rhizobiaceae, BIOVAR1 primers to identify members of Agrobacterium biovar group I, and a third set, VIRG, to determine presence of virG (only present in pathogenic Agrobacterium strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of Agrobacterium–plant interactions (p ≤ 0.001) and molecular biology (p ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores (p ≤ 0.0001) and attainment of capstone experience learning outcomes.

The Bean Beetle Microbiome Project: A Course-Based Undergraduate Research Experience in Microbiology

“…While still in its early stages, microbiome research has grown rapidly and garnered the attention of both scientists and non-scientists alike, providing an excellent and engaging topic area for undergraduate research experiences. Inquiry-based activities for student microbiome research have previously been developed for aquatic ecosystems ( Boomer et al, 2002 ; Gibbens et al, 2015 ; Agate et al, 2016 ), soil ( Martinez-Vaz et al, 2015 ; Finer et al, 2016 ; Rahman et al, 2016 ), public spaces ( Muth and McEntee, 2014 ; Weber and Werth, 2015 ), and the human microbiome ( Wang et al, 2015 ; Debelius et al, 2016 ; Garbarino and Mason, 2016 ; Lentz et al, 2017 ; Weber et al, 2018 ). Here we present a CURE using the bean beetle ( Callosobruchus maculatus ) as a model animal system for undergraduate microbiology laboratory courses.…”

Microbial characterization and fermentative characteristics of crop maize ensiled with unsalable vegetables

“…However Surette et al . 35 identified several genera on carrots, including Agrobacterium and Variovirax , bacteria typically isolated from soil 36,37 that were not present in the current samples. This difference could be explained as a result of commercial washing of the vegetables with Sodium hypochlorite, which has been previously demonstrated to reduce bacterial counts of Salmonella and Listeria by 2 log at 200 mg/L 38 .…”

Ecological performance of multifunctional pesticide tolerant strains of Mesorhizobium sp. in chickpea with recommended pendimethalin, ready-mix of pendimethalin and imazethpyr, carbendazim and chlorpyrifos application

“…The purity of cultures was tested using the Gram staining technique (Ouma et al 2016). Further, for differentiation of Mesorhizobium from Agrobacterium and other contaminants, different con rmatory tests were performed on the Hoffer's alkaline (Ansari et al, 2014), the ketolactose, (Finer et al 2016), the congo red yeast extract mannitol agar (CRYEMA) and the glucose peptone agar media (Gaur and Sen 1981). The cultures of mesorhizobia and rhizobacterium were maintained on the Yeast Extract Mannitol Agar (YEMA) and the Nutrient Agar (NA) media slants, respectively and stored at 4ºC for further use during the study period.…”