Mushroom DNA barcoding project: Sequencing a segment of the 28S rRNA gene.

DNA barcoding is an important molecular methodology for species identification that was developed over the last two decades and it should be covered in the biology bachelor curriculum. Here, we present an example of DNA barcoding by sequencing a segment of the 28S nuclear ribosomal large subunit rRNA gene of wild mushrooms and framing the education in a project form for undergraduate students in biology. Students perform this project in 6-8 weeks, which also includes preparing a poster, writing a report and presenting a paper related to the work in a journal club format. First, fieldwork in the Netherlands was carried out, during which students collected mushrooms under supervision of a professional mycologist with the goal to (a) verify morphologically based identifications with a molecular method and (b) assess phylogenetic relationships of the different species collected. Next, DNA extractions and quantitation were performed, PCR amplification was done, and samples were sent out for Sanger sequencing. Students aligned and analyzed the sequences using BLAST and Geneious and subsequently created a phylogenetic tree. In case of collecting DNA barcodes of an earlier sequenced species, students could upload the data to a repository established for facilitation of future research projects. The method described is very robust, reagents and equipment are readily available, and costs are relatively low. In addition, the results can be compared to published fungal phylogenetic trees.

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents.

Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163.

During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide new insight into the changes occurring when Hp and Hb bind, the present study provides a new potential tool for measuring and removal of Hp-Hb complexes from plasma/serum.