Abstract Bachelor Project FBT 2018-2019: Development of identification techniques for plant pathogenic fungi and bacteria
Phytopathogens can cause enormous losses in yield and quality of crops. Different crops are threatened by various plant diseases. The increasing problem provides the access to novel findings to characterize fungi and bacteria. Taxonomic identification is important for controlling plant pathogens.
Morphological approaches are routinely used in taxonomic studies for classification of fungi and bacteria. Using morphology alone is too limited, that’s why researchers are using a combination of morphology and molecular data. The goal of this study is to develop a general polymerase chain reaction (PCR) for the identification of fungi and bacteria. In parallel, there was also made a flow chart for a general identification of bacteria.
First, desoxyribonucleic acid (DNA) is extracted from a pure culture of fungi or bacteria. By using universal primers, a selected locus is amplified. PCR results were checked by capillary electrophoresis. When successful, the 16S ribosomal DNA (rDNA) genes and the internal transcribed spacer (ITS) region are ready to be sequenced. Sequencing is performed by using the Sanger method, where the sequence is determined using a radioactive primer. The obtained sequences were aligned against a database.
The flow chart is based on four selective biomedical tests: MacConkey agar, oxidase test, King B agar and Yeast dextrose calcium carbonate agar (YDC).
By using five known fungi and bacteria species an optimized PCR protocol was obtained. The ITS region of fungi was amplified and the primer pair ITS5/4 was selected. The primer pair 27F/1492R was chosen for the amplification of the entire 16S rRNA region of bacteria. The effectivity was tested on unknown fungi species. It is necessary to check the GenBank identifications and to isolate the primary infection.
The theoretical flow chart was tested on known bacteria species. Most test results were as expected, but the color on MacConkey is not always pronounced.
An optimized protocol was developed for the identification of fungi and bacteria. When possible, both morphology and molecular data should be used for correct identification. Otherwise, the optimized protocol is used when the morphology is too unclear. The flow chart gives a general idea of the identification on genus level.
Abstract Bachelor Project 2017-2018: Determination of metals in surface-, ground- and drinking water using ICP-MS
The increased knowledge of the effect of metals in water has led to a raising demand for the determination of metals in water. Therefore, Inagro wants to obtain a recognition from an accreditation institution.
Inductive Coupled Plasma – Mass Spectrometry (ICP-MS) is used to measure the concentrations of metals. A development of the calibration curves, control samples and internal standards will lead to a usable method. Interferences must also be investigated in order to be able to assess the influence on other results. Eventually a validation can be performed to quantify the accuracy of the method.
Results show that some elements are difficult to measure. Especially mercury, aluminium, selenium and boron (only for ground and surface water) are the most problematic. Linearity, Limits of detection and trueness are reached for almost all elements with the ground and drinking water method. Some deviant linearity’s or high reproducibility’s lead to problems. However, the method for surface water shows more problems where especially repeatability has to improve. The measurement uncertainty depends on good results for trueness and repeatability. For this reason, some elements have obtained too high measurement uncertainties.
Taking all results in count, the surface and drinking water method comes up as the best method. For the majority of the elements recognition will be possible. The other method for surface water and some elements of drinking and surface water will require improvements before the method can enter routine measuring.