3 Material and methods
5.6 Methodological considerations
The number of target gene copies g-1 DW sample was used as an indication of the biomass of AM fungi in the different substrates. It was assumed that the differences in copy numbers was a sign of difference in abundance of AM fungi. Of course, the primers used in the qPCR did not amplify all AM fungi and there is no one primer pair that will amplify only the wanted species (Kojima et al., 2004; Redecker et al., 2000; Sato et al., 2005). However, Sato et al. (2005) found in a Nucleotide BLAST search that the specific primers used in this assay amplified almost all AM fungi.
The Nucleotide BLAST search done in this study also only showed species of the Glomeromycota phylum, which form arbuscular mycorrhiza (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Redecker et al., 2013). The variation in number of gene copies within each treatment was rather large and small differences between the treatments may not be possible to detect. This large range is possibly partly due to the standard deviation, SD≤0.5, that was allowed for the technical rep-licates in the qPCR assay. This SD allowed for a maximum of one qPCR cycle dif-ference among the technical replicates run for a sample, which corresponds to a doubling in gene copies. The qPCR method is a very sensitive method since tiny amounts of DNA can be amplified (Bio-Rad Laboratories, n.d.) but might not be very precise as samples contaminated with foreign DNA during the laboratory pro-cesses could give misleading results. However, the blanks (control samples) were empty, supporting the validity of the results. The variation among replicates is most likely related to real biological variation in the system. There are a range of factors that could affect how well the samples reflect the plot-level AM fungal abundance e.g. sampling spatial organization, sample size, sub-sampling for analyses etc.
Another limitation was the microscope investigation since the staining was difficult to perfect and resulted in AM structures being difficult to discern, or simply my lack of experience made me miss structures. The latter is not very likely as several prac-tice runs were made before the actual investigation and many pictures of AM struc-tures were used to compare with. More likely there was low AM colonisation in the field.
The conclusion of this investigation is that application of waste-based fertilizers af-fects AM fungi in various ways. According to the results soil-associated AM fungal biomass are associated with a large host biomass, while root-associated AM fungal biomass are positively affected by a combination of digestate and ash. The mineral fertilizer had a positive effect on the wheat biomass but not on the root-associated AM fungal biomass. In order to explain the large variation within treatments, studies with more replicates could be conducted. Furthermore, community analyses could give further insight into the effects on AM life strategies.
Conclusions
I would like to thank my supervisors Sigrun Dahlin and Karina Engelbrecht Clem-mensen for always being available for questions and guidance, and all the personnel at the department of Forest mycology and plant pathology for giving of their time to help me with my experiments. I would also like to thank Elias Bäckström for the pH-values. Last, but not least, I would like to thank Formas for financing the project.
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