LB 23-203 - Testing viability of RT-PCR for in situ measurements of fungal decomposition of wood

Measuring fungal activity in situ is a significant challenge facing mycology today. Most methods for measuring fungi in the wild rely on culturing samples in a lab or using DNA sequencing to determine presence/absence of specific species. The first excludes fungi that cannot be cultured well and may overestimate the contribution of species prone to culturing, and the second cannot measure activity, only presence/absence, and may include dead or trace species not contributing to decomposition at all. We instead attempted to measure fungal activity by sampling for RNA and investigating whether real-time polymerase chain reaction (RT-PCR) is a viable method for measuring fungal RNA expression. Samples of wood shavings were collected from decomposing logs of three species (Pinus taeda, Populus tremuloides, and Betula papyrifera) placed in a Minnesota forest ten months and eleven years earlier. Logs were laid as part of the FACE Wood Decomposition Experiment, a long-term NSF-funded project that also gives us long-term data on the climatic conditions and log characteristics over the course of those time spans. Two methods of RNA extraction (Qiagen RNEasy and Machery-Nagel NucleoSpin minikits) were tested. RT-PCR was run on 280 samples representing every substrate species and length of decomposition. Two primers coding to specific lignin-degrading enzymes, three primers coding to cellulose and hemicellulose enzymes, and three housekeeping genes were tested in a RT-PCR setup. Overall, the method showed promise. Pitfalls mostly stem from testing samples collected in the field, because the genetic material used is from an assemblage of fungal species, leading to messy or incomplete signals in the RT-PCR. Further, none of the three housekeeping genes were consistently amplified for samples collected in the summer, a common problem with fungal samples. This lack of a housekeeping gene prevented us from producing quantitative gene expression assessments. However, one primer each related to lignin, hemicellulose, and cellulose decomposition, worked consistently across all wood species and lengths of decomposition, which will allow us some insight into enzymatic activity within sample logs. Degree of amplification was greater than expected for the primers that did give a response. Overall, we’ve shown that RT-PCR, though not optimized for RNA analysis of complicated fungal assemblages, is a viable option for measuring enzymatic levels. This technique will allow for examination of fungal activity in sample logs, thus enabling us to relate decompositional pathways to climate, substrate species, time since death, log orientation, and more.