Can brown rot and white rot fungi work together to clean up spilled diesel?

Methods

Summary

This project will facilitate the interaction of a brown rot fungus (Fibroporia vaillantii) and a white rot fungus (Stropharia rugosoannulata) on a layer of compressed soil that has been contaminated with diesel.

Wood blocks made of oak and pine will be colonized by each fungus.  To do this, we will add sterilized wood blocks to 2-week-old cultures on agar and let them grow for 5 weeks.  These will be the inocula we use to introduce the fungi to each other on the soil.

I will collect topsoil from local woodlands, separate course debris, and spike it in the lab with diesel fuel to a concentration of 4% w/w.  I will then compress layers of this contaminated soil on 9" x 9" (about 23 cm square) tin cake pans, and place a wood block of each species on either end of the tray.  Because the soil is compressed, the fungi will primarily grow on the surface, and we'll be able to observe their growth and interaction when they meet in the middle of the tray.  They will grow on these trays for 10 weeks.

We'll observe the following pairings:

1. Fibroporia + Fibroporia

2. Stropharia + Stropharia

3. Fibroporia + Stropharia

4. Fibroporia alone for 5 weeks, then removed and replaced with Stropharia

5. Stropharia alone for 5 weeks, then removed and replaced with Fibroporia

6. No fungi 

Treatments 1-3 examine effects of fungal interactions, treatments 4-5 examine effects of one species arriving before the other ("priority effects"), and treatment 6 is a fungus-free control.

At weeks 0 and 10, we will analyze composite soil samples from each tray for diesel content using gas chromatography and mass spectrometry (GC-MS).  With this information, we will calculate the hydrocarbon removal ratio per carbon chain length detected-- this will tell us which species pairing was most successful in diesel degradation.

At weeks 0 and 10, we will also perform high-throughput Illumina DNA sequencing of soils from each tray to identify the initial composition of the soil microbial community, and then how that community changed after becoming colonized by the fungi.