Sampling the Fungi That Drive Soil Carbon Sequestration.

By Oliver Lindsay, research assistant at Royal Botanic Gardens, Kew

Laura, Oli, and Heather on site. Prof. Martin Bidartondo is part of the team too, but he missed this trip!

On the 20th of June 2022, Dr. Laura M. Suz and I set off from the Jodrell lab at Kew Gardens towards Carmarthenshire in Wales. It was our first visit to the site since I started the research assistant position and we were armed with soil corers, hundreds of plastic bags but most importantly, antihistamines (just in case). Our destination was Glandwr Forest and our goal was to peer below the surface to reveal the communities of symbiotic fungi associating with the trees planted for The Carbon Community’s landmark carbon study.

Mycorrhizal fungi are immensely important for trees by increasing the functional surface area of their root-network. They provide their host plant with nutrients and water from the soil and in return, they receive sugars and other products of photosynthesis from the plant - it’s a win-win situation.

These symbiotic relationships are critical to plant health and development, with an estimated 250,000 plant species forming them – this represents approximately 90% of all the plants we’ve described on Earth!

There are two main types of mycorrhizas that are important for The Carbon Community study: ectomycorrhizas (ECMs) and arbuscular mycorrhizas (AMs). Both types are important in taking carbon from the tree and moving it into the soil, either via the physical biomass of the fungal mycelium, or through exuding carbon-containing chemicals into their environment.

These chemicals also help the fungi to structure and stabilise soil by clumping it together and forming aggregates. That’s not all the tricks up their sleeve: mycorrhizal fungi can also suppress the release of carbon, by outcompeting decomposers in the soil.

The main part of our research project at Kew is to compare the mycorrhizal communities between the different treatments applied to the study plots, with special interest on the impact of the soil inoculation from nearby mature woodland (see below for a picture of Laura and Martin sampling here). This information, coupled with the work carried out by the volunteers at the site and the other research partners will be important in informing future reforestation efforts in the UK, with a focus on enhancing biodiversity and carbon storage capacity both above and below ground (thanks to the fungi).

An oak root from the site colonised by at least two different ectomycorrhizal fungi.

Our first trip was a pilot – we needed to figure out how we were going to sample the saplings in the study and how long it would take, there are a lot of trees and sampling them all would have been a mammoth undertaking! Needless to say, Heather was invaluable in helping us navigate the site and providing expertise around tree identification - especially when my hay fever began to kick in, so thank you Heather! However, despite the allergies, there was plenty of time to admire the truly magnificent scenery of the surrounding countryside when we’d finished sampling. Patchwork fields and rolling hills, with the peaks of Bannau Brycheiniog in the distance – it was a privilege to be able to work in such a beautiful landscape.

Apart from the occasional mushroom, truffle or crust, mycorrhizal fungi are predominantly hidden from view below the surface, so in order to capture the community of fungi forming symbioses, we needed to get directly at the roots of the trees.

We did this with a soil corer – the tool of the trade for the mycorrhizal team at Kew. Individual trees for sampling were chosen using an arcane and complex set of rules that I won’t divulge here, but once a tree was chosen, we took four ~25cm soil cores from around the base of the stem, each one receiving its own bag. The hope was that there would be roots colonised by mycorrhizal fungi within those cores, so we could select and identify the fungi and determine the mycorrhizal communities present. At the end of the day, our efforts yielded 84 soil cores taken from 21 trees from across the site, and one from a control plot. Now the real work could begin!

Closeup of anectomycorrhizal root colonised by Xerocomellus pruinatus (the mattbolete).

After transporting our bounty back to London, we could start processing the samples. To reveal the roots, each soil core was washed in a sieve with a tight enough mesh to prevent any of the smaller root fragments from falling through. We then took the roots to the dissecting microscope for a closer look. The trees in the study that form ECMs are oak, birch, alder, Sitka spruce and aspen (the last one is a special case as it can form both types of mycorrhizal relationship, ECM and AM). For these trees, it is possible to see the symbiotic fungus colonising their fine roots, sometimes even with the naked eye, but a microscope makes it easier.

In ECMs, the fungi encase the ends of the very fine roots in a net of specialised hyphae called a mantle. This structure makes the roots look thicker and is often a different colour and texture to the plant tissues. Once you know what to look for, it becomes possible to isolate and pick out the colonised roots from the rest of the rabble.

The sheer diversity of colours and morphology of ECMs made sure this job was never boring (why are they so colourful when they live in the dark?!). We recorded their morphology as it can give us clues about their role in the uptake of nutrients and how much carbon they pump into the soil.

Stained arbuscules and hyphae within the cells of a Rowan root. Viewed under a compound light microscope (x60 magnification)

Once colonised roots were picked from cores, it came time for a bit of root surgery. In order to find out what species were colonising the roots, we needed to extract and sequence their DNA.

From each sampled tree, we excised 12 individually colonised ectomycorrhizal root tips. However, before taking the tips, they were each meticulously cleaned of soil and potential contaminants using a tiny paintbrush. This became quite a delicate operation at times! Once all the cores and roots were processed, the DNA of the mycorrhizal fungi could be sequenced, and their secrets revealed.

The AM roots were a slightly different story. There are three trees in the study that can form this type of mycorrhiza: wild cherry, rowan and the dual host aspen. Unlike ECM fungi, arbuscular mycorrhizal fungi are shy and don’t form any visible external structures on the roots they colonise. We had to instead rely on the fact that we sampled from directly around the trees, so any woody root would be from that individual rather than anything else that was growing nearby (mainly grass or other herbs with non-woody roots).

This may change and become more challenging in the future as the trees and their roots grow. The collected roots were destined for one of two things:  to either be subjected to a 60 °C bath of potassium hydroxide (a kind of potash), followed by a 100 °C cocktail of vinegar and ink; or frozen and crushed to extract the DNA of the fungi within the roots.

The first method briefly describes the root staining process, which reveals the intracellular structures the fungus makes within the root by staining them blue with ink. The key structures here are the arbuscules, from which the AM fungi get their name. These are incredibly fine, branched hyphae occurring within the root cells that form the interface of symbiotic nutrient exchange with the host plant. Looking at these tree-like structures under a microscope allows us to gauge the levels of colonisation within the roots, and by proxy, the magnitude of nutrient exchange occurring between the trees and the soil. The other roots collected were for DNA sequencing, to figure out what fungal species are colonising the trees and whether they differ between treatments.

Laura and Martin sampling roots at the mature woodland site.

I went up to the carbon study site four times between June 2022 and February 2023, accompanied mostly by my colleague Ben Underwood, and one time Brigid Wong, a student from Imperial College London. Altogether we collected a total of 784 soil cores. We sampled 184 seedlings from across all the 72 treatment replicates; we took cores from six of the hedgerows bordering the study; and we took 16 cores from the control treatments. All these cores were washed and scoured for roots, again with a lot of help from my colleagues in the mycorrhizal lab at Kew – many collective hours were spent doing this!

That’s not the end of the story though, each of the 1,700 individual EM root tips we collected have been sequenced and the fungal data are being thoroughly analysed and combed through, along with the fungal data from the nursery seedlings which I haven’t even touched on here, and the many AM roots assessed for their colonisation level. We’re excited to see how it all comes together and what it reveals! Does soil inoculation from mature woodlands impact the mycorrhizal communities that associate with the trees? Do these communities work synergistically with basalt to increase carbon storage and accelerate tree growth? The answers to those questions will impact the way forests are regenerated in the UK, and it’s been an absolute privilege to come along for the ride.

A well-placed bench at the site, with views towards Bannau Brycheiniog national park.

Further reading:

van der Heijden MGA, Martin FM, Selosse MA, Sanders IR. 2015 Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytologist 205(4), pp. 1406–1423.

Suz LM, Bode J, Byrne A, van der Linde S, Bidartondo MI. 2022. Nutrients, carbon, mycorrhizas and tipping points in forests. Quarterly Journal of Forestry 116: 36-43.

Bidartondo MI, Suz LM. 2020. Fungi, nitrogen deposition and forests: challenges and changes. In Practice 110: 26-30.