The forest BFF's (belowground fungal friends)

The mystic idea of fungi controlling the world beneath our feet has always been appealing to us humans, especially in these times where we all seek to reconnect with each other.

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Ectomycorrhizal fungi (EMF) have been shown to contribute to trees' health as well as to connect several trees creating common mycorrhizal networks (CMN's) belowground. The interaction between each tree and its mycorrhizal partner is based on the mutual exchange of carbon for nutrients. Carbon has been previously documented to move through these networks from one tree to its neighbor, despite the autotrophic nature of trees. While some studies supported the idea of these networks, direct evidence for the involvement of mycorrhizal fungi in carbon transfer was still scarce. Moreover, we were interested to know what governs this transfer? Whether the identity of the trees aboveground or the identity of the fungi below? 

The presence of fungi in our experimental mesocosms
the presence of fungi in our experimental mesocosms.
In this example, Suillus collinitus fruiting body
is emerging next to a Pinus halepensis sapling.

To answer these questions, we at the Weizmann Institute of Science Tree Lab (Stav Livne-Luzon, Rotem Cahanovitc, and Tamir Klein), aided by Roey Angel of Czech Biology Center, constructed a unique "three trees in a pot" experimental system. In this system, two trees could connect belowground (i.e., a Donor and a Recipient), separated by a fine mesh excluding the roots but not hyphal connections. The third tree served as a control, separated by a full plastic barrier. We labeled our middle tree (the Donor) using 13CO2 and for the next 36 days, we examined the δ13C values of the different plants' tissues and the respiration from the soil compartment of each tree. We used different combinations of Pinus halepensis and Quercus calliprinos saplings, growing on forest soil, enabling the plants to interact with various fungal partners creating diverse CMN's.

 We saw that, while the control plants’ tissues (leaf, stem, and roots) remained unlabeled throughout the duration of the experiment, in the Recipient trees, the roots were labeled well above natural δ13C variation (while the leaves remained unlabeled). Furthermore, the Recipients' stem tissues were slightly labeled, suggesting that the transferred carbon was further distributed in the recipient plant and that the label was not restricted to the EMF mantle that surrounds the root tips. The pulse labeling, coupled with a repeated sampling strategy of tissue and respiration allowed us to trace how the 13C was distributed through the Donor tree belowground and onto the Recipient tree. Six out of eight tree pairs, including all pair combinations, demonstrated a transfer of 13C to some extent, indicating that the transfer is not strictly dictated by phylogenetic relatedness among the trees.

 Subsequently, we were interested to know what governs the transfer of carbon belowground. If it is not the tree's identity aboveground, can it be the fungal identity below? To identify the fungal species active in the transfer, mycorrhizal fine root tips were used for DNA-stable isotope probing (SIP) with 13CO2 followed by sequencing of labeled DNA. Combining the sequencing and DNA-SIP results, we could create a novel taxonomic list of the main EMF genera involved in C transfer among neighboring trees, pinpointing the exact taxa involved. The EMF genera Pustularia, Terfezia, Tomentella, Tuber, Sphaerosporella, Geopora, and Suillus have been found to directly receive 13C from the pine Donor saplings and integrate the 13C into their DNA. Separate sequencing of the paired Recipients found Pustularia, Terfezia, Tomentella, and Tuber enriched 13C-DNA. While many fungal species were found to interact with either the Donor or Recipient trees, only a few species were indeed enriched with 13C-DNA on both the Donor and the Recipient sides. This suggested that while Pines and Oaks have some shared fungal taxa, only a few species engage in the transfer of carbon among these phylogenetically distant trees. Why do some fungal species "share the wealth" and others don’t? This is one question that is remained to be answered.

The experimental scheme
the experimental scheme included labeling of trees with 13CO2, repeated sampling of the plants' tissues followed by DNA-SIP, and sequencing of labeled DNA, which allowed us to identify the fungal partners engaged in carbon transfer between neighboring trees. 

 We detected carbon transfer among neighboring trees, and the absolute amounts of carbon transferred through these networks were small, yet significant. The fate of this carbon, the form at which it is being transferred, and the ecological significance of its transfer are all questions yet to be unraveled. By improving our knowledge of these key players' identity and ecological role, we better comprehend the interactions shaping forest biomes. It may well be that the mystic ideas of fungi controlling the forest architecture from below are not that imaginary after all. In these times where we seek to reconnect with each other, we can all learn from the forest BFF: belowground fungal friends!



Post-doc, Weizmann Institute of Science