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© Peter Werner
Original publication: Mycena News, December 2011

Mycorrhiza, the relationship in which fungi trade mineral nutrients, water, and other soil resources with plants for food in the form of carbohydrates, is often thought of as a model of co-operative mutualism. But is it necessarily? After all, there are many kinds of fungal root parasites that have at least some traits that resemble mycorrhizal fungi, and there are a broad range of plants that are myco-heterotrophs, “mycorrhizal cheaters” that function in many ways like mycorrhizal plants, but in fact, parasitize mycorrhizal fungi and their associated plants by taking up minerals and food without giving anything in return. Furthermore, not all mutualistic symbiosis is of equal benefit to both partners, and in many cases there is a dominant partner that controls the symbiosis. Lichens are a prime example of this, where the fungi are the dominant partner and, in a sense, hold captive the algae or cyanobacteria they live in intimate association with. Mutualisms in which one partner can easily turn the relationship into an exploitative one without pushback from the other tend to degenerate into parasitic relationships over time unless the other partner evolves control mechanisms of its own to keep the benefits of the relationship mutual.

And yet, mycorrhiza is a very old association, dating back to (and playing a key role in) the colonization of land by the first non-aquatic plant life. Fossil arbuscular mycorrhizas that resemble living mycorrhizas today have been found in the Early Devonian Rhynie chert, some 410 million years old. This would imply that this association between fungi and plants is very stable and that there are mechanisms on both sides to keep it mutually beneficial.

Recently, this idea was put to the test by an international group of scientists led by Toby Kiers of Vrije Universiteit, Amsterdam. The researchers hypothesized that for the mutualism to be truly stable, the relationship should be regulated by both partners. Plants should be able to allocate less carbon to fungi that offered the plant less phosphorus (thought to be the major and most critical nutrient provided to plants by arbuscular mycorrhizal fungi), and fungi should be able to offer less phosphorus to plants that offered less carbon.

The experiment was conducted using arbuscular mycorrhizal (AM) fungi, which are largely microscopic fungi in the Glomeromycota that form associations with the majority of the world’s plant species. (Note that this is a different kind of mycorrhizal association from what we’re used to as mushroom hunters—the ectomycorrhizas, in which asco- and basidiomycetes, often mushroom-forming ones, are associated with a select few plant groups, typically certain families of trees and shrubs.) The fungi chosen for the experiment were closely-related species of Glomus with differing phosphorus-sharing abilities, tested with the plant Medicago truncatula, a laboratory workhorse in the bean family (and closely related to alfalfa) often used in experiments examining mycorrhizal as well as nitrogen-fixing bacterial symbiosis.

The series of experiments involved providing stable heavy isotopes of carbon or phosphorus to one partner and testing how much ended up in the symbiont. Plant-to-fungus nutrient flow was tested using carbon-13 and -14, and fungus-to-plant flow was tested using phosphorus-32 and -33. In the first set of experiments, Medicago were inoculated with the “cooperative” species of Glomus intraradices that readily shared phosphorus, and several other “less cooperative” species (G. aggregatum and G. custos) that were less forthcoming with phosphorus. The plants were given 13CO2 during their growth. The roots and associated fungi were later harvested and RNA extracted, and selective primers used to isolate out RNA coming from each species of Glomus. From here, the amount of 13C that found its way into the RNA of each species could be quantified. (This is thought to be well-correlated with the amount of 13C that goes into the plant’s biomass overall.)

The results supported the hypothesis that plants rewarded AM fungal species that were known to provide more phosphorus, with the more cooperative Glomus species ending up with a higher proportion of 13C in its RNA than the less cooperative species. However, further testing needed to be done to understand how the fungi treated differing contributions from the plant, and whether the plant’s reaction was truly a reaction to different amounts of phosphorus being offered, or whether the plant simply rewarded different Glomus species differently, regardless of the amount of phosphorus the plant gets.

To answer this, an experiment was done using a sophisticated Petri dish design. Fungal cultures and Medicago root-tissue cultures were grown side-by-side in three isolated compartments. Later, the three subcultures were brought together so the plant and fungal cultures could associate and inoculate the roots with AM fungi. In one set of experiments, plants were fed 14C-tagged sucrose in one compartment, and no extra sucrose in another; fungi grew in a third compartment, and the fungi were later paired either with high-sucrose or low-sucrose roots, the latter being expected to provide little if any carbon to the fungi. In a reciprocal set of experiments, fungi were provided with 32P-tagged orthophosphate or no orthophosphate, with a root culture grown in the third compartment. In each set of experiments, the fungi were represented by two subsets consisting of “cooperative” G. intraradices and “less cooperative” G. aggregatum.

Again, the results supported the hypothesis that both plants and fungi directly reciprocated with less reward to partners providing less sugar or orthophosphate, and plants providing less reward to fungal species that were less sharing with available phosphorus. Cooperative Glomus that had been given (and presumably passed along) phosphate ended up with significantly more 14C than those without phosphate, while such was not the case with less-cooperative Glomus, regardless of available phosphorus. Similarly, roots given high levels of sucrose, and hence providing more to the fungi, ended up with significantly more 32P allocated to the fungal arbuscular compartment within the plant’s root cells. However, this was true with both the cooperative and less-cooperative Glomus. Closer analysis of the orthophosphate held in the arbuscular compartments told the whole story – the cooperative species stored more of the phosphate compound in a short-chain form readily available to the plant, whereas the less cooperative one stored more of the phosphate in a long-chain form available to the fungus, but not to the plant. The less-cooperative fungus hence “hoarded” available carbon and the plant responded accordingly with less of a sugar reward.

This series of experiments establishes that in the case of arbuscular mycorrhizas, mycorrhiza is indeed tightly regulated by both partners in a kind of “biological market”. This two-way control of the symbiosis should have a stabilizing effect on the relationship, and would explain its long persistence, as indicated by the fossil record. Further experimentation will be needed to see if this two-way control is true of ectomycorrhiza and other mycorrhizal systems as well.

The experiment raises other interesting questions that will also require further lines of inquiry. Like other types of mycorrhizas, there are a number of myco-heterotrophic “cheater” plants that parasitize AM fungi, not only getting phosphate and other minerals without providing carbon in return, but actually reversing the normal nutrient flow and getting carbon from the fungus. There is also at least one known case of a “cheater” AM fungus, in which the normally mutualistic Glomus macrocarpum acts as a parasite of tobacco, causing a wilting disease. How do these species overcome the normal tendency of plants and fungi to turn off reciprocation when the other partner is not donating its share? And in the case of myco-heterotrophs, which are known to have independently evolved a number of times, how does the fact that cheater plants commonly emerge within mycorrhizal evolutionary lines not destabilize the relationship over evolutionary time? As always, answering a scientific question raises many more.

Further reading

Peter Werner is a mycologist, a microscopy and imaging specialist, a biological photography enthusiast, and all-around science and natural history buff. After having earned his bachelor’s degree in botany from University of Washington, he studied fungal taxonomy for several years as a graduate student at SFSU. He has been an active mushroomer for the last 30 years and is a long-time member of MSSF.