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Unraveling the ecology of a widespread fungal group by genomic, isotopic and physiological analyses

Periodic Reporting for period 1 - Mycena (Unraveling the ecology of a widespread fungal group by genomic, isotopic and physiological analyses)

Reporting period: 2015-08-01 to 2017-07-31

"In my project ""MYCENA"", I have been working on elucidating the life strategy of Mycena (or Bonnet mushrooms), a widespread group of macrofungi with important functions in forests and other terrestrial ecosystems. Like animals, mushrooms cannot do photosynthesis themselves, and must obtain their carbon from plants. Basically, mushrooms can do that either by degrading already dead plant material (wood, debris and litter), or by associating with a live plant source. This latter group of mushrooms can again be roughly subdivided into those that form a parasitic relationship, where they harm or kill the plant by sucking out carbon without providing anything in return; and those that form a mutualistic (mykorrhizal) relationship, where the plant receives phosphorous and nitrogen in return for the carbon allocated to the mushroom.

All the thre types of fungal relationships with plants are highly important to every ecosystem on Earth, as to the forestry, agriculture and biotechnological industries. The degraders help clear the earth of dead leaves and twigs, providing the foundation for the life of multiple other organisms´ lives, and many enzymes produced in large quantities by biotech industries originate from degrading fungi. The mykorrizhal fungi enhance the growth of their tree hosts significantly, while parasitic fungi are responsible for enormous losses to forestry and agriculture. While knowledge of the lifestyle of fungal species is very important, a major limitation is that only relatively few mushroom species have a well-known lifestyle.

The genus Mycena is one of the largest in the macrofungi (those with a stipe and a cap), and its members had traditionally been uniformly assumed to be degraders of dead plant material. However, modern DNA sequencing studies have shown that surprisingly, many living plant roots actually contain Mycena DNA, suggesting that several members of the genus could in fact be ""biotrophic"" This finding was itself a byproduct of other research, but my MYCENA project directly targeted the members of the genus - in plant roots, in direct growth experiments with one Mycena species and one plant (to see if and how they affected each other), and in full sequencing of the DNA in their genomes (to see if the mushrooms had a genetic makeup that were mostly fit for interaction with living plants, or with breaking down dead plant material), taking advantage of the most modern and cutting-edge molecular biological technologies."
I used 4 lines of work (termed WP1-4):

In WP1, gathered a large dataset of DNA sequences derived from roots of 9 putative host plant species from temperate and Arctic, and analysed them for their content of Mycena sequences. I created a small database (about 400) of high-quality DNA sequences frown known Mycena species that I could use as a reference to identify the root sequences to specific Mycenas.

In WP2, I collected Mycena species and used them to create a culture collection of pure cultures. I grew up to 50 plates of 25 species, and harvested DNA and RNA from them in order to sequence their genomes and predict their genes. We applied for extra funding and successfully obtained it, in collaboration with Dr. Francis Martin in Nancy. The sequencing itself is being done at the Joint Genome Institute in California.

In WP3, 10 selected species from these same abovementioned pure Mycena cultures were used to grow together with pine seedlings in order to study the interactions with Mycena species and pine. Much of the practical work here was done by Msc Ella Thoen in a collaboration as part of her PhD thesis.

In WP4, I used stable nitrogen and carbon isotopes to analyse wild Mycena collections in the context of whole ecosystems. I collected plants, other known (non-Mycena) degraders and mykorrhizal mushroom, and Mycenas in order to compare their relative content of heavy carbon-13 and nitrogen-15. I analysed 5 different biotopes resembling those where the host plants for WP1 had been collected. I analysed around 350 samples.

An overview of the main results at the end of the project is a follows:

• Mycena species are commonly found inside plant roots of all sorts of plants known to be ectomykorrhizal host species
• However, they are much more frequently found in studies analysing the whole root system or at least not just the root tip) than in studies targeting the root tip specifically
• Thus, they are likely not associated with the root tip and thus not with the primary feeding root like most ectomykorrhizal species
• Many of these “root Mycena species” (or OTUs) are not known from already described species
• Growth experiments show no evidence of a symbiotic (mykorrhizal) relationship
• This is also corroborated by the isotopic evidence
• Growth experiments do, however, show evidence of a strongly negative impact of Mycena on plant hosts for certain species – not merely parasitic, but even necrotrophic
• Mycenas in roots are slightly more common in Arctic than in temperate zones

Summing up, our preliminary conclusions are that most known Mycenas are indeed saprotrophs, but that some of them appear to be opportunistic biotrophic root invaders that prey upon living plants. Our hypothesis is that this is the reason that they are frequently found in roots. That this is more common in the harsh Arctic environment than in more benevolent climates might suggest that this is a trait that is developed as a survival response to stressful/extreme environmental conditions and short growth seasons. There are no indications that being associated with Mycenas offer any type of benefit to the plants.
As all Mycenas have hitherto been considered uniformly saprotrophic, this project has shown that this genus has a much broader nutritional range than previously thought.
When all results are in by 2018, we expect to have revealed their genetic makeup of the Mycena lineage, shown that the ecology of this group has many more facts than previously believed, and put together the story of the evolutionary history of the Mycena mushrooms as a group. The fact that Mycenas can be opportunistic biotrophs will be of interest to anyone working with ecology, with adaptation to life in extreme environments, and especially to all mycologists.

The project was conceived mostly as a pure scientific endeavour. However, there will be an interesting result of the project from an economical-biotechnological point of view which is actually a byproduct not mentioned in the original application: Among the 25 genome sequenced species, there are generalist degrader species that grow on just about any carbon source offering itself, slightly specialised degraders who prefer conifer wood, hardwood or litter, and some that are very highly specialised that only grow on litter/debris from one or a few species. The genes associated with the degradation of these substrates will provide the direct DNA code for a lot of new enzymes that can be used by the biotechnological industries that are always looking for new enzymes with highly specific abilities in degrading or modifying substrates for the industry.
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6 pure cultures