Fungi

Endophytes – literally ‘inside a plant’ – are micro-organisms (bacteria or fungi in most cases) that carry out all or part of their life cycle inside a plant in a symbiotic manner, but with no systematic mutual benefit. Discreet, because they cause no apparent symptoms of disease, they are actually ubiquitous and can be found in almost every plant species studied to date. The endophytic species already known are very diverse, and only a small proportion have probably been identified, characterised and named. There may be over a million species of endophytic fungi, but only 75,000 had been described by 2005, and the number of species is still largely unknown. A single leaf can harbour numerous endophyte species, both bacterial and fungal.

In the Life On Trees programme, we are only investigating endophytic fungi and not bacteria. Accepted by their host plant’s immune system – probably as a result of a long process of coevolution – endophytic fungi colonise the interior of the living tissues of all or part of the plant.

They can be identified in several ways, increasingly by amplification (PCR) and sequencing of a small piece of DNA. Some endophytes can be cultured from a fragment of their host plant (wood or leaf) in an appropriate growth medium. These two approaches are used in the Life On Trees programme.

In the first approach, the tissues of the tree and its epiphytic plants are taken, sterilised and preserved. The DNA is then extracted in a single operation (analysis of a set of genomes), without the possibility of extracting it species by species. The advantage of this procedure is that it gives access to a large part of the fungal community. The disadvantage is that nothing more than DNA sequences are obtained, and identifications to species are questionable after bioinformatics analysis.

The other approach is to culture these endophytic fungi directly in the field, in order to isolate each fungal line in pure culture. Once the individual strains of fungi are sufficiently grown, samples are taken and placed in a buffer solution to keep the DNA intact for future molecular analysis. Determining the number of genera and species harvested is impossible on the basis of the morphology of the asexual form, which is the only form that develops on agar medium. The morphological characteristics that enable species to be identified can only be observed in the sexual form. This is why the fungus barcode gene (ITS, for Internal Transcribed Spacer) is sequenced and the names of genera and species are obtained by searching for sequence similarities in global databases (GenBank, UNITE).

The advantage is that the identification work is carried out on a strain-by-strain basis and that, after processing, pure cultures of individual species/strains are always available, which can be reused for future work or additional DNA analyses if there is any doubt about the species. The disadvantage is that part of the fungal community is inaccessible: some endophytic fungi cannot be cultivated, and for those that can be cultivated, only fast-growing ones can be cultivated in the relatively short time available for a field mission.

Fungi are collected directly from the tree and its epiphytes (in the mosses in particular), as well as from dead wood removed from the tree. Among the ‘large fungi’ collected, i.e. those that can be seen with the naked eye or a magnifying glass, there are two main groups: the Ascomycetes, which form their spores inside a reproductive cell (an ascus), and the Basidiomycetes, which form their spores outside a reproductive cell (a basidium). Among the collections obtained from dead wood or directly from the crowns of the trees studied, the most common fungi belong to the Basidiomycetes group.

Myxomycetes (Amoebozoa)

Myxomycetes were initially grouped with fungi because their life cycles include a macroscopic vegetative sporulation stage. This grouping turned out to be polyphyletic, comprising several independent lineages of protists, none of which are related to the ‘true fungi’ of the Fungi kingdom. Their name comes from ‘myxo’, meaning gelatinous or slimy, in reference to the texture of the plasmodia. They are also known as ‘amoeboid fungi’. The core of the historical group (Myxomycetes in the strict sense or Mycetozoa) has been integrated into the phylum (or supergroup) Amoebozoa, which also includes many amoebae. However, they are still traditionally studied by mycologists, even though they tend to be ‘collective amoebae’. Several collections of Myxomycetes have been obtained from dead wood taken from trees in the LOT programme.