Molecular ecological analyses of specific interactions between symbionts in the arbuscular mycorrhizal symbiosis

Arbuscular mycorrhiza is an ancient symbiosis between the majority of land plants and fungi
from the phylum Glomeromycota. Arbuscular mycorrhizal fungi (AMF) colonize plant roots
and contribute to the mineral nutrient uptake of the hosts in exchange for carbohydrates. AMF
species diversity and identity was reported to have a decisive influence on the composition
and productivity of natural plant communities. Only around 200 glomeromycotan species
described so far were thought to colonize the majority of higher plant species and thus, their
host specificity was thought to be very low.
In this thesis, molecular methods were used to investigate ecological aspects of root
colonizing AMF. The community composition of these fungi was analyzed in two plant
species-rich grasslands facing different environmental conditions and harboring different
plant communities. One site consisting of two meadows located close to each other was
situated in the upper montane zone of the Swiss Alps. The other was located in the lowland in
France on the edge of the Jura mountains. The roots were analyzed using AMF-specific
nested PCR, RFLP screening and sequencing of rDNA small subunit and internal transcribed
spacer regions. AMF sequences were analyzed phylogenetically and used to define
monophyletic sequence types.
Overall, 27 different AMF sequence types were detected in the root samples from both
field sites. The overlap between the AMF communities in the alpine and lowland site was
relatively small - they shared just six sequence types. These results indicate strong
geographical differences in the AMF community composition, reflecting different
environmental conditions and plant species communities in each site.
The question was adressed, whether different host plant species co-occurring in the same
area host distinct or similar AMF communities. Gentiana verna, G. acaulis and Trifolium spp.
growing in two alpine species-rich meadows harbored significantly different AMF
communities, whereas the differences between the two sites were negligible. These results
indicate that within a relatively small area with similar soil and climatic conditions, the host
plant species can have a major influence on the AMF communities within the roots. In these
alpine sites, there was also a focus on green plants from the family Gentianaceae. In contrast
to their mycoheterotrophic relatives, the green gentians did not show a high level of
specificity towards AMF. The plants sampled harbored AMF communities comprising
multiple phylotypes from different fungal lineages.
In the lowland site – a calcareous grassland – different culturing methods and their
influence on the AMF community composition in the roots were adressed. Four plant species
were sampled i) directly in the field, ii) in a bait plant bioassay conducted directly in that
grassland and iii) in a greenhouse trap experiment using soil and a transplanted whole plant
from that grassland as inoculum. The community composition in their roots was strongly
influenced by the experimental approach, with additional influence of cultivation duration,
substrate and host plant species in some experiments.
Some fungal phylotypes, e.g. Glomus mosseae and several members of Glomus group B,
appeared predominantly in the greenhouse experiment or in bait plants. These phylotypes can
be considered r strategists, rapidly colonizing uncolonized ruderal habitats in early
successional stages of the fungal community. Other phylotypes as Glomus badium and
GLOM-A16 were detected almost exclusively in roots sampled from plants naturally growing
in the grassland or from bait plants exposed in the field, indicating that they preferentially
occur in late successional stages of fungal communities and thus represent the K strategy. The
only phylotype found with high frequency in all three experimental approaches as well as in
the alpine site was GLOM A-1 (Glomus intraradices), which is assumed to be a generalist.
These ecological strategies of different AMF species or phylotypes should be considered
in AMF experimental work. In greenhouse trap experiments it is difficult to establish a rootcolonizing
AMF community reflecting the diversity of these fungi in the field roots, because
fungal succession in such artificial systems may bias the results. However, the field bait plant
approach might be a convenient way to study the influence of different environmental factors
on AMF community composition directly under the field conditions.
Finally, the co-existence of the Glomeromycota in the root samples with fungi from the
basidiomycotan order Sebacinales was addressed. These fungi are widely distributed and
known to form various types of presumable mycorrhizal associations of different morphology
(ecto-, ectendo-, orchid, ericoid, jungermannoid) with a broad range of host plant species.
However, their presence in plants forming arbuscular mycorrhiza has not been reported yet.
Therefore, the root samples originating from the two species-rich grasslands mentioned above
were analyzed with Sebacinales-specific primers for the D1/D2 region of the nuclear 28S
rDNA subunit. Fungi from the order Sebacinales were present in the majority of the root
samples from both sites, where they co-existed with the Glomeromycota. In agreement with
studies targeting the Sebacinales in ericoid plants, the phylogenetic analysis of sebacinoid
sequences from our samples did not reveal any patterns according to their host plant species
or geographical origin.