Endophytic fungi of most woody trees are poorly studied due to their cryptic existence, the
complexity of the community and the poor ability of traditional tools to characterize them.
This study characterised the endophytic community present in commercially important
Eucalyptus grandis trees in South Africa. Two different experimental approaches were used.
Firstly environmental bar-coding, or metagenetics, was done using 454-pyrosequencing
parallel sequencing of the barcode amplicons of all the fungal isolates present in the plants
from the total DNA of the plant. Secondly, conventional DNA bar-coding of was done of
fungal endophyte isolates. Isolates of the Botryosphaeriaceae family of latent endophytic
pathogens were further characterised using a multi-gene phylogenetic approach from both
E. grandis and related native S. cordatum that grew in close proximity.
The endophytes within the three E. grandis trees were hyper diverse. A total of 1 281
Molecular Operational Taxonomic Units (MOTU) was identified based on 454-
pyrosequencing of the E. grandis fungal endophyte infections. Only 85 fungal endophytic
species were identified amongst isolates from one of these trees, using the conventional
DNA bar-coding approach. Fifteen times more species/MOTU was thus recovered using a
metagenetics compared to an isolation approach. Despite this high diversity the species
accumulation curves indicate that more endophytic diversity is to be discovered. The multigene
analysis of Botryosphaeriaceae isolates obtained from the E. grandis and S. cordatum
trees show that three species co-infect both these hosts. Two novel host associations are
also reported. This approach of verifying identities of cryptic species with appropriate multigene
analyses is most likely needed for other diverse species complexes associated with
A very thorough sampling strategy is required to adequately characterize the endophyte
diversity in trees. The experimental approach, the 454-pyrosequening identification workflow
and database described in this study will be useful to study these endophyte communities
over time and space in future. Using these techniques and workflows described, questions
related to host association, diversity and spatial distribution within hosts, and geographical
delimitation of endophytes can be addressed.