7. Molecular studies of hyperparasitic fungi
Hyperparasitic fungi belong to different phylogenetic lineages and have
different morphologies, and as a result, no specific set of molecular
methods has been developed to study hyperparasites. Yet, despite these
differences, researchers frequently encounter similar problems when
studying them. Some hyperparasites are minute in size and require
non-standard micromanipulation techniques. In addition, many have
melanin in their cell walls, which provides rigidity but inhibits PCR
amplification and the ability to get high quality DNA (Bermúdez-Cova et al., 2022; Eckhart et al., 2000; Haelewaters et
al., 2015).
Because they are part of multitrophic networks, it is common to find
hyperparasites intermingled with tissue of the primary parasite and
other organisms present in a given sample. This makes the isolation of
DNA exclusively from the hyperparasite difficult. Moreover, many
hyperparasitic fungi are biotrophs and cannot be grown axenically. The
hosts themselves may also be biotrophic, further complicating DNA
isolation from either partner. These factors have contributed to a lack
of reference sequences for taxonomic and systematics research and also
have ramifications even for genomics research; for mycoparasitic
hyperparasites, in silico attempts at de-novo genome sequencing
derived from metagenomic data can be unfeasible because the methods used
for separation of host and hyperparasite sequences cannot easily
discriminate between the two fungi (Quandt et al., 2017).
Due to the challenges described above, publicly accessible databases are
notably lacking in their representation of hyperparasites. As an
example, in the latest version of the UNITE database (version 9.0, 27
October 2022) (Nilsson et al., 2018), out of almost 8.4 million
ITS sequences, there are only 35 of Laboulbeniales—a taxon with over
2,300 described species and many more yet to be described (Haelewaters et al., 2021a). Not all species in this order are hyperparasites,
but many of them are, and as UNITE is the primary database used in
environmental microbiome studies (Tedersoo et al., 2022), the
paucity of taxa that are represented leads to an underreporting of their
presence in nature and therefore our understanding of the natural world.
Generalizations about the genetic “toolkit” that hyperparasitic fungi
use are difficult if not impossible to make, due to the phylogenetic and
morphological diversity of both the primary parasite and the primary
host. However, the nature of individual hyperparasitic relationships can
and should be investigated. In one such example, Koch and Herr (2021)
used transcriptomics (RNA-seq) to examine the differential expression of
genes in both the hyperparasite, Entoloma abortivum, and its
host, a plant-pathogenic Armillaria, during their parasitic
interaction compared to expression in their respective sporocarps.
Transcripts obtained from the interaction interface are mainly from E. abortivum, the hyperparasite, and contain genes hypothesized
to be involved in mediating recognition of Armillaria and
detoxification of compounds produced by the pathogen. Modern techniques
such as these now allow for examining the nature of the interaction
between the hyperparasite, its primary parasite, and the primary host.