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.