6. Evidence of hyperparasitic interactions

Studies of hyperparasitic interactions between fungi and their hosts have been observed both in the field and by microscopy (Kim and Vujanovic, 2018; Moore et al., 2020; Smith et al., 2008). However, in most cases, the antagonistic activity of the hyperparasite is not evident in the field, and the exact interactions may only be revealed under laboratory conditions, when the cultivation of the hyperparasite is possible or when infected primary and/or secondary hosts can be reared.
The associations of hyperparasites and their hosts can be visualized by molecular techniques that employ expression of fluorescent proteins (Hasan et al., 2022). For example, the gene-encoding green fluorescent protein (GFP) was expressed in Trichoderma species, which helped to elucidate their interactions with Pythium ultimum, the invasion of the hyphae and sclerotia of Rhizoctonia solani, and the penetration of the plant-parasitic nematode Globodera pallida (Contina et al., 2017; Lu et al., 2004; Sarrocco et al., 2006). Also, Németh et al. (2019) used a GFP marker to visualize the life history strategy of Ampelomyces quisqualis.
Hyperparasitic interactions may be assumed if the parasite causes distinctive morphological or physiological alterations of the primary parasite, with the latter showing signs of phenotypic changes, such as deformation of cells, growth impairment, and changes in color (Gams et al., 2004Jeffries, 1995Zheng et al., 2017). For example, urediniospores of Puccinia striiformis f. sp. tritici collapse and lose viability after being colonized by hyphae of Alternaria alternata and Cladosporium cladosporioides (Zhan et al., 2014; Zheng et al., 2017). Parasitism may also be assumed when parasites affect the reproductive rate of the hosts, e.g., by decreasing levels of sporulation of fungal hosts. This has been observed for hyperparasites of black mildews, powdery mildews, and rusts and smuts (Bermúdez-Cova et al., 2022; Legler et al., 2016Zhan et al., 2014Zheng et al., 2017). The incapability of fungi growing on parasites to be cultured on axenic media, i.e., without their hosts, also serves as an indication that they are obligate hyperparasites (Jeffries, 1995).