Gymnopus fusipes (syn. Collybia fusipes), is an understudied basidiomycete fungus commonly found in woodlands across Europe. G. fusipes is a slow-growing primary pathogen, with the ability to destroy whole root systems, and has been linked to episodes of oak decline in Europe and the UK. Orange lesions on large central roots are characteristic of G. fusipes infection and are often
accompanied by white mycelial fans. Above the ground level, typical decline symptoms (such as poor crown condition and presence of fruiting bodies) are not always correlated with infection status, leading to ineffective detection and diagnosis, leaving young and mature trees at higher risk of being wind thrown due to a lack of anchoring roots. To address the paucity of information on the infection biology and ecology of G. fusipes, the current study focused on four main objectives; (i) to conduct a systematic literature review to highlight existing knowledge and identify key knowledge gaps in order to collate and analyse existing information on G. fusipes into an up-to-date resource on the species, (ii) to optimise methods for isolation, culture, nucleic acid extraction and phylogenetic analysis of G. fusipes, (iii) to develop a rapid molecular-based diagnostic assay, suitable for use on field samples, to allow for accurate diagnosis of G. fusipes, without the need for pure culture, and
(iv) to investigate disease progression at a molecular level using transcriptomic analysis of G. fusipes at different infection stages, including an early infective state (through a seedling inoculation trial), an established infective state (through sampling at a site heavily impacted with G. fusipes) and a non-infective state (through vegetative mycelial cultures).
A systematic review of 96 publications revealed that G. fusipes is associated with numerous species of tree host, mainly Quercus spp. and is suggested to have a presence across the northern hemisphere, although a lack of molecular validation of its identity and occasional spurious citation in the small amounts of focused literature makes this unclear. Optimisation of culture dependant methods and molecular analyses identified the best practice for isolation of G. fusipes from
environmental samples and cultivation in the lab. Optimal methods for nucleic acid extraction were also documented. A growth rate study of five geographically diverse G. fusipes strains across five ecologically relevant temperatures suggested that temperature and the G. fusipes isolate selected both had a significant effect on the growth rate of G. fusipes, however there was no significant interaction between the temperature and isolate, indicating that there is no localised temperature adaptation in this species. A G. fusipes specific qPCR assay, based on the fungal 18S rRNA gene was developed to be applicable to a range of environmental sample types without the need for pure culture, resulting in accurate detection of this species in mycelial plate cultures, fruiting bodies and
infected woody tissues. Genome sequencing combined with comparative transcriptomic analysis of G. fusipes in different infective stages highlighted differences in gene expression profiles between a non-infective agar culture, and an active infection in living tissue, illustrating that genes involved with processes such as enzyme production and transcription promotion are highly expressed in
active infection, and those encoding carbohydrate binding and chitin production being highly expressed when in a non-infective state.
This body of work represents important progression in understanding the biology, ecology and infection biology of G. fusipes. The data presented in this study are crucial to informing management and potential methods to combat this important root rot pathogen.