Wheat fungal endophyte communities are inseparable from the host and influence plant development.

Plants harbor complex and highly diverse fungal endophyte communities (FECs), making it difficult to evaluate the functional role of individual taxa, subsets of the community, or the FEC as a whole. To reduce the complexity of this system, we aimed to produce fungi-null wheat () plants. To this end, we treated seeds with heat and fungicides and generated plants from rescued embryos and callus tissue. A culture-based approach and reverse transcription PCR analysis were negative, indicating that all treatments produced plants apparently free of fungi. However, the analysis of DNA using digital droplet PCR and next-generation sequencing revealed that tissues from all treatments retained low levels but diversity-rich FECs. While the FECs varied in composition across treatments and tissues, they all included core taxa of the mycobiome. The reduced fungal biomass, along with the changes in FEC composition, negatively affected plant development, supporting a FEC contribution to proper plant development and fitness. Our discovery that a large part of the FEC cannot be separated from plants and can be transmitted through seeds and tissue culture calls for reevaluation of particular microbiome paradigms, such as core taxa concepts, transmission modes, and functional species.IMPORTANCEThe native microbiome in a given plant must be considered when evaluating the effect of a single taxon or synthetic community. The pre-existing microbiome can interact with artificially added microbial cargo, which affects the final outcome. Such issues can be at least partially solved by the use of endophyte-free plants, which provide a clean background that should be useful in determining the effect of a single taxon, taxa combinations, or the entire microbiome on plant performance. Previous reports regarded plants as endophyte-free or axenic by the lack of fungal growth on culture media or the generation of plants from tissue cultures. We showed here that while fungi could not be isolated from fungicide-treated or tissue culture-regenerated plants, nevertheless, all plants contained rich fungal endophyte communities; namely, it was impossible to create fungi-free wheat plants. Our results call for rethinking fundamental microbiome-related concepts, such as core taxa, transmission mode, and functional species.