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Fungal endophytes develop inside plants without visible external signs, and they may confer adaptive advantages to their hosts. Culturing methods have been traditionally used to recognize the fungal endophytic assemblage, but novel metabarcoding techniques are being increasingly applied. This study aims to characterize the fungal endophytic assemblage in shoots, rhizomes and roots of the tall grass Brachypodium rupestre growing in a large area of natural grasslands with a continuum of anthropized disturbance regimes. Seven out of 88 taxa identified via metabarcoding accounted for 81.2% of the reads (Helotiaceae, Lachnum sp. A, Albotricha sp. A, Helotiales A, Agaricales A, Mycena sp. and Mollisiaceae C), revealing a small group of abundant endophytes and a large group of rare species. Although both methods detected the same trends in richness and fungal diversity among the tissues (root > rhizome > shoot) and grasslands (low-diversity > high-diversity grasslands), the metabarcoding tool identified 5.8 times more taxa than the traditional culturing method (15 taxa) but, surprisingly, failed to sequence the most isolated endophyte on plates, Omnidemptus graminis. Since both methods are still subject to important constraints, both are required to obtain a complete characterization of the fungal endophytic assemblage of the plant species.

Background and Aims In highland ecosystems, global change processes are intense and foster vegetation shifts that may have an impact on soil functioning. Soil bacterial communities may be particularly sensitive to these changing scenarios. The aim of this research is to determine whether the loss of floristic diversity caused by the unusual dominance of a native component -the perennial grass Brachypodium rupestre (L.) Beauv., which is expanding aggressively in natural grasslands of the Western Pyrenees-, parallels a decrease of the soil bacterial functional diversity and their potential for nutrient transformations.Methods. We conducted the study in eight grasslands exposed to different degrees of B. rupestre spreading. Soil community physiological profiles of the heterotrophic bacteria, enzymatic activities related to C, P and N cycles, C and N microbial biomasses, N components and soil physical and chemical properties were determined.Results Soils below low-diversity grasslands had lower bacterial functional richness and diversity but greater urease activity, pH and nitrate than soils in diverse grasslands. Ammonium pools, C and N microbial biomasses and enzymatic activities related to C and P did not differ between grasslands. ConclusionsThe expansion of B. rupestre and the decrease of plant diversity coincided with a significant decline of bacterial functional diversity and an alteration of the N cycle. Not only plant composition but the prevailing disturbance regime may account for the results. Results also suggest that B. rupestre may rely on its capability to use N efficiently rather than on a soil bacteria-mediated N availability.

The plant microbiome is likely to play a key role in the resilience of communities to the global climate change. This research analyses the culturable fungal mycobiota of Brachypodium rupestre across a sharp gradient of disturbance caused by an intense, anthropogenic fire regime. This factor has dramatic consequences for the community composition and diversity of high-altitude grasslands in the Pyrenees. Plants were sampled at six sites, and the fungal assemblages of shoots, rhizomes, and roots were characterized by culture-dependent techniques. Compared to other co-occurring grasses, B. rupestre hosted a poorer mycobiome which consisted of many rare species and a few core species that differed between aerial and belowground tissues. Recurrent burnings did not affect the diversity of the endophyte assemblages, but the percentages of infection of two core species -Omnidemptus graminis and Lachnum sp. -increased significantly. The patterns observed might be explained by (1) the capacity to survive in belowground tissues during winter and rapidly spread to the shoots when the grass starts its spring growth (O. graminis), and (2) the location in belowground tissues and its resistance to stress (Lachnum sp.). Future work should address whether the enhanced taxa have a role in the expansive success of B. rupestre in these anthropized environments.

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