High diversity of root endophytes in the pioneer endemic Betula aetnensis Raf. colonizing volcanic lavas

Introduction Betula aetnensis Raf. is an endemic tree species native to Sicily of particular conservation value, that establishes on nutrient-poor and water-limited lava of Etna volcano. In these harsh conditions plant-microbe interactions could play a crucial role in plant growth, resource uptake and resistance to abiotic stresses. The aim of this study is to investigate the root-associated microbial community of B. aetnensis growing in its natural habitat. The comparison between individuals from natural habitats and nursery-grown seedlings aims to assess the symbiotic status of plant material used for afforestation purposes. Materials and Methods Betula aetnensis individuals were collected in its natural habitat (NAT, NE slopes of Etna volcano, Sicily) and in a forest nursery (NURS). Plant age was determined and soil analysis were carried out to measure soil pH, organic matter, C, N, and P content. Root tips were analyzed under the microscope for Ectomycorrhizal (EM) structures, and root fragments where stained and observed for arbuscular mycorrhizal (AM) and endophytic fungi. The automated ribosomal intergenic spacer analysis (ARISA) was performed to investigate the diversity of prokaryotic and eukaryotic root endophytes. Denaturing gradient gel electrophoresis (DGGE) was applied further to investigate the fungal diversity and endophytes were identified by sequencing of DGGE bands. Results Betula aetnensis root tips exhibited a high level of EM colonization (average 75% NAT, and 94% NURS) with clear morphological differences suggesting the occurrence of different fungal species. The typical AM structures were observed with high frequency of colonization (> 93%). Fungal endophytic structures were also observed. All the symbiotic structures were found in all NAT and NURS plants regardless the plant age and the higher OM content of the NURS soil. Bacterial diversity, investigated by ARISA, was higher than fungal diversity, and the endophytic fungal diversity was higher in NAT roots than in the NURS roots. Surprisingly, the typical ECM and AMF fungal sequences were not obtained by molecular analysis (DGGE) as most sequences were affiliated to Ascomycetes (mostly Helotiales) and Basidiomycetes. Helotiales, referred to as dark-septate endophytes (DSE) with an as yet undefined ecological role, appeared the dominant components of the fungal community inhabiting both NAT and NURS B. aetnensis roots. Conclusion This is the first report of the occurrence of a four-way symbiotic interaction in B. aetnensis. This complex co-occurrence of ECM, AMF and other endophytes in the same root system has rarely been observed. The results suggest that B. aetnensis finds in the harsh lava of the Etna volcano enough propagules to establish such complex multipartite interactions. The role played by these symbionts for B. aetnensis growth and survival is to be elucidated as they could be of key importance for the conservation of this endemic threatened species.