Determination of plant-fungal bio-indicators of Moroccan cork oak forest functioning

The Cork oak (Quercus suber), an emblematic Mediterranean evergreen sclerophyllous tree, constitutes a major ecological and socioeconomic resource for Mediterranean populations, but suffers various forms of decline. National politic directives aiming at a better conservation and development of cork oak stands were strengthened, bringing to the foreground new research fields, such as the development of ecological indicators (e.g. floristic and microbial diversity) and the set up of ecological engineering strategies for ecosystem conservation and restoration strategies.Deciphering ecological indicators is crucial to survey the health of ecosystems and to ensure a suitable response to environmental changes. The functioning of cork oak forests is closely tied to the formation of symbioses between the cork oak and ectomycorrhizal fungi. Unraveling the complexity of fungal community structure and diversity, a key parameter in sustaining plant productivity, thus appears as one of the major issues to determine innovative ecological indicators.The current study aims at determining multiple ecological indicators based on the high throughput characterization of fungal diversity in three different Moroccan cork oak forests (Maâmora, Benslimane, Chefchaouen), composed of degraded and non degraded stands. Fungal communities associated to cork oak and accompanying shrubs were investigated by using high-throughput Illumina sequencing and morphotyping.The study was conducted in three habitats of the Moroccan cork oak forest, located in the Moroccan Northern Mountains known as “Chefchaoun” (35°15'5.14"N 005°30'6.68W, 1534 m elevation), and in the central plateau bordering the Atlantic Ocean (North-west of Morocco) known as “Maâmora” (34°17'06.186"N 6°28'30.792"W, 27 m elevation) and “Benslimane” (33°41'9.85''N, 6°54'7.26''W; 326 m elevation). The three habitats are under a Mediterranean-type climate characterized by hot and dry summers and mild and wet winters. They are characterized by an abundant understory, notably Cistus salviifolius, Lavandula stoechas, and Thymeleae lythroides for Maâmora, and Arbutus unedo, Erica arborea, and Pistacia lentiscus for Benslimane and Chefchaoun. Fifty four cork oak trees were sampled between February and June 2013. The sampling design was based on the selection of 3 degraded plots and 3 non degraded plots per forest, spaced 100 meters apart, each composed of three trees 20-30 meters away from any other (54 samples in total).The Internal transcribed spacer ITS1 of the nuclear ribosomal RNA was amplified using the primers ITS1FI2 (5’-GAACCWGCGGARGGATCA-3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’)(Schmidt et al., 2013). The amplification reaction was performed in a final volume of 25 µl with the primers ITS1FI2 and ITS2 (0.6 µM each), 2 µl of DNA extract, 200 µM of each dNTP, 200 ng/ml BSA, GoTaq® DNA Polymerase (2 units) and 1X Green GoTaq® Reaction Buffer (Promega, Charbonnieres, France), with the following cycling conditions: 95°C for 15 min; 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s; a final elongation step at 72°C for 5 min. To increase richness recovery and to limit PCR biaises, three PCR replicates per sample were pooled and purified using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Velizy-Villacoublay, France) following manufacturer’s guidelines. All amplicon products were subjected to paired-end Illumina MiSeq sequencing (2×300 bp) by Molecular Research LP (MR DNA, TX, USA).