We use the term fungal community or mycota aware that we isolated only part of the culturable fungi and missed uncultivable fungal species. Amplification and sequencing of the fungal isolates ITS1-5.8S-ITS2 rDNA (ITS) region Amplification and sequencing of the ITS of the fungal isolates was performed with the primers ITS1F (or ITS1) and ITS4 (the sequences of these primers are available at: http://www.biology.duke.edu/fungi/mycolab/primers.htm). Direct PCR was performed using a sterile pipetor tip (10 μl) to transfer aseptically a very small amount of mycelium in a PCR tube and to squash it manually with the tip in the
PCR mix (25 μl mix, reagents and conditions Vorinostat order of the Taq PCR core kit (QIAGEN, selleck chemical Inc., Valencia, California, USA). Sequencing used the amplification primers, reagents and conditions of the BigDye ® Terminator v3.1 Cycle sequencing Kit and an automated capillary sequencer ABI 3700 DNA analyzer (Perkin Elmer, Applied Biosystems, Foster City, CA, USA). Fungal diversity and species accumulation curves Nomenclatural issues follow Mycobank. We estimated the species
diversity in asymptomatic, esca-symptomatic, and nursery plants by calculating the Simpson index of the fungal community identified in each plant sample. The community composition was assessed based on the relative abundance of species in the culturable part of the fungal community. The expected total species diversity in the different plant categories was estimated by resampling the available plant samples. Based on 1000 replicates without replacement, we calculated the total recovered diversity within each plant category. Species accumulation Janus kinase (JAK) curves were estimated using the vegan package implemented in the R statistical software (R Development Core Team 2006). Principal component analyses (PCA) A principal component analysis was performed in order to eventually identify differentiated fungal communities between symptomatic, asymptomatic and nursery plants. Each plant was considered as an independent replicate and the isolated fungal community on each plant
sample was recoded as presence-absence data. We assessed the fungal community based on incidence data rather than on relative frequencies to reduce the bias introduced by species that may be more easily brought into culture than others. The R package vegan was used to calculate the main https://www.selleckchem.com/products/ly333531.html ordination axes 1 and 2 based on Euclidean distances (R Development Core Team 2006). Biplots were produced based on the PCA to show both the relationship of the fungal species and the plant samples in respect to the main axes. Results Delimitation and classification of the operational taxonomic units (OTUs) based on ITS sequences of the fungal isolates The isolates were grouped based on their vegetative macro-morphology.