In addition, we will increase the number of specific oligonucleotides that are spotted onto the phylochip (up to 10,000) to adapt to the taxonomic IWR-1 clinical trial diversity found
in soils at the study sites. Small-scale phylochips, so-called “”boutique”" arrays, such as the one designed in this study, are a time-saving and cheap approach for monitoring specific fungal species over years and/or in several hundred of samples. At the present time, the detection of a single species with our custom phylochip cost only one sixth of the price paid for the cloning/sequencing approach. The upscaling of detectable species on the phylochip (up to 10,000) will further lower the cost (by a factor of twenty).
Thus, the phylochip approach should be an attractive Milciclib purchase method for routine, accurate and reproducible monitoring of fungal species on specific sites, in which a high sample throughput is required. Methods Site description and root sampling The Breuil-Chenue experimental site is a temperate forest located in the Morvan Mountains (47°18’10″”N, 4°4’44″”E, France) at 650 m. The parent rock is granite and the soil is an alocrisol that is characterised by a pH ranging selleck kinase inhibitor between 4 and 4.5, with moder type humus and micro-podzolisation features in the upper mineral horizon. In 1976, a part of the original stand, composed mainly of beech Dapagliflozin (90% of the stems), oak and young birch on a homogeneous soil type, was clear-cut. Subsequently, beech (Fagus sylvatica L.) and spruce (Picea abies (L.) H. Karsten) were planted separately in 20 m by 20 m adjacent stands . Sampling of the root tips was performed in each stand (beech and spruce) in October 2007. A drill was used to obtain three soil cores (4 cm diameter × 10 cm depth) from each of the two treatments, along 18 m transects in the middle of each of the two plantations. The distance between the soil cores was 6 m, and the samples were collected at distances of more than 0.5 m from the trees or the stumps.
Soil cores were immediately transported to the laboratory in isotherm boxes and stored at 4°C. Within five days, the roots were manually separated from the adhering soil, gently washed, and then examined under a stereomicroscope at 40×. Morphological typing of all of the ECM tips (approximately 50-250 tips per sample) was performed according to Agerer . ITS sequencing An individual ECM root tip from each ECM morphotype was selected for molecular characterisation by ITS sequencing. The remainders of the ECM root tips in each sample were used for ITS amplification, cloning and sequencing, and phylochip analysis (Figure 2). The samples were conserved at -20°C.