Succession process of rhizosphere fungal community in different diameter families of Pinus massoniana seed orchard

Three different families with 20 year-old seedlings (with the same cut-off time), and the row spacing between individual plants was more than 5m.The diameter at breast height (DBH) was surveyed in different family lines. The selection of the wood class was divided according to the relative DBH rank of the trees, with those of large DBH in the family lines being the dominant wood (labeled H), those of average DBH being the medium wood (labeled M), and hose of small DBH is the weak wood (labeled R), which were repeated as three trees. Take the range around the roots of the selected wood to make a vertical section of soil, 20-30 cm from the trunk and 10-30cm in depth (A layer), extract the root system within the soil layer, first shake off the loose, large pieces of soil on the roots that do not contain roots, and then use a brush to obtain a soil sample around the inter-root soil of 0-5mm around the inter-root soil, and then mix the inter-root soil samples from all four sides of the single tree species to form a composite sample. The soil samples of layer A without Pinus massoniana roots were taken as non-rhizosphere soil (marked as N), and the soil samples of 30-60cm (layer B) were taken as deep soil samples.All homogenized samples were immediately field-screened (2mm) to remove debris and roots transferred to a sterile refrigerator.Soil samples were subjected to DNA extraction, DNA extraction quality was detected by 0.8% agarose gel electrophoresis, and DNA was quantified by UV spectrophotometer30. Conservation region primers were designed, sample-specific barcode sequence was added, and the product was cut and recovered after PCR amplification, and the recovered product was fluorescence quantified, and then the product was mixed in the corresponding ratio according to the required sample sequencing volume. After library preparation and inspection, the recovered products were fluorescently quantified and mixed according to the sample sequencing volume and then sequenced on the Illumina Miseq platform.