A moisture gradient exists in containers filled with soilless substrates where the substrate is wetter at the bottom of the container and becomes drier towards the top of the container. This moisture gradient affects other substrate chemical properties and therefore may affect biological properties including the microbial communities present. Microbial communities in soilless substrates have only recently been studied and little is known about their uniformity throughout the container. This research aimed to evaluate how the bacterial and fungal portions of the microbial community may change along the vertical profile of a container substrate. A substrate was mixed that consisted of manure compost, peat moss, and perlite (20:65:15 v/v) and planted with a single sunflower seedling. After 0, 3, and 6 weeks in a greenhouse environment, samples of soilless substrate from the top, middle, and bottom of the container (approx. 4.3 cm depth for each layer) were collected for DNA extraction. Bacterial and fungal communities were characterized by sequencing PCR amplified 16s rRNA genes and ITS regions, respectively. We found that the phyla Pseudomonadota, Bacteriodota, and Ascomycota were present throughout the container profile in all three layers. However, bacterial genera Paucibacter, Pseudomonas, and Iodophanus, and fungal genera Cercophora and Mortierella differed in abundance within each layer. Pseudomonas tolerant to negative abiotic factors were greater in the bottom layer after 6 weeks. Likewise, Coprinellus , responsible for lignin and cellulose degradation, was also present only in the bottom layer. The diversity of bacterial communities differed between layers, with the greatest in the middle and the lowest in the top layer. The percentages of all bacterial amplicon sequence variants (ASVs) that were shared among the three layers at 3 and 6 weeks were only 16% and 28%, respectively. The diversity of fungal communities was less affected by layer and time, but the percentages of shared fungal ASVs among layers were still only 27% and 28% at 3 and 6 weeks, respectively. In consequence, it is necessary to consider sampling technique and location when collecting a DNA sample from a container substrate.
