Root-associated microbiota of decline-affected and asymptomatic Pinus sylvestris trees

Study Extent

Three mountainous regions were selected for sample collection: Sierra de Almijara, Sierra Nevada and Sierra the Baza, all of them located in Granada (Andalusia, southeast Spain). In Sierra de Almijara, 12 unaffected P. sylvestris trees were chosen and marked (ASH01-12), while just decline-affected Scots pines were found in Sierra de Baza, and 12 individuals were also selected (BSD01-12). In contrast, 12 asymptomatic and 12 affected P. sylvestris trees were considered for the analyses in Sierra Nevada, which were named NSH and NSD, respectively. Those trees showing stunted growth, yellowing of the crown and/or defoliation were classified as symptomatic. All the samplings were performed in spring of 2022.

Sampling

In each experimental area, rhizosphere soil samples and young roots were collected from the same tree, and processed in parallel as described below. Thus, after sample processing, for each of the 12 replicates rhizosphere and root endosphere microbial communities came from the same tree, obtaining 96 bacterial and 96 fungal samples (12 x ASH, 12 x BSD, 12 x NSH, 12 x NSD x 2 plant compartments). After the sequencing data processing, four samples were removed from the analysis due to the low quality of the sequencing reads or due to the inadequate number of quality reads.

Method steps

1. At a distance of less than 50 cm from the trunk of the trees, the topsoil (25-50 cm) was removed by digging. Then, the main roots of the selected tree were followed until young poorly suberized roots were found. The soil closely adhered to these roots was collected by rubbing them manually (rhizosphere soil samples). Furthermore, the roots were also collected and kept at 4ºC along with rhizosphere samples until they were processed in the laboratory. It should be marked that two samples of rhizosphere soil and roots were taken from two different parts of the same root system of each tree (2 x 48 trees). Roots were surface esterilized as described Lasa and co-workers (2024; https://doi.org/10.1016/j.scitotenv.2024.171858). Then, total DNA from rhizosphere soil and sterilized roots was extracted by using the DNeasy® PowerSoil® Pro and DNeasy® Plant Pro Kits (Qiagen; Hilden, Germany), respectively, following the manufacturer's instructions.The hypervariable regions V3-V4 of the bacterial 16 rRNA gene and the fungal ITS2 were sequenced following a 2 × 275 PE strategy at the genomics service of the Institute of Parasitology and Biomedicine López-Neyra (CSIC; Granada, Spain). Sequencing reads were processed in R by using the packages and functions, and the methods described in Lasa et al., (2024). Lasa, A.V., et al. (2024) Mediterranean pine forest decline: A matter of root-associated microbiota and climate change. Science of the Total Environment 926, 171858. https://doi.org/10.1016/j.scitotenv.2024.171858

All bacterial and fungal ASV were classified at Kingdom, Phylum, Class, Order, Family and Genus level where possible.

1. Bacteria
2. Fungi

Three mountainous protected areas in Andalusia (southeast Spain): Sierras de Tejeda, Almijara y Alhama; Sierra de Baza, and Sierra Nevada. Several Pinus sylvestris stands (asymptomatic and decline affected pines) were selected.