FBIP: Linking ecosystem processes and soil microbial diversity in Rooibos and Honeybush

Study Extent

South Africa, Western Cape, Eastern Cape

Sampling

Sampling sites were selected in the Western Cape, South Africa and included one commercial and one natural population at each site. Each site was sampled in triplicate during the cold, wet winter (May 2014) and the dry, warm summer (January 2015) seasons. Bulk soil samples were collected up to a depth of 10 cm. For the rhizosphere samples, soil surrounding the plants was carefully removed to depths of 10–20 cm until roots were found. Root fragments, at least 15 cm in length, together with about 200 g of closely surrounding soil were placed in a sterile plastic bag. All samples were stored on ice directly after sampling.Triplicate samples were pooled and homogenized. Soil samples were air dried and sieved (2 mm mesh) to remove roots and organic debris. DNA was extracted within 24 h of sample collection using the ZR Soil Microbe DNA kit (Zymo Research, CA, USA). Details of the DNA sequencing process is provided in the two publications (Postma et al.)

Method steps

1. Soil samples At each site, soil cores will be taken at depths of 0-5cm and 5-10cm (microbes, DNA analyses, soil processes) and 20-30cm (nematodes). Soil will be sampled using an auger or graduated spade, depending on the soil type. At each site, soil properties will be measured on bulk and rhizosphere soil. Standard methods will be used to measure soil particle size, pH, electrical conductivity, total N, total C and C/N ratios; and will be performed by a commercial laboratory. iButtons will be used to monitor soil temperature over time. Isolation and culturing Five plants will be sampled from each site at each sampling time. Plants/roots will be carefully removed from the soil to keep roots intact. Rhizosphere soil will be collected from roots in the laboratory by shaking to obtain soil loosely adhered to the roots. Rhizosphere soil firmly adhered to the roots will be used for root-associated culturing studies and for soil processes. Roots and root nodules will be used to isolate mycorrhizal and bacterial symbionts, and for DNA analyses. In addition, roots, stems and leaves will be washed, surface sterilised, rinsed twice in sterile distilled water, and used for the isolation of endophytic fungi, yeasts and bacteria. Arbuscular mycorrhizal (AM) fungal diversity will be assessed by extracting spores from soil samples and separating these into morphotypes. The identification of AM fungi is based on the structure and morphology of spores, therefore no culturing will be done. Soil dilutions will be done according to standard protocols and plated onto selective media supplemented with antibiotics. Diazotrophic bacteria capable of root nodulation and N2 fixation will be isolated directly from surface sterilised roots. Entomopathogenic nematodes will be trapped from soil using different insect hosts. Harvested infective juveniles will be stored at 14 C and recycled through wax moth larvae for morphological, morphometric and molecular identification. Other nematodes will be isolated from soil using Cobb’s decanting and sieving method. For the sampling of arthropods a vacuum sampler (18cm diam. nozzle) will be held vertically over branches for 1 second. Organic litter samples will be collected under individual plants by taking one litre of surface organic litter (ca. 5 cm in depth). Arthropods will be extracted from soil samples using the Berlese–Tullgren funnel extraction method. All collected arthropods from above and belowground samples will be sorted into morphospecies. A subset of individuals of the ten most abundant arthropod species collected using the above-mentioned methods will be used to determine the associated microbial taxa. Fungi and yeasts will be isolated from both the surface and the internal structures of these arthropods. Insects will be allowed to walk on media in order to isolate surface associated yeasts, fungi and bacteria; and macerated to isolate internal microbial species. Fifty-liter air samples will be taken with a Mass-100 Eco® sampler on fungal selective media at opposite sides of the plants. Identification All isolated and collected samples will be identified using standard lab manuals and recent monographs of the genera of interest. Non-sporulating fungi and those not identifiable according to morphological techniques will be identified by molecular means. DNA extractions, PCR and sequencing will be done using standard protocols. The fungal genera identified by morphological characteristics will determine which gene regions will be targeted. Resulting sequences will be compared to those on the NCBI GenBank database. Barcodes generated from these sequences will be deposited in GenBank and linked to reference material at the ARC-PPRI in Pretoria. Bacteria will be identified using the 16S and other gene regions, depending on genera. Indices used to measure ecosystem function will include coloniser-persister values on which the maturity index is based. CP triangles will be used as a graphical description of faunal composition. Real time PCR (qPCR) will be used to quantify the number of specific indicator species within a sample, and the 16S rRNA gene will be used to identify the entomopathogenic nematode associated bacteria. Community analysis ARISA will be used for microbial community profiling. Labelled eubacterial and fungal specific primers will be used to determine community diversity. The PCR products will be run on an ABI 3010xl Genetic analyser. ARISA data will be analysed using Genemapper 4.1 software. Bacterial 16S-rRNA and fungal ITS gene regions will be amplified using 454 titanium sequencing the Ion PGM sequencer. Sequencing and analysis of data will be conducted at the RU Facility, CAF at SU, and UWC. Soil processes Fine roots, nodules, leaves and rhizosphere soil will be sampled from each site and enzymatic activities related to N and P acquisition and metabolism will be analysed. Both bulk soils and root-associated soil will be subjected to enzymatic analyses and include: substrate induced respiration to determine microbial biomass, acid and alkaline phosphatase activity, urease activity, soil mineralization (aerobic incubation) Plant physiology Plant and soil microbial enzyme activities will be determined separately. Rhizosphere microbial enzyme activities for phosphatase, phytase, rna-ases and, GS, GOGAT, GDH and glucosidase will be assessed. Biological nitrogen fixation of the hosts will be determined by stable isotope analyses to determine the proportion of N fixed from atmospheric N. As an index of plant vigour, we will determine two of the plant’s most essential resource acquisitions: minerals and water. Plant species composition and species richness (alpha and beta diversity) will be determined using standard floristic survey methods. Statistical analyses will be done using standard methods and the appropriate software packages.

Most specimen identified to Genus level, Some to Species and Kingdom level.

1. Fungi
   rank: kingdom
2. Bacteria
   rank: kingdom

South Africa, Western Cape, Eastern Cape