FBIP: Actinobacterial diversity associated with rooibos plants

Study Extent

Four farms located near Clanwilliam and Citrusdal

Sampling

Rooibos samples were collected from four farms located near Clanwilliam and Citrusdal – two farms were natural (non-cultivated), organic plants, while the other two were large commercial farms which treated the plants with pesticides (plants were not irrigated). At each site leaves, roots, rhizospheric and bulk soil were collected.Actinobacteria were isolated from soil, leaves and rhizosphere. The number of actinobacteria selected from each site was limited to 100 isolates.In total 1426 actinobacteria were isolated and glycerol stocks have been prepared for all these isolates (stocks are stored in 96 well format). These strains have been stored in the Institute for Microbial Biotechnology and Metagenomic (IMBM) culture collection. Strains were de-replicated based on morphological features and antibiotic activity, which resulted in 139 strains being selected for full polyphasic characterization. Note: The culture collection will be screened in upcoming research projects. In addition, the collection is available for other researchers. The 16S rRNA gene for the 139 strains selected for full characterization isolates were amplified and the isolates were identified to the genus/species level. In highly speciated genera, such as the genus Streptomyces, the 16S rRNA gene lacks the sensitivity to resolve some species relationships. Therefore, for strains where 16S rRNA could not clearly distinguish a strain as being unique (based on phenotypic characteristics) it was decided to amplified the gyrB gene. Metagenomic analysis was very problematic. While it was relatively easy to extract metagenomic DNA from the soil samples, it was very difficult to extract DNA from rooibos leaves. When DNA was extracted the concentration was very low and the 454 amplicon workflow requires 500 ng of input DNA. In addition, the DNA contained PCR inhibitors (likely to be plant phenolics) which inhibited the emulsion PCR. After several months of optimization and one failed pyrosequencing reaction it was decided to rather use the Illumina MiSeq as the metagenomic protocol for this sequencer had just been published. The MiSeq sequencing was successful and based on preliminary data analysis at least 80 000 sequence reads pass quality filters.

Method steps

1. The project will be undertaken in the Western Cape Province and will be supervised by Drs Kirby and Le Roes-Hill, and conducted by students registered at UWC and CPUT for BSc (Honours) degrees and ND:Biotechnology, respectively. All the equipment, facilities and research expertise for the microbiological part of the project exist within the two research groups. Collaborations have been established with Prof Pieter Gouws (a member of the Rooibos Council) and Prof Jeanine Marnewick, who will assist in sample collection. Culture-based and culture-independent (metagenomic) analyses of the plants and surrounding soils will be conducted, as culture-based techniques only detect a fraction (less than 1%) of the bacterial diversity in an environment [1]. Samples will be collected from several organic and non-organic rooibos farms located within the Cedarberg Region (Citrusdal, Clanwilliam and Niewoudltville). Sampling At each site, approximately 200g of bulk soil will be collected at a depth of 5-10cm for soil analysis (soil particle size, pH, electrical conductivity, total N, total C; which will be conducted at BemLab). Six plants will be sampled at each location. Plants will be carefully removed from the soil to limit disturbance of the roots. Rhizosphere soil will be collected by shaking the root the plants in sterile plastic bags to dislodge the soil loosely associated with the roots. Root samples will also be collected and processed in the laboratory to obtain the rhizospheric soil adhering tightly to the roots. Fresh leaves will be picked from the plants and placed in sterile plastic bags. All samples will be processed within 24 hours of sampling. Isolation and culturing Actinobacteria will be isolated by placing 1 g of soil in 10 ml of sterile distilled water and vortexing vigorously to dislodge the bacteria from the soil particles. The soil suspension will be serially diluted and plated on a selection of agar media known to favour the growth of actinobacteria. All isolation plates will contain cycloheximide to limit fungal growth. For the isolation of endophytic actinobacteria from rooibos leaves, leaves will be surface sterilized with 3% bleach and 70% ethanol, rinsed twice in sterile water. Sterile leaves will be ground up in phosphate buffer using a pestle and mortar; the resulting extract will be serially diluted and plated on plant extract and tap water media [2]. Plates will be incubated at 30°C for up to 8 weeks. Actinobacteria will be identified by colony morphology. Actinobacterial identification The 16S-rRNA gene will be amplified using published protocols. Isolates will be identified to the genus level by BLAST analysis using the EzTaxon-e server [3]. Interesting isolates (i.e. those belonging to rare genera or known PGPRs) will be characterized further. Isolates which represent novel species will be characterised by a full polyphasic taxonomic approach (phylogenetic analysis, phenotypic testing, chemotaxonomic analysis, DNA-DNA hybridization, scanning electron microscopy (SEM) (4,5,6); and their descriptions will be published. Novel isolates will be deposited in curated culture collections (DSMZ and NRRL). SEM will be performed on roots and leaves to visualize actinobacterial mycelium directly associated with plant tissue. Detection of actinobacteria by metagenomic analysis Total metagenomic DNA will be isolated from the soil samples using a soil-DNA isolation kit (ZR Soil Microbe DNA kit; Zymo Research, USA). Actinobacterial 16SrRNA gene sequences will be amplified by nested PCR using universal 16S-rRNA gene PCR primers [7] and actinobacterial-specific 16S-rRNA gene primers [8,9] which have been adapted to include the 454 titanium A/B adaptor sequences. Amplicons generated from several soil/leaf samples will be pooled, sequencing libraries will be generated using Roche 454 LibL kits (unidirectional sequencing) and sequenced on a Roche GS Junior.

All specimen identified to Species level

1. Actinomycetales
   rank: order

Four farms located near Clanwilliam and Citrusdal