FBIP: Endophytic bacterial communities in the fynbos biome

Study Extent

Fernkloof Nature Reserve

Sampling

The plant material was washed with tap water, cut into sections and surface sterilized by immersion into 2% NaOCl (3 min), 2% sodium thiosulphate (3min) and sterile distilled water. The samples were divided in two equal portions, one used for molecular analyses, and the other for bacterial isolation. For bacterial isolation, the tissues were homogenized by using sterilized mortars and pestles in sterile potassium phosphate buffer. The supernatant was used for serial dilution plating on R2A solid medium (Merck) and the plates were incubated at 25 °C and screened for colonies at 2-21 days after plating. Individual colonies were picked to produce pure cultures, which were preserved in liquid R2 medium with 50% glycerol at -80 °C. Isolates were compared and grouped by BOX-PCR (Versalovic et al., 1994), then identified by amplification and subsequent sequencing of bacterial 16S rRNA genes with primers 27F and 1492R and assigned to species using the closest relative by using EzTaxon-e (http://eztaxon-e.ezbiocloud.net/).

Method steps

1. We will isolate and identify endophytic bacteria associated with 3 fynbos plant species representing 3 disparate families (Disa graminifolia, Leucadendron gandogeri and Senecio burchellii). We will study their phenotypic traits potentially associated with plant-growth promoting activities. Finally, as it has been shown that a great proportion of bacteria are unable to grow under laboratory conditions, we will use culture-independent approaches (i.e. T-RFLP and pyrosequencing) to study the impact of site, soil and plant species on endophytic bacterial community diversity and composition. Sampling Participants: Dr. A. Valverde, Prof. D. Cowan, Honour student Timeframe: January 2014 Samples will be collected in the Fernkloof Nature Reserve (FNR). Plants will be randomly sampled from 3 sites. At each site, five individual plants per species will be collected.Healthy-looking plants will be collected with sterile spades and gloves. Spades will be sterilized between different sampling sites and/or plant species. The material will be stored at 4 °C and processed within 48 h after harvesting. Soil chemical analysis Participants: outsource Timeframe: February-April 2014 Soil samples will be sieved (2 mm mesh diameter) and analysed by the Central Analytical Facilities (Stellenbosch University) using standard methods. Endophytic bacteria isolation and identification Participants: Dr. A. Valverde, Prof. D. Cowan, Honour student Timeframe: February-July 2014 The plant material will be thoroughly washed with tap water, cut into sections and surface sterilized by immersion into 2% NaOCl (3 min), 2% sodium thiosulphate (3min) and sterile distilled water (dH2O; 3x3 min) The samples will be divided in two equal portions, one used for molecular analyses, and the other for bacterial isolation. The material for the DNA-based analyses will be snapfrozen and stored at -80 °C prior to analysis. For bacterial isolation, the tissues will be homogenized by using sterilized mortars and pestles in sterile potassium phosphate buffer. The supernatant will be used for serial dilution plating on R2A solid medium (Merck) and the plates will be incubated at 25 °C and screened for colonies at 2-21 days after plating. Culturable bacterial densities will be estimated by dilution plating. Individual colonies will be picked to produce pure cultures, which will be preserved in liquid R2 medium with 50% glycerol at -80 °C. Isolates will be compared and grouped by BOX-PCR (Versalovic et al., 1994), then identified by amplification and subsequent sequencing of bacterial 16S rRNA genes with primers 27F and 1492R and assigned to species using the closest relative by using EzTaxon-e (http://eztaxon-e.ezbiocloud.net/). Testing for phenotypes Participants: Dr. A. Valverde, Prof. D. Cowan, Honour student Timeframe: March-July 2014 - Phoshate solubilization ability will be assessed by growing the strains in NBRIP medium (Nautiyal 1999). Phosphate solubilization is evidenced by the presence of a transparent halo around the strain. - ACC deaminase activity will be characterized by measuring the production of α-KB as described by Honma and Shimomura (1978). - IAA synthesis ability will be assessed as previously described (Khalid et al., 2004). After colour development the absorbance at 535 nm will be measured, and the IAA synthetized estimated (μg/ml) by comparison with a standard curve based in IAA solutions (Sigma). Community fingerprinting (T-RFLP) and pyrosequencing analysis Participants: Dr. A. Valverde, Prof. D. Cowan, Honour student Timeframe: April-September 2014 Metagenomic DNA isolation of endobacterial communities will be carried out with the DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. For T-RFLP analysis the 16S rRNA genes will be amplified using primer pair 799F-1492R, as primer 799R is reported to exclude plant chloroplasts (Chelius & Triplett, 2001). The forward primer will be labeled with 6’ carboxyfluorescein (6-FAM). T-RFLP analysis will be performed as described elsewhere (Valverde et al., 2012). To identify organisms present in each sample, 454 sequencing of the 16S rRNA genes will be performed. A total of 12 samples (4 per plant species) will be selected on the basis of T-RFLP analysis. The SSU rRNA genes present in each sample will be amplified with the primers 1114F and 1392R containing the 454 adaptors (Engelbrektson et al., 2010). Using QIIME (http://qiime.org/) short reads will be removed and the remaining reads will be trimmed to 220 bp. Low-quality reads will be removed from the analysis using default quality settings (http://qiime.org/scripts/split_libraries.html). These high-quality sequences will be clustered into OTUs at 97% identity. The consensus sequence of sequences in each OTU will be used as a representative sequence. Each representative sequence will be affiliated using the RDP classifier. Statistical analysis Participants: Dr. A. Valverde, Prof. D. Cowan, Honour student Timeframe: throughout the project, but especially October-December 2014 Redundancy analysis (RDA) will be used to correlate bacterial community composition (expressed as abundance of bacterial T-RFLP derived OTUs) with host-plant species, sampling site and soil properties. Significance of these correlations will be assessed by 999 permutations. RDA analysis and visualization will be performed with the “vegan” package in R (http://www.r-project.org/). Intra- and intercommunity analysis will be performed using QIIME. Bacterial diversity of the samples will be calculated using rarefaction, ACE and the Shannon-Weaver diversity indices. Intercommunity OTU composition will be evaluated with UniFrac distance. UniFrac distances between samples are based on the fraction of branch length that is unique to each sample in a shared phylogenetic tree. Phylogenetic clustering is a good proxy for habitat associations that have likely emerged through evolutionary adaptation (Wang 2013).

Identified to Genus level

1. Bacteria
   rank: kingdom

South Africa; Western Cape; Hermanus; Fernkloof Nature Reserve