FBIP: Endophytic bacterial communities in the fynbos biome

Angel Valverde1, Angel Valverde2, Mahlatse Kgatla3

1 University of Pretoria, Private bag X20, Hatfield, 0184, Pretoria, South Africa; 2 University of Pretoria, Private bag X20, Hatfield, 0028, Pretoria, South Africa; 3 SANBI, 2 Cussonia Avenue, Brummeria, 0184, Pretoria, South Africa

Corresponding author(s): Angel Valverde (angel.valverde@up.ac.za), Angel Valverde (angel.valverde@up.ac.za)

Received {date}; Revised {date}; Accepted {date}; Published {date}

Citation: Combination of authors, year of data paper publication (in parentheses), Title, Journal Name, Volume, Issue number (in parentheses), and doi of the data paper.

Resource Citation

Valverde A (2019): FBIP: Endophytic bacterial communities in the fynbos biome. v1.1. South African National Biodiversity Institute. Dataset/Occurrence. http://ipt.sanbi.org.za/iptsanbi/resource?r=fbip_endophytic_bacterial_communities_in_the_fynbos_biome&v=1.1

Abstract

Endophytic bacteria associated with 3 fynbos plant species  (Erepsia anceps, Leucadendron laureolum, Phaenocoma prolifera); with collection locality data, Genbank accession numbers and Identification of bacteria based on the 16S rRNA gene sequence. The data is found in the NCBI Sequence Read Archive and are available under the project number SRP059346.

Keywords: Endophytic bacteria, fynbos plants, Observation

Project details

Project title: Endophytic bacterial communities in the fynbos biome
Personnel: Angel Valverde
Funding: Funding from Foundational Biodiversity Information Programme (FBIP)
Study area descriptions/descriptor: South Africa; Western Cape; Hermanus; Fernkloof Nature Reserve

Data published through GBIF: http://ipt.sanbi.org.za/iptsanbi/resource?r=fbip_endophytic_bacterial_communities_in_the_fynbos_biome

Taxonomic coverage

General taxonomic coverage description: Identified to Genus level
Taxonomic ranks
Kingdom: Bacteria

Spatial coverage

General spatial coverage: South Africa; Western Cape; Hermanus; Fernkloof Nature Reserve
Coordinates: 34°53'9.6''S and 33°3'50.4''S Latitude; 17°53'9.6''E and 22°6'14.4''E Longitude 

Temporal coverage: June 24, 2014

Methods

Method step description: 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).
Study extent description: Fernkloof Nature Reserve
Sampling description: 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/).

Datasets

Dataset description
Object name: Darwin Core Archive FBIP: Endophytic bacterial communities in the fynbos biome
Character encoding: UTF-8
Format name: Darwin Core Archive format
Format version: 1.0
Distribution: http://ipt.sanbi.org.za/iptsanbi/archive.do?r=fbip_endophytic_bacterial_communities_in_the_fynbos_biome
Publication date of data: 2019-09-30
Language: English
Licences of use: Creative Commons Attribution (CC-BY) 4.0 License

Metadata language: English
Date of metadata creation: 2019-05-13
Hierarchy level: Dataset