Elucidating key plant growth-promoting (PGPR) traits in Burkholderia sp. Nafp2/4-1b (=SARCC-3049) using gnotobiotic assays and whole-genome-sequence analysis.

Burkholderia sp. Nafp2/4-1b (=SARCC-3049) is a plant growth-promoting rhizobacteria (PGPR) initially isolated from the rhizosphere of pristine grassland in South Africa, and its ability to enhance growth was previously evaluated on maize (Zea mays L.). Here, the bacterium was tested with the aim of investigating its role in improving the nodulation and growth of the forage legume lucerne (Medicago sativa L.) when it is co-inoculated with the rhizobial symbionts of this legume in the glasshouse. When the co-inoculation resulted in a statistically significant (P = 0·05) increase in the number of nodules and improved plant biomass compared with single inoculation, we sequenced and analysed its genome to gain a better understanding of the genetic determinants responsible for the observed PGPR traits. The Illumina HiSeq 2500-sequenced genome resulted in 92 scaffolds, with an N50 of 322 407 bp, a total draft genome size of 7 788 045 bp and GC content of 66·2%. Analysis of the genome sequence confirmed the presence of a number of essential genes that code for various PGPR traits. The main plant beneficial genes associated with PGPR traits in Burkholderia sp. Nafp2/4-1b include pyoverdine siderophores biosynthesis gene (PvdF); acdS that codes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase; the tryptophan synthase genes involved in auxin biosynthesis (TSA1, TSB1) and the pqqABCDE operon related to phosphate solubilization. This study generated valuable information on the potential of the PGPR Burkholderia sp. strain Nafp2/4-1b as an effective commercial inoculant, which warrants further formulation and field application studies before developing it into a low cost, environmentally safe and effective biofertilizer.

MinION 16S datasets of a commercially available microbial community enables the evaluation of DNA extractions and data analyses.

New advances in sequencing technology and bioinformatics analysis tools have significantly supported the culture-independent analysis of complex microbial communities associated with environmental, plant, animal and human samples. However, previous work has shown that DNA extraction can have a major influence in the community profile. As such there is a constant need for new methods to efficiently and rapidly prepare and analyze DNA for microbiome research, especially in the case new and emerging technology like the Oxford Nanopore Technologies (ONT) MinION. A commercial standard was used, in triplicate, to evaluate three DNA extraction protocols, including two commercially available and one "in-house" DNA extraction method. All DNA extractions were done as per manufacturer's instructions and prepared with the same commercial ONT 16S sample preparation kit, prior to being analysed using MinION sequencing. Eight MinION 16S datasets of this microbial reference community were obtained. Reads were initially base called and demultiplexed using ONT's Guppy™ sequencing software (version 3.2.4), filtered using NanoFilt and then classified using Usearch. A set of R scripts are presented to process sintax files generated from Usearch and produce an OTU table that can be used for further analyses. All datasets were deposited into the SRA (NCBI) database. These datasets will allow future extraction kit comparisons using MinION sequencing since a standardize laboratory process using commercially available components, such as the MinION 16S sample preparation kit, microbial reference community and extraction kits, were used. The current ONT 16S workflow making use of the Epi2me agent only provides QC metrics and the ID's of the main genera identified and does not provide any tools currently for further downstream community comparison. The analyses scripts provided in the supplementary material will thus further enable the testing of new datasets against these reference sets and provide users the ability to compare their workflows with ours, thus standardizing comparisons and workflows.

Microbial and functional diversity of Cyclopia intermedia rhizosphere microbiome revealed by analysis of shotgun metagenomics sequence data.

Cyclopia spp., commonly referred to as honeybush due to the honey scented flowers, are indigenous legumes mainly growing in the Cape Floristic Region of the Western Cape, South Africa. Dozens of species, including Cyclopia intermedia, C. subternata, C. plicata, C. genistoides are used to make the well-known, popular and widely enjoyed beverage called 'honeybush tea'. In the past, most rhizosphere microbial studies associated with Cyclopia spp. focused mainly on the taxonomy and diversity of the root nodule associated symbiotic nitrogen fixing rhizobia. The work presented here is the first report on the microbial and functional diversity of rhizosphere microbiome associated with Cyclopia intermedia. Metagenomic shotgun sequencing was performed on the rhizosphere soil sample collected from this Cyclopia sp. using illumina Hiseq 2500 platform which resulted in an α- diversity of 312 species. Analysis of the metagenome sequence using the Metagenomic analysis server (MG-RAST) indicated that bacteria constitute the dominant domain followed by Eukaryota, Archaea and other sequences derived from fungi and viruses. Functional diversity of the metagenome based on analysis using the Cluster Orthologous Group (COG) method showed metabolism as the most important function in the community. The raw sequence data is uploaded in FASTQ format on MG-RAST server with ID mgm4855911.3 which can be accessed at http://www.mg-rast.org/linkin.cgi?project=mgp90368. The data on the microbial and functional diversity of the rhizosphere community of Cyclopia intermedia generates a baseline information about the microbial ecology of this indigenous legume. The microbial profile data can also be used as indicators of soil health characteristic of the rhizosphere of this important legume.

Draft Genome Sequence of Mesorhizobium sp. Strain SARCC-RB16n, an Effective Nodulating and Nitrogen-Fixing Symbiont of Rooibos [Aspalathus linearis (Burm. f.)] in South Africa.

The draft genome sequence of Mesorhizobium sp. strain SARCC-RB16n reveals the presence of major symbiotic (nod and nif) and additional plant growth-promoting (PGPR) genes associated with enhanced growth of Aspalathus linearis (Burm. f.) in South Africa. The genome sequence provides vital information for the development of a commercial inoculant for rooibos cultivation.

The Draft Genome Sequence of Burkholderia sp. Strain Nafp2/4-1b Confirms Its Ability To Produce the Plant Growth-Promoting Traits Pyoverdine Siderophores, 1-Aminocyclopropane-1-Carboxylate Deaminase, and the Phytohormone Auxin.

Burkholderia sp. strain Nafp2/4-1b is a rhizobacterium isolated from the rhizosphere of grassland in South Africa. This draft genome report confirms the presence of genes related to iron acquisition, alleviation of abiotic stress in plants, and other essential traits of plant growth-promoting rhizobacteria (PGPR) that signify the potential of this strain as a plant growth-promoting agent.

Limited endosymbiont variation in Diuraphis noxia (Hemiptera: Aphididae) biotypes from the United States and South Africa.

Symbiosis allows an insect access to imbalanced food sources on which other organisms cannot survive. A bacterial endosymbiont, Buchnera aphidicola, gives aphids the ability to feed on phloem depleted of certain essential amino acids by producing those required. Pseudogenes and lower plasmid copy numbers of essential amino acid genes in B. aphidicola, endosymbiont of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), suggest that this symbiotic relationship is degenerating. The complete endosymbiont assemblages, copy numbers of plasmids (important in essential amino acid production), and sequence variation in B. aphidicola, from 10 Russian wheat aphid biotypes, were investigated. B. aphidicola was found to be monosymbiotic in the Russian wheat aphid biotypes and other Diuraphis species examined. An insert, occurring in an inverted repeat region on the leucine plasmid, was the only variation found in the approximately 10-kb B. aphidicola sequence analyzed from each Russian wheat aphid biotype. This inverted repeat was shown previously to be conserved within the family Aphididae. The insert occurred in B. aphidicola sequences isolated from four Russian wheat aphid biotypes. Copy numbers of the leucine plasmid differ between the South African and U.S. biotypes and were similar to previously reported values for biotypes from the same geographic regions. These results suggest that B. aphidicola may still contribute to Russian wheat aphid fitness when the aphid feeds on a variety of hosts.