Diversity and distribution of Tylosema esculentum (Marama bean) endophytic bacteria communities in Omitara, Harnas and Otjinene, eastern Namibia

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University of Namibia
Tylosema esculentum is a nutritious drought avoiding plant endemic to the Kalahari Desert. Our study assessed the density, diversity and distribution of endophytic microbial community structures associated with leaves, stems and tuberous roots of T. esculentum in Eastern Namibia. Culture-dependent and PCR-based 454 pyrosequencing methods were used. ANOVA with pairwise comparison revealed a significant difference in bacterial density between below and above ground. Endophytic bacterial isolates (605) were identified, grouped into 24 genera and three phyla. Proteobacteria was the most represented (67.4%) followed by Firmicutes (23.7%) and Actinobacteria (4.3%). Shannon diversity index, revealed a significant difference between the tuberous roots and leaves (p = 0.005) and stems (p = 0.006) microbial communities. The cluster analysis revealed a separation between the above and below ground microbial communities. The PCA and the Jaccard diversity indices confirmed these findings. Our results suggested that the microbial community composition was mainly governed by the plant parts rather than the location or sampling time. The phylogenetic analysis showed that all these microbial communities fell into two clades distinct from known cultivated bacteria from NCBI. All isolates associated with T. esculentum were positive for the nifH gene amplification. Only 42% nested with known strain in the NCBI GeneBank Database. This finding showed the presence of putative novel nitrogen-fixing bacteria associated with T. esculentum. Ten samples (leaves, stems and tuberous roots) from Omitara were examined using 16S rRNA gene pyrosequencing method. The presence of the three phyla Firmicutes (50.3%), Proteobacteria (38.32%) and Actinobacteria (4.46%) was confirmed. Two more phyla Fusobacteria (5.7%) and Bacteriodetes (1.368%) were revealed. Strikingly, 2 phyla (Firmicutes and Proteobacteria) of the five phyla represented 89 % of the total sequences. Similarly, four families (Enterobacteriaceae, Bacillaceae, Pasteurellaceae and Fusobacteriaceae) of the 18 recorded, represented nearly 91% of the total T. esculentum bacterial community assemblage. The genus Bacillus was predominantly found in the tuberous roots though shared across all samples. The class Clostridiale was exclusively found in leaves and stems. Within the Gammaproteobacteria class, the sequence grouping showed the Enterobacteriaceae family and the yet to be identified Enterobacteriaceae dominated. Unlike the Enterobactericeae that are mostly found in the tuberous roots, the Pasteurellaceae family was preferably found in the leaves and stems. Actinobacteria have shown a ubiquitous colonization compared with the Bacteriodetes that colonized the above ground part of the plant. T. esculentum organs exerted selective pressures on their associated bacterial communities. Only 68% of all reads could be classified at the phylum level. Firmicutes are the most dominant phylum in the current study with 46.9% sequences that have not yet been classified to any existing family, order or genus. Also, the rarefaction curves predicted that additional sampling will lead to significantly increased estimates of diversity. Sequences in this study, have shown similarities with sequences occurring in water-stressed environments with plant growth promoting traits. In conclusion, T. esculentum bean lives in community with a large diversity of potentially plant growth promoting bacteria
A dissertation submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy (Microbiology)
Diversity, Tylosema esculentum, Distribution