Ecological studies of indegenous bambara groundnut rhizosphere bacteria and their metabolic activities

Abstract

With the rise in world population and decrease in food supply due to global climate change, food security becomes very pertinent. Malnutrition, food scarcity and poverty have consistently affected population growth. This issue has driven scientists to seek out other plants that have been understudied but have potentials for food security. Metabolites from rhizobacteria have also been found to be important in improving crop yield and most especially rhizobacteria from legumes like Bambara groundnut. This study was designed to harness the interaction between Bambara groundnut, various rhizobacteria in the soil , their metabolites and their roles in biocontrol and biofertilization. This, in turn, will help to increase crop yield by resisting pests and diseases. This will also improve plant growth and productivity. In this study, soil from the rhizosphere of Bambara groundnut was screened for Plant growthpromoting properties (PGP) of iso lated rhizobacteria . They included indole acetic acid (IAA), hydrogen cyanide (HCN), phosphate solubilization (PS) and ammonia production (NH3P) activities using standard methods . In addition, antifungal assay using a dual culture method was used to analyze the biocontrol properties of the isolates and their phylogenetic identification were carried out using the I 6S gene. Forty three ( 43) bacterial isolates from the Bambara groundnut rhizosphere were screened for their plant growth-promoting and biocontrol potentials. Out of this, 41 .87% showed positive actions in one or some of the PGP tests, 27.91 % were all able to produce these enzymes: catalase, oxidase and protease. All the iso lates were able to produce ammonia while 4.65%, 12.28% and 27.91 % produce HCN, lAA and solubilize phosphate , respectively making them important target as biocontrol and biofertilizer agents. Among the isolates, the species identified included Bacillus, Kocuria, Arthrobacter and Enterobacter. Growth of Fusarium graminearum was suppressed in vitro by 9.3% of the isolates while 16.3% were antagonistic against Bacillus cereus and Enterobacter faecalis. This study reveals the PGP and biocontrol potentials of rhizobacteria from Bambara groundnut rhizosphere. Due to the underutilized nature of Bambara groundnut and its resistant qualities to drought and harsh environmental conditions, some of the bacterial isolates were analyzed for their production of volatile organic compounds (VOCs). Volatile organic compounds are secondary metabolites produced by living organisms including bacteria in response to metabolic activities in the environment. In order to assess the production of these bioactive compounds, isolates were screened for the production of new compound or known compounds from Bambara groundnut rhizosphere. The antibacterial activities of crude extracts from three selected isolates were determined against E. faecalis, Pseudomonas aeruginosa, Microbacterium cryophilus and B. cereus. The butanol, hexane, ethyl acetate and petroleum ether extract of BAMr, BAMhi and BAMli were active when subjected to gas chromatography-mass spectrometry (GC-MS) analysis to ascertain the chemical components and structure of the bioactive compounds. Some of the VOCs released are Phthalan, p-xylene, tropeolin, tropone, fumaronitrile, tridecane and isocarboxazid . It was observed that not much work has been carried out to extract these compounds from Bambara groundnut rhizosphere. So far this is the first time that these bioactive compounds are extracted from Bambara groundnut rhizobacteria and are very potent antibacterial compounds. The functional diversity of the rhizospheric bacterial in growth stages of Bambara groundnut can be used to assess its impact on crop production. The rhizosphere of Bambara groundnut at different growth stages was also assessed for catabolic diversity and the pattern of metabolism by the bacterial community as a function of the carbon source utiIization profile (CSUP) of the rhizosphere using BIOLOG™. Soil samples were analyzed using a 96-well carbon source plate to determine the mostly utilized source of carbon by mi crobes in the soil samples. Cluster analysis revealed a shift in soil microbial community diversity and activity over the plant growth stages. Bacterial abundance and diversity were higher at 4 and 8 WAP and lowest in the bulk soil before planting. The highest utilization of alcohols, amides, amines, aromatic chemicals, brominated chemicals and phosphorylated chemicals was found in the control treatments. The highest utilization of carboxylic acids, ester, amino ac ids and polymers and carbohydrates were found in the treatments across growth stages. This implies that the soil samples between 4 WAP and 12 WAP were richer in diversity of microbial species and their abundance making the soil important in crop production. With the structure of carbon source utiIization in the rhizosphere of Bambara groundnut, the diversity of bacterial that enhanced the richness and diversity in the soil was determined and planting strategies can be formulated. The bacterial communities at the different growth stages of Bambara groundnut and the bulk soil was also determined. Paired end illumina Miseq sequencing of 16S rRNA was carried on the soil samples of the bacterial community. The phyla operational taxonomic units (OTU) were dominated by actinobacter (30.1 %), proteobacter (22%), acidobacter (20.9%), bacteroides (8.4%), chJoroflex (4.5%) and firmicutes (4.4%) in all soil samples. Samples from bulk soil had the least average percent phyla (0 I and 04) while samples at 16 W AP (Fl and F4) had the highest average percent phyla. Rubrobacter was the most predominant genera, after which is Acidobacterium and Skermanella. It was observed from the analys is of OTU that there was significant change in the bacterial structure of the rhizosphere with a higher abundance of potential plant growth promoting rhizobacteria, at the different growth stages which included genera such as Bacillus and Acidobacterium. These results demonstrated that the bacterial communities of Bambara groundnut rhizosphere in the field are dynamic and changes with abundance at growth stages of the plant. Given the knowledge above of the bacterial community, isolated bacteria, their metabolites and activities in vitro, BAMr, BAMhi and BAMli were applied as biofertil izers to Bambara groundnut grown on the field. Bambara groundnut seeds were coated with bacteria and the isolates left for 48 hours before planting them in the field using complete randomized block experimental design with three replications. The Null hypothesis stated that there is no significant difference between the treatments BAMli, BAMhi, BAMr and control while the alternative hypothesis emphasized that there exists a significant difference between the treatments. Growth parameters such as length of plant, number of leaves, number of branches and number of seeds were measured . The results were significant for both the treatment/bacterial effect and the block/week effect at a 5% level of significance (treatment: F = 12.028, p = 0.00 I; block: F = I 05.350, p = 0.000). Since p-value is less than 0.05, the Null hypothesis is rejected in favour of the alternative hypothesis. However, the overall model used for this data was significant (F = 196.068, p = 0.000) signifying that this model fits the data. Characterization of BAMhi, BAMli and BAMr using the 16S rRNA gene reveals their identity as B. amyloliquefaciens, B. thuringiensis and Bacillus spp respectively. These Bacillus strains have proved to be plant growth-promoting agents that can be used as biofertilizers to enhance the growth of crops in order to improve yield. This is the first time that rhizobacteria from Bambara groundnut rruzosphere is applied as biofertilizer.