A total of 27 different colonies of bacteria were isolated from leaves, stems and roots of the three agroforestry trees. More bacterial endophytes were recovered from the leaves compared to the stems while roots had the least percentage recovery (Table 1). Most bacteria were recovered from C. calothyrsus followed by L. diversifolia.
Phenotypic characterization of the bacterial isolates
Based on phenotypic characteristics, the isolates were characterized using colony appearance, morphology such as elevation, type of margin, opacity and appearance after Gram staining (Table 2). Yellow raised colonies with entire margins, opaque, cocci in cell shape and Gram negative were recovered from all the three plant parts. White colonies lying flat on the media with an entire margin, translucent, rod shaped and Gram negative colonized the roots, leaves and stems of L. diversifolia, roots and leaves of C. Calothyrsus and leaves of S. sesban. Filamentous white colonies with irregular margins, opaque, rod shaped and Gram negative were present in the leaves of all the plants. Cream colonies that were raised with entire margins, opaque and Gram negative bacilli were found to colonize leaves, stem and roots of S. Sesban, C. Calothyrsus and stem of L. diversifolia. White colonies, raised with undulated margins, opaque, rod shaped which stained purple, were recovered from the three parts of C. Calothyrsus. Raised yellow light colonies with entire margin, opaque in opacity, rods in shape and Gram negative were found in the leaves and stems of L. diversifolia and C. Calothyrsus while white colonies that are flat on the media surface with entire margins, translucent, cocci in shape and Gram positive were recovered from stems of S. sesban and leaves of L. diversifolia.
Molecular characterization
A total of 27 pure bacterial isolates were successfully amplified and sequenced using 16S rRNA primers. Analysis of 16S rRNA gene sequence revealed closely related bacterial species belonging to 10 genera. Genus Bacilli (33.3%) was dominant compared to genus Staphylococcus (22.2%) and Alcaligens (11.2%). Genus Xanthomonas and Sphingomonas each had 2 isolates. Others with 1 isolate each included Pseudomonas, Pantoea, Bacterium, Enterobacteria and Acinetobacter. Isolates (BLS1, BLS2, BLS3, BRC3, BRC5, BSS3, BLS5, BRS1, BSC2, BSC5, BSL3 and BRL5) constituted 66% of the isolates and belonged to phylum proteobacteria. A total of 44% of the isolates (BLL4, BLL6, BSL1, BLC4, BLC5, BLC6, BSS1, BSS2, BRS3, BLC1, BLC3, BLL5, BRC1, BRC3 and BSC1) belonged to phylum firmicutes (Table 3). All isolates had sequences with ˃97.00% identity match with gene bank sequences apart from isolate BLS2and BLS1 whose match identity was 87.08% and 91.07% respectively. Sequences of the isolates were registered in the NCBI Bankit with accession numbers ranging from MW251519.1 to MW251545.1 (Table 3).
Phylogenic analysis
The phylogenetic tree established using the neighbour joining method clustered the isolates into 6 clades (Figure 1). The clades represented orders which included Bacillales, Xanthomonadales Sphingomonadeles, Burkholderiales, Pseudomonodales and Enterobacterales. Bacillales comprised of isolates belonging to two genera; Bacilli and Staphylococci. Genus Bacilli had 9 sequences compared to Staphylococci that had 6 sequences clustering at 100% bootstrap. Isolates in the order Bacilllales colonized all 3 plants (S. Sesban,C. Calothyrsus and L. Diversifolia). Order Xanthomonadales and Sphingomonadeles comprised of 2 isolates each with 100% bootstrap support. Order Enterobacterales comprised of genus Bacterium, Pantoea and Enterobacteriaceae with 100% bootstrap support. Order pseudomonodales comprised of genus Pseudomonas and Acinetobacter while genus Alcalgenes belonged to order Burkholderiales. Bacterial endophytes in the order Pseudomonodales were isolated from S. Sesban and C. Calothyrsus while endophytes in the order Burkholderiales were recovered from L. diversifolia and C. calothyrsus.