Molecular identification and characterisation of endophytesof indigenous trees in Zimbabwe

Abstract

Endophytic bacteria of genera Bacilli, Klebsiella, Enterobacter and Serratia and Ascomyta endophytic fungi of genera Fusarium, Debaryomyces, Schizosaccharomyces and Microsporomyces were isolated from Pterocarpusangolensis, Colophosperm mopane, Scherocaryabirrea and Vernonia amygdalin using culture methods. Genomic DNA extracted from the isolates was amplifiable by the polymerase chain reaction (PCR). A 16S rRNA gene amplification on 55 bacterial isolates gave rise to an approximately 1500 bp fragment. Similarly an 18S rRNA gene amplification on 56 fungal isolates gave rise to an approximately 1500 bp fragment as well. In both cases these PCR fragments were digested with the restriction enzyme Rsa 1 to eliminate redundancies in preparation for sequencing. A total of 37 endophytes made up of 30 bacteria and seven fungi weresequenced and fully identified through sequence alignment in the GenBank database. Phylogenetic trees were drawn for both bacteria and fungi and clustered into two major families of Bacillaceae and Enterobacteriaceae for bacteria and a single phylum Ascomycota for fungi.Gram staining of the 30 bacteria that were sequenced revealed eight gram positiveand twenty two negative- rods which were in complete agreement with the families of Bacillaceae and Enterobacteriaceae identified by phylogenetic respectively. Preliminary screening for bio-active substances identified a Bacillus substilis isolate that had both indole -production and anti-microbial activity against Staphylococcus aureus and Escherichia coli. Furthermore cultivation on different carbon sources revealed four Bacillus sp. with extracellular cellulase and amylase activities and a Serratiasp with extracellular amylase activity. Of the 56 fungal isolates five had both extracellular amylase and cellulose activity and one had and extracellular xylanase activity. In conclusion, the endophytes identified in this study already reveal a host of enzymatic and plantgrowth-promoting activities that could be exploited for biotechnological uses.