Identification and phylogenetic analysis of in vitro banana fungi contaminants based on ITS regions sequence
Plant tissue culture offer a means for the rapid production of disease-free plants in large quantities, however fungi contamination is a major constraint to its successful application. This study characterized, identified and conducted phylogenetic analysis on fungi contaminants of in vitro cultured banana based on inter-space (ITS) regions sequence. Genomic DNA were extracted from pure culture of fungi contaminants. Polymerase chain reaction amplification and Illumina short sequence were conducted using ITS1 and ITS4 primers. The nucleotide sequences were aligned for consensus and compared with NCBI GenBank using Basic Local Alignment Search Tool (BLAST). Analysis of the sequences using MEGA 7 Software at higher similarity sequence identified five Aspergillus spp., three Penicillium spp., 1 each of Fusarium, Trichoderma and Cladosporium species as the contaminants. The overall genetic distance between the fungi species was 0.205 and the Maximum Composite Likelihood of nucleotide substitution showed Thyiamine is the most stable. The fungi were clustered in three major groups at 0.10 genetic distance which subdivided into five clusters. A cluster and sub-cluster of five Aspergillus strains; a major cluster of three Penicillium strains; a cluster comprising of Fusarium chlamydosporum and Trichoderma viride; and, a sole fungi Cladosporium tenuissimum. The Aspergillus group were phylogenetically related to A. flavus and A. parrisclerotigenus, the identified Penicillium spp were closely related to Penicellium citrinum while the detected Cladosporium aligned with Cladosporium tenuissium and Phoma multirostrata. The study concludes that molecular identification of the fungi contaminants covers the setbacks of conventional methods and the information provided could be helpful in development of specific and effective sterilization protocol to minimize contamination during in vitro culture procedure.