Molecular Identification of Rhizospheric Fungi Associated with ‘Saba’ Banana via the Amplification of Internal Transcribed Spacer Sequence of 5.8S Ribosomal DNA

Background and Objective: The banana ranks among the top fruits in the world. Production of bananas is beset with problems of pest and disease infestation. The local Philippine cultivar Saba has been reported to possess resistance to major diseases affecting other banana cultivars. This study assessed fungal species present in the soil rhizosphere of Saba banana. Materials and Methods: Fungal isolates obtained and purified from the soil and were characterized morphologically in a previous study. These were identified by amplifying the ITS-5.8S rDNA sequences. Prior to amplification, the isolation of fungal DNA was optimized by the freeze-thaw method where mycelia were collected and stored at -80EC for 48 h. Then, a modified CTAB method was used to extract DNA. The PCR fragments were amplified using the primers Forward: ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and Reverse: ITS 4 (5-TCCTCCGCTTATTGATATGC-3). The cycling conditions were as follows: Initial denaturation at 95EC for 2 min, denaturation at 95EC for 1 min, annealing at 60EC for 1 min, 35 cycles, extension at 72EC for 1.5 min and final extension at 95EC for 5 min. Multiple sequence alignment and phylogenetic analyses were performed. Results: Fungal DNA has been successfully isolated. Two out of three fungal species whose morphological characteristics were earlier reported to conform with Aspergillus were validated in this study. Isolate 2, Aspergillus niger strain was deposited and assigned Genbank Accession No. KX093813. Isolate 3 (Genbank Accession No. KX073814) was identified as another strain of Aspergillus. They were shown to belong to a common clade with Aspergillus strains that were derived from rhizospheric soil. Strains of Aspergillus have been reported to possess various roles among which are as causal organisms of fruit rot, agents for bioremediation, antagonists of Fusarium species as well as producers of organic acids, enzymes and nutraceuticals. Conclusion: The amplification of ITS-5.8S rDNA sequences is a powerful tool in the identification of fungal species. Knowledge of fungal communities associated with plants are key to managing their health and future coping mechanism against potential pests.