Extra-cellular Enzymes Assay and Genetic Identification of Banana (Musa spp) Endophytic Fungal Isolates Ng’ang’a

Abstract

Banana fungal endophytes are known to produce extracellular enzymes that hydrolyze polymeric compounds, including chitin, proteins, lipids cellulose and hemicelluloses that are primary components of pest cuticle. It is the hydrolytic activity of the extracellular enzymes that enables nematode antagonistic fungi to accomplish direct parasitism. Fusarium spp occur in different forms and lack of clear morphological traits separating different species in this genus. This is often confusing making morphological observations alone unable suffice for complete identification of this group. DNA-based identification techniques have had often been greatly useful in distinguishing such closely related species. The objective of the study was to screen production of extra-cellular enzymes to appraise the suggested mode of action by endophytes and to use DNA sequence analysis to confirm the morphological identification. Seventeen Fungal endophytic isolates were used in screening for extra-cellular enzymes activity in substrate amended solid media. Gelatin agar, tween20 and chitin powder were used to amend the media so as to mimic nematodes cuticle structural components. All the isolates produced protease as a zone of inhibition starting from 24 hours after inoculation and lasted until the entire gelatin was hydrolyzed. There was significant difference on area of inhibition among the isolates (P≤0.05) isolates 4MR45,7SIPB34, 5JTO22 and 2MR24 had marginally higher protease activity rate than other tested isolates while isolate 10IJT43, 5MR52, 5IC112 and 2MR23 had the lowest rate. No lipase and chitinase activities were observed on the isolates used in the study. There was zone of inhibition around the isolates that were inoculated on the control plates. The results suggested that the fungal endophytes have the ability to degrade collagen, which is a major component of exoskeleton thus employing protease to attack nematodes cuticle to gain penetration. The absence of lipolytic and chitinolytic activities suggest the fungal endophytes banana isolates are incapable of attacking the nematodes eggs. The phylogenetic analysis of the isolates 18s rDNA gene grouped all the isolates in the genus Fusarium under four species; Fusarium oxysporium, F solani, F aquiseti and F culmorum. Thirteen isolates grouped together with Fusarium oxysporum and formed the major clade of specie (clade 1). Clade 2-4 contained isolates with species similar or closely related to Fusarium oxysporium. The molecular phylogenetic analysis confirmed the isolates to be fusarium isolate with scores of 99% to 100% in similarity.