Evaluation of rhizospheric Trichoderma species strains for producing cell wall-degrading and defense related enzymes in response to Fusarium oxysporum f. sp. lentis

Abstract

Biocontrol strategies have been mainly focused on proposing the use of biocontrol agents (BCAs) isolated from the rhizospheric region of the plant for protection against phytopathogens. In the present study six isolates of Trichoderma sp. were isolated and identified. Among them, two isolates were identified as Trichoderma harzianum, two as T. asperellum, one as T. virens, and the rest one identified as T. hamatum. These isolates were identified molecularly by using Internal Transcribed Spacer (ITS) primers amplification by polymerase chain reaction and submitted to National Center for Biotechnology Information (NCBI). All identified isolates along with isolate ITCC-5593 were screened for their antagonistic activity using dual culture method. Trichoderma harzianum isolates (ThL-1, ThL-4 and 5593) were found more potent antagonists against Fusarium oxysporum f. sp. lentis (Fol) NFLRJ-3. Selected isolates of Trichoderma sp. after dual culture assay were further studied for the production of cell wall-degrading enzymes (chitinase and β-1,3 glucanase) against NFLRJ-3 along with production of phyto-hormones i.e. Indole Acetic Acid (IAA) and Gibberllic Acid (GA3) two isolates namely ThL-4 and 5593 gave maximum production of hormones. The elicitation of the defense related enzymes in the plants were analysed using biochemical assays for important enzymes, i.e. Peroxidase (PO), Polyphenol oxidase (PPO) and Super oxide dismutes (SOD) level in challenged plants over untreated and unchallenged control plants. It was found that T. harzianum secretes two major cell wall-degrading enzymes, i.e., chitinase and β-1,3 glucanase. Optimum production of both the enzymes were found at 96 h of incubation and maximum activity was showed by isolate 5593 i.e. 9.16 and 2.86 U/mg protein, respectively. The role of T. harzianum isolates in inducing defense enzymes (PO, PPO and SOD) in lentil when exposed to Fol was also observed. It was found that T. harzianum (ThL-4 and 5595) induced higher levels of enzymes. This study proposes the use of rhizospheric Trichoderma sp. isolates for better and all around protection of plants against soil and seed borne phytopathogens.