Morphological, biochemical and molecular characterization of Trichoderma isolates from tuber crop ecosystems

Abstract

Elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson) is an important tuber crop popular as a food security crop and as a remunerative cash crop. Collar rot caused by Sclerotium rolfsii is the most destructive and predominant disease causing great crop loss in elephant foot yam. Application of Trichoderma spp is being recommended as the eco-friendly strategy to combat the crop loss. The present study, ―Morphological, biochemical and molecular characterization of Trichoderma isolates from tuber crop ecosystem‖ was conducted at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram during October 2018 – August 2019. The study was performed to evaluate 43 Trichoderma isolates obtained from tuber crop ecosystem for their bio-control potential against S. rolfsii, to characterize the isolates using morphological, biochemical and molecular approaches and to analyse the molecular diversity. The differential antagonistic potential of the isolates were assessed by adopting three in vitro screening methods. The screening methods executed were dual culture/ direct confrontation method, antibiosis test for production of diffusible metabolites and volatiles. Based on the additive effect of each mode of inhibition, it was concluded that the isolates viz., T38, T36, T32, T40 and T6 have excellent antagonistic potential. Twenty six best isolates were selected for further study based on the ranking of additive result. The efficiency of 26 isolates for induction of chitinase and β-1,3-glucanase was studied against the cell wall of S. rolfsii as carbon source. There was no direct correlation observed between antagonistic potential of isolates and induction of chitinase enzyme. Whereas, positive correlation was observed between antagonistic potential and induction of β-1,3-glucanase enzyme. The effect of volatile organic compounds (VOCs) on plant growth was studied using mustard seeds. The isolates 94

showed differential response to various growth parameters like fresh weight, number of leaves, root length, shoot length and number of shoot lets. For morphological characterization, the macro and micro morphological characters such as growth rate, colony color, reverse colony color, odor of culture and branching pattern of conidiophore, size of conidia and phialides of isolates were studied. Morphological identification of Trichoderma isolates up to species level was found difficult due to the overlapped expression of these characters. The molecular characterization was done by amplifying and analyzing the sequences of ITS gene 1 and 2 and tef1 gene. The six different species identified are T. asperellum, T. virens, T. hamatum, T. reesei, T. longibrachiatum and T. erinaceum. The variability was studied using SSR markers and it was found that Jaccard’s similarity coefficient of 10 SSR primer banding patterns varies from 0.31 to 1.00. Fourteen T. asperellum isolates (T1, T2, T3, T5, T11, T13, T15, T16, T17, T18, T19, T20, T42, T43) were clustered in cluster A and the remaining three T. asperellum isolates (T32, T34 and T41) were clustered into cluster B. It showed the significant variability even within the same species. Considering the antagonistic potential, high chitinase and glucanase production and plant growth promotion, isolates T2, T15, T32, T34 (T. asperellum) and T40 (T. erinaceum) were selected as effective bio-control agents. The present study helped in identifying the Trichoderma isolate with high antagonistic potential against S. rolfsii and the entire process ensured more précised and targeted application of Trichoderma isolate in field condition. The outcome of the study will be a key factor in developing appropriate management strategy to mitigate collar rot disease in elephant foot yam.