Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management

PLOS ONE RESEARCH ARTICLE Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management Ramangouda G. ID1,2, M. K. Naik2, Rahul B. Nitnavare3,4, Richa Yeshvekar5, Joorie Bhattacharya1,6, Pooja Bhatnagar-Mathur1¤, Mamta Sharma ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, India, 2 Department of Plant Pathology, University of Agricultural Sciences, Raichur, Karnataka, India, 3 Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Sutton Bonington, United Kingdom, 4 Plant Science Department, Rothamsted Research, Harpenden, United Kingdom, 5 Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, United Kingdom, 6 Department of Genetics, Osmania University, Hyderabad, Telangana, India ¤ Current address: International Maize and Wheat Improvement Center (CIMMYT), México-Veracruz, El Batán Km. 45, Mexico * OPEN ACCESS Citation: G. R, Naik MK, Nitnavare RB, Yeshvekar R, Bhattacharya J, Bhatnagar-Mathur P, et al. (2023) Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management. PLoS ONE 18(1): e0280064. https:// doi.org/10.1371/journal.pone.0280064 Editor: Abhay K. Pandey, Tocklai Tea Research Institute, INDIA Received: October 14, 2022 Accepted: December 20, 2022 Published: January 18, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0280064 Copyright: © 2023 G. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 μg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 μg/ml) and N2-1 (16.41 μg/ml); and succeeded to sporulate even at 2000 μg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of β-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. PLOS ONE | https://doi.org/10.1371/journal.pone.0280064 January 18, 2023 1 / 16 PLOS ONE Funding: a) ICRISAT supported the study b) “The organization/funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) “The authors received no specific funding for this work.” Competing interests: The authors have declared that no competing interests exist. Genetic enhancement of Trichoderma for chickpea dry root rot disease management Introduction Trichoderma spp. are among the most promising biocontrol agents used against numerous plant pathogenic fungi [1]. Trichoderma is known to stimulate plant health by protecting the plant via mycoparasitism, anti-microbial secondary metabolite production, as well as local and induced systemic resistance against invading pathogens [2]. There are several reports on Trichoderma species as biocontrol agent, however, only a handful of species like T. harzianum, T. afroharzianum, T. viride, T. asperellum, T. koningiopsis, and T. virens based formulations are used to manage the plant diseases [1, 3]. Often Trichoderma species exhibits greater variability in antagonistic capacity and bio-stimulant action on plant pathogens and plants [4]. Some strains are more suitable for biological control of disease and others for stimulating crop growth and nutrient uptake [5]. The limited efficacy and inconsistent performance against targeted pathogens remains a major drawback in the field conditions [6]. Various strategies have been employed to enhance the efficacy and consistency of biological control activity. Recurrent selection of Trichoderma strains, and random mutations with UV light or chemical mutagenesis followed by subsequent selection has been a successful strategy to enhance efficacy and fungicide tolerance of Trichoderma species [7]. There have been several successful reports regarding the development of novel Trichoderma strains by using mutagenesis which has led to enhanced fungicide tolerance and biocontrol potential [1]. Pesticide tolerance in Trichoderma would be a prerequisite to reduce the fungicide resistance in pathogens and to promote the plant health [8]. Pesticide-resistant Trichoderma in combination with reduced fungicide application would help in reducing the detrimental effects of the latter on soil along with ensuring satisfactory levels of crop protection [9]. Moreover, reduced amount of chemical usage could weaken pathogen propagules for subsequent attack and give additive/synergistic effect on plant growth [10]. Among fungicides, carbendazim is one of the most extensively used benzimidazole (methyl benzimadazol-2-yl carbamate) for control of soil-borne diseases through seed treatment and foliar application [8]. Carbendazim resistance in pathogen populations have been detected in the field shortly after an intensive and exclusive exposure [11]. However, reversion of resistant populations has not been reported even after the selection pressure was removed [12]. Primarily, benzimidazole functions b (...truncated)