Isolation and molecular identification of Trichoderma species from wetland soil and their antagonistic activity against phytopathogens

Journal Pre-proof Isolation and molecular identification of Trichoderma species from wetland soil and their antagonistic activity against phytopathogens Kandasamy Saravanakumar, Myeong-Hyeon Wang PII:

S0885-5765(19)30203-6

DOI:

https://doi.org/10.1016/j.pmpp.2020.101458

Reference:

YPMPP 101458

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Physiological and Molecular Plant Pathology

Received Date: 18 July 2019 Revised Date:

31 December 2019

Accepted Date: 5 January 2020

Please cite this article as: Saravanakumar K, Wang M-H, Isolation and molecular identification of Trichoderma species from wetland soil and their antagonistic activity against phytopathogens, Physiological and Molecular Plant Pathology (2020), doi: https://doi.org/10.1016/j.pmpp.2020.101458. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Kandasamy Saravanakumar :Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Visualization; Roles/Writing – original draft; Writing review & editing. Myeong-Hyeon Wang: Funding acquisition; Project administration; Resources; Software; Supervision; Validation; Writing review & editing

Graphical abstract

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Isolation and molecular identification of Trichoderma species from wetland soil and their

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antagonistic activity against phytopathogens

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Kandasamy Saravanakumar, and Myeong-Hyeon Wang*

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Department of Medical Biotechnology, College of Biomedical Sciences, Kangwon National

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University, Chuncheon, Gangwon do, 24341, Republic of Korea.

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*Corresponding author

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Myeong-Hyeon Wang

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Phone: +82-33-250-6486; Fax: +82-33-241-6480

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Email: [email protected]

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Abstract

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Trichoderma species are known to protect the plants from pathogen infections through

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multifunctions, such as secondary metabolism, mycoparasitism, hyperparasitism, nutrient

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competition, enzymes and induced systemic resistance (ISR). Herein, we isolated a total of 18

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Trichoderma strains including nine species such as T. atroviride, T. virens, T. velutinum, T.

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harzianum, T. asperellum, T. koningiopsis, T. aureoviride, H. lixii, and T. koningii from the soils

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samples, collected from the wetland ecosystem of South Korea. These strains were screened

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against the pathogens- Macrophomina phaseolina (MP), Fusarium graminearum (FG), and

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Botrytis cinerea (BC) - by in vitro antagonistic assay. Amongst, T. aureoviride (SKCGW013)

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showed higher antagonistic activity against the targeted pathogens than other isolates did. The

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strain SKCGW013 was further used for extraction, purification and analysis of the metabolites

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by using column chromatography (CC) and gas chromatography mass spectroscopy (GC-MS).

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The expression of secondary metabolites regulatory genes of non-ribosomal peptide synthetase

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(NRPS), polyketide synthase (PKS) were studied by RT-qPCR. The results showed the presence

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of eight dominant compounds in the ethyl acetate fraction of the strain SKCGW013 and these

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compound were then screened by molecular modeling method against phytopathogens. In

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addition, RT-qPCR study revealed the significant expression of metabolites related genes.

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Further molecular docking study showed that the compounds from strain SKCGW13

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synergistically inhibited the targeted pathogens. Among the compounds - 2H-Pyran, 3-bromo-2-

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butoxytetrahydro-, cis - exhibited high docking inhibitory energy against the targeted proteins,

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FgSwi6 and Bcpmr1 from FG and BC respectively. Overall this study concluded that T.

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aureoviride SKCGW013 was an excellent source for discovery of novel metabolites as bio-

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control agents as evident by its metabolite profile with antifungal activity. 2

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Keywords: Biocontrol, Trichoderma, Phytopathogens, Enzymes, metabolites.

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1. Introduction

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The ubiquitous fungi Trichoderma species belong to the Ascomycota are present in a wide range

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of geographical locations. They can be isolated from various ecological sources including soil,

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water, plant parts and delaying woody materials, etc., by applying the conventional

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microbiological methods of culture in laboratory or industrial scale production for the generation

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of various bioactive metabolites and enzymes [1, 2]. Trichoderma strains are rich in the synthesis

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of various microbial molecules with promising bioactivities [3]. The molecules reported from

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Trichoderma species act as the elicitor to interact with the phytopathogens or plants to induce the

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biocontrol activity through the molecular mechanism such as systemic acquired resistance (SAR),

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and induced systemic resistance (ISR)[4-7]. Moreover, the enzymes and metabolites derived

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from Trichoderma can synergistically induce the biocontrol activity against various pathogens

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[8-10]. Universally, it is claimed that Trichoderma species are potent biofertilizer or bio-control

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agents to enhance the productivity of the agricultural crops [11-14]. In addition, Trichoderma

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strains have recently received a greater attention in bio-nanotechnology, specifically in the

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synthesis of various bioactive inorganic nanoparticles [15-20].

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The plant diseases caused by various pathogens including the Macrophomina phaseolina

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(MP), Fusarium graminearum (FG), Botrytis cinerea (BC), Rhizoctonia, phythium (RP),

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phytophthora (P) and Curvularia lunata (CL) that lead to significant economic loss in various

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agricultural crops [21]. Trichoderma strains are promising biocontrol against pathogens (MP, FG,

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BC, RP, P and CL) and also stimulating plant growth [22]. They are recognized as economically

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important fungal groups, involved in biocontrol of various phytopathogens and nematodes 3

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through mycoparasitism, hyperparasitism, nutrient competition [23]. Being avirulent and

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endophytic plant symbionts, Trichoderma strains penetrate in plants via roots and trigger

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beneficial effects through activation of plant innate immunity and nutrients uptake [13, 24].

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Remarkably, the antibiotic metabolites and enzymes produced from Trichoderma species

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synergistically inhibit the plant disease incidents [25]. Apart from the agricultural applications,

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Trichoderma strains are utilized in biotechnology as cell factory for the production various

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enzymes with industrial importance [26, 27]. Trichoderma strains do produce a number of

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industrially important molecules (for the review see [28]), a few of which are available in the

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market, such as cellulase from T. reesei, cellobiohydrolase from T. viride and T. reesei, pectin

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lyases from T. reesei, xylanases from T. reesei and T. konignii and hydrophobin from T. reesei

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[29]. Moreover, Trichoderma are also known producer of carbohydrate active enzymes

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(CAZymes), cellulase, exoglucanase, endoglucanase, β-glucosidase, xylanase, pectinase,

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amylase, glucose isomerase, glucoamylase, protease, phytase, β-glucanase, lipase, phospholipase,

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and lysophospholipase with extensive biotechnological applications; but, the level of enzyme

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production from the naturally occurring strains is low for industrial application. Therefore, some

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of Trichoderma strains are genetically modified to increase the production of targeted molecules

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especially proteins in large scales [30]. The biotechnological and economical importance of

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Trichoderma has increased the interest of searching the novel strains from various ecological

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niche. However, only very few works are demonstrating the isolation and screening of

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biotechnologically important Trichoderma strains from the wetland soils of Republic of Korea.

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Hence, the present work was undertaken on isolation, molecular identification and screening of

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antagonistic Trichoderma from wetland soil, collected from Republic of Korea against various

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phytopathogens. 4

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2. Materials and Methods

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2.1. Collection of soil samples, isolation and Molecular identification Trichoderma.

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A total of 92 soil samples were collected from two different locations, namely (i) wetland forest,

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Chuncheon

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(37°24'33.64"N; 129°12'12.89"E) (Fig.1). The collected soil samples were kept in the ice box

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(4°C) and transported to the laboratory of Kangwon National University, Chuncheon for the

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isolation of Trichoderma strains. The strains were isolated using the selective medium modified

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potato dextrose agar according to the methods described in earlier studies [1, 31]. The strains

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were purified by repetitive colonies picking and culturing in potato dextrose agar (PDA). Then

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they were identified by applying the conventional morphological properties and molecular

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internal transcribed spacer (ITS) and translation elongation factor 1 alpha (tef1a) gene

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sequencing analysis according to the methods described elsewhere [32-34]. All the Trichoderma

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isolated were preserved in 20% glycerol stock in -80°C.

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2.3. Screening active biocontrol strain against phytopathogens

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The phytopathogens Macrophomina phaseolina (MP), Fusarium graminearum (FG), and

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Botrytis cinerea (BC) were obtained from Korean culture center of microorganisms, Seoul,

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Republic of Korea. To select the potent biocontrol strain, a total of 18 Trichoderma strains were

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screened against the three phytopathogens by antagonist assay described earlier [35, 36]. In brief,

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the 5 mm of the growing edge of the Trichoderma and phytopathogens were placed on opposite

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direction of PDA plates and incubated at 27±2 °C in incubator for 5 days. Then the growth

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inhibition was measured using the roller and percentage of the inhibition rate was calculated

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using the formula described elsewhere [8, 36, 37] as I = (Control-Test)/control x 100, where I-

si

(37°51'19.84"N;

127°44'50.28"E), (ii)

5

coastal wetland,

Gangwan

do

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percent of inhibition, control-pathogens radial growth (cm), and test-pathogens radial growth

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(cm) in dual culture plate. Followed by the cell wall degrading enzyme activity from the potent

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antagonist strain was analyzed using the methods reported elsewhere [38, 39].

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2.4. Extraction and GC-MS analysis of secondary metabolites

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Among the tested Trichoderma strains, T. aureoviride (SKCGW013) was selected as potent

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biocontrol strain and used for the extraction of metabolites. The strain was cultured in

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Trichoderma biomass production medium described elsewhere [8] at 28±2°C in 180 rpm for 10

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days in shaking incubator. After the incubation period the extracellular products and fungal

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mycelia were separated by filtration using the Whatman No. 4 and then the extracellular products

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was extracted with 250 ml of ethyl acetate for overnight at 180 rpm. The ethyl acetate phase and

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water phase were separated using a separating funnel. The ethyl acetate phase containing

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metabolites was concentrated using a rotary evaporator at 40 °C. Finally the ethyl acetate extract

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was re-extracted and then subjected to the gas chromatography and mass spectrophotometry

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(GS-MS; HP Agilent Technology, 7890A California, USA) analyses. Secondary metabolites

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from the extract of T. aureoviride (SKCGW013) were identified by matching the GC-MS results

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with electronic searches of the National Institute of Standard and Technology (NIST) GC-MS

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chromatogram and mass electronic library W8N05ST.L.

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2.5. Antifungal activity of metabolites

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The antifungal activity of T. aureoviride extracts (TAE) was tested against the targeted fungal

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pathogen (FG) in PDA plates according to the methods described earlier [8]. In brief, the

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different concentrations of TAE (50-500 µg.mL-1) was sterilized by filtration and then

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incorporated into cooled PDA. After solidification, the FG was inoculated on the PDA medium, 6

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incorporated with TAE and the plates were incubated in 27±2 °C for 4 days and then the

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percentage of growth inhibition was measured using the standard formula by comparing the

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growth of the FG on PDA plates containing with or without TAE and the results are presented as

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FG growth inhibition (%) calculated according to the formula described above.

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2.6. Virtual screening of active metabolites

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The protein FgSwi6 from FG is known to be involved in the growth, pathogenicity carbendazim

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sensitivity, cellulose utilization, lithium tolerance, deoxynivalenol production and virulence of

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filamentous fungus FG [40]. Another protein Bcpmr1 from BC is also known to be involved in

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the pathogenicity and growth of BC [41, 42]. These two proteins were targeted using the

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metabolites identified from TAE by applying the computational modeling study. For the

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computational study, the 3D structure of the proteins, FgSwi6 and Bcpmr1 was prepared by

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retrieving their sequences from the NCBI (https://www.ncbi.nlm.nih.gov/protein/). These protein

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sequences

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(https://www.swissmodel.expasy.org/). The proteins were then pretreated according to the

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protocols described earlier [43]. The structure of the ligands (compounds identified from TAE),

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such as 6-Pentyl-2H-pyran-2-one, Propionamide, 2-Aminooctane, Bicyclo[2.2.1]hept-5-ene-2,3-

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dicarboxylic

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ethanamine,

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phenylpropinoic acid were generated using the ACD/ChemSketch using the canonical SMILES

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retrieved from the PubChem (https://www.ncbi.nlm.nih.gov/pccompound). The ligand and

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receptor interactions based on their docking energy score were measured by computational

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modelling using the ArgusLab 4.0.1 (Mark Thompson and Planaria Software LLC). Finally the

were

used

acid,

to

generate

2H-Pyran,

the

3D

structure

using

3-bromo-2-butoxytetrahydro-,

cis,

the

SWISS

2,4-Cyclopentadiene-1-

1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene,

7

MODEL

and

3-

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interactions between the receptor and ligands were observed by BIOVIA Discovery Studio 2016

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(Accelrys Software Inc., San Diego, CA, USA).

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3. Results and discussion

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3.1. Isolation and identification of Trichoderma

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Generally the identification of the Trichoderma spp. usually accepted based on the two DNA

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gene fragment sequence analysis, while the new Trichoderma spp. can be accepted by at least

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three DNA barcode fragments analysis [44-46]. Thus present work a total of 18 isolates divided

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into nine species of Trichoderma were isolated from two different sampling sites of Republic of

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Korea (Fig.1) and identified based on two DNA gene sequencing analysis including internal

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transcribed spacer (ITS) and translation elongation factor 1 alpha (tef 1α) based NCBI blast

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analysis (Fig.2). However, a number of the classification studies have shown that the individual

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sequence such as ITS, or tef- 1α gene sequences based phylogenic tree was not able to

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distinguish all Trichoderma spp. Thus combination of multi-loci sequences based phylogenic

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tree analysis is suggested for better distribution of Trichoderma spp. [47, 48]. Therefore, in the

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present work was contracted the phylogenetic tree using the concatenated dataset of ITS- tef 1α

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inferred by maximum parsimony method [49]. The same or closely related species were

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clustered on a clade on the resulting tree and the test and conference taxa was compared clearly

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according to earlier report [1, 2, 46, 50, 51]. The present results revealed the similarities between

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Trichoderma species while the out-group sequence of Nectria berolinensis formed the non-

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similarity clusters. Interesting the T. harzianum was formed a group of cluster in association with

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reference sequence while another group was formed for same clades association with other

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species such as T. virens, T. velutinum and Hypocrea lixii. Similar results were obtained for the 8

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Viride clades, which indicated the similarity of the species within clades of Trichoderma species.

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Although, the concatenated dataset of ITS- tef 1α of Trichoderma spp. formed the similar groups

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according to their species but in case of clades were formed the two different group that's

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indicated the difference within the clades (Fig.2). For instance, the present study observed two

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different cluster for Viride and Green/Harzianum from the concatenated dataset of ITS- tef 1α

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inferred by maximum parsimony tree, which indicated the requirement of further depth

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molecular assessment for better understanding of the Trichoderma taxonomy. Among the two

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sites of the coastal area, the site II showed high species diversity and richness (Table.1). The

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dominant species recorded were T. harzianum. T. atroviride, T. virens, T. velutinum,

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T.harzianum, T. asperellum, T. koningiopsis, T. aureoviride, and T. koningii (Table 1).

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3.2. Screening of active biocontrol strains

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Trichoderma isolates were tested against three phytopathogens (MP, FG and BC) by

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antagonistic assay. All the strains showed the potential antagonist activity against the targeted

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pathogens. Among the strains, T. aureoviride (SKCGW013) showed a high inhibition activity

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against MP (92.5%), FG (94.5%) and BC (89.32%) (Fig.3). Similarly, earlier reports also

192

evidenced the potent inhibitory effect of Trichoderma on pathogens such as Botrytis cinerea[52],

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Fusarium graminearum [25, 53] and Macrophomina phaseolina [54] through the production of

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antibiotic metabolites and enzymes mediated competition for nutrients and space[55, 56].

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Moreover, the strain SKCGW013 was showed the higher enzyme activity such as chitinase

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(71.21±1.44%), cellulase (68.45±2.32%), protease (48.65±0.12%) and β-(1-3) glucanase

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(78.15±1.84%) and it was higher than other strains tested in this study. Further study analyzed

198

the metabolites profile of the strain SKCGW013 by applying the chromatography assay. The

199

results showed a total of the 185 secondary metabolites including the polyketides, esters, nitriles, 9

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alkanes, benzenes, olefins, acids, alcohols and aldehydes in the ethyl acetate extract of

201

SKCGW013 (Fig.4a). Similarly, Trichoderma strains are known to produce chemically

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diversified antifungal metabolites as the biological weapon against various phytopathogens [57-

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60]. The antifungal activity of unbounded metabolites of Trichoderma was then tested against

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fungal pathogen FG. The results showed significant inhibition of FG at the dose depended

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manner (Fig.4b&c). Similarly the crude extracts of Trichoderma strains are reported to inhibit

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the growth of F. graminearum and F. oxysporum f. sp. cucumerinum in the PDA plates [8, 25].

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Thus the present results confirmed the biocontrol potential and stability of unbounded

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compounds of Trichoderma against phytopathogens.

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3.3. GC-MS based identification of dominant compounds and computational studies

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Based on the chromatography, the dominant metabolites from TAE of SKCGW013 were

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identified as 6-Pentyl-2H-pyran-2-one, Propionamide, 2-Aminooctane, Bicyclo[2.2.1]hept-5-

212

ene-2,3-dicarboxylic acid, 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis, 2,4-Cyclopentadiene-1-

213

ethanamine,

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phenylpropinoic acid (Fig.5a). These compounds were selected for the molecular inhibitory

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interaction towards the proteins - FgSwi6 from the FG [40] and Bcpmr1 from BC [41, 42] by

216

using the computation approach. The docking results showed that all the tested compounds

217

showed good docking score against the targeted proteins, evidencing the synergetic antifungal

218

activity of metabolites from TAE (Table 2). Among the compounds,

219

butoxytetrahydro-, cis (Fig. 5b) displayed higher interaction and inhibitory capacity against the

220

targeted proteins, as indicated by promising docking energy of -8.812 Kcal/mol against FgSwi6

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and that of -9.808 against Bcpmr1. The active compound 2H-Pyran, 3-bromo-2-

222

butoxytetrahydro-, cis

1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene,

inhibited the expression of 10

and

3-

2H-Pyran, 3-bromo-2-

FgSwi6 by establishing the bond with

223

aliphatic hydrophobic side chain Ile 590, Ile725, Ile 600, Leu593, Ile 368, Ala 724, Leu 362, Ile

224

721, Met 711, Met 677, Val 695 and Ala 676, aromatic hydrophobic side chain Phe 718, and

225

polar neutral side chain Thr 678 (Fig.6a-b). In the case of Bcpmr1, the active compound

226

interacted through bond with aliphatic hydrophobic side chain Leu 436, Leu 435, Leu 396, Ile

227

400, Val 438, and Leu439, and electrically charged side chain Arg 397 (Fig.6c-d). Similar kind

228

of the molecular docking approaches are previously applied to screen the active compounds from

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Trichoderma against fungal pathogens, such as F. graminearum and F. oxysporum [8, 25]

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3.4. qRT-PCR Analysis of the secondary metabolites regulatory genes

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The ketosynthase domain of PKSI gene and adenylation domain of NRPS gene were

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detected through PCR amplification as these genes are involved in the antimicrobial activity [61].

233

The qRT-PCR results indicated that both genes were expressed but the level of expression was

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higher in NRPS gene (relative gene expression 2.41) than that in PKS1gene (relative gene

235

expression 9.22). The gene expression indicated the presence of metabolites in TAE belonging

236

to NRPS and PKS1 families that resulted in enhanced antifungal activity [62]. Similarly, the

237

previous results have evidenced the correlation between the expression of the NRPS and PKS1 in

238

the endophytic fungi and their bioactivities including antimicrobial, biomedical and biocontrol

239

activity [63-65].

240

4. Conclusion

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This work reported the potential of newly isolated T. aureoviride (SKCGW013) on the inhibition

242

of phytopathogens (MP, FG, and BC). The strain synthesised novel metabolites such as 6-Pentyl-

243

2H-pyran-2-one, Propionamide, 2-Aminooctane, Bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid,

244

2H-Pyran,

3-bromo-2-butoxytetrahydro-,

cis, 11

2,4-Cyclopentadiene-1-ethanamine,

1,3,3-

245

Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene, and 3-phenylpropinoic acid as evident

246

by the preliminary metabolism analysis. This calls for critical study on transcriptomes to

247

understand molecular mechanisms adapted by Trichoderma strain for antifungal activity.

248

Therefore, further study will be focused on purification and molecular mechanism of synthesis

249

for the secondary metabolites, produced by T. aureoviride (SKCGW013).

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Conflict of interest

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The authors declare that they have no conflict of interest

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Acknowledgment

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This work was supported Korea Research Fellowship Program through the National Research

254

Foundation of Korea (NRF) funded by the Ministry of Science, ICT (2017H1D3A1A01052610).

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Reference

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Table 1. Description of List of Trichoderma strains collected by this study and their sources Strain code

Culture collection

Identification

Source

KNUP001

NCBI accession ITS1,ITS2 tef-α MG552067 MN513281

CMTCC KNU001

Soil, Wetland forest, Chuncheon

KNUP002

MG552068

MN513282

CMTCC KNU002

SKCGW001

MG552069

MN513283

CMTCC KNU003

SKCGW002

MG552070

MN513284

CMTCC KNU004

SKCGW003

MG552071

MN513285

CMTCC KNU005

SKCGW004

MG940956

MN513286

CMTCC KNU006

SKCGW005

MG940957

MN513287

CMTCC KNU007

SKCGW006

MG940958

MN513288

CMTCC KNU008

SKCGW007

MG940959

MN513289

CMTCC KNU009

SKCGW008

MG940960

MN513290

CMTCC KNU010

SKCGW009

MG940961

MN513291

CMTCC KNU011

SKCGW010

MG940962

MN513292

CMTCC KNU012

SKCGW011

MG940963

MN513293

CMTCC KNU013

SKCGW012

MG940964

MN513294

CMTCC KNU014

Trichoderma atroviride Trichoderma virens Trichoderma velutinum Trichoderma harzianum Trichoderma asperellum Trichoderma harzianum Trichoderma harzianum Trichoderma harzianum Trichoderma harzianum Trichoderma harzianum Trichoderma harzianum Trichoderma koningiopsis Trichoderma koningiopsis Trichoderma

Soil, Wetland forest, Chuncheon Sediemnt, Coastal wetland, Gangwan Sediemnt, Coastal wetland, Gangwan Sediemnt, Coastal wetland, Gangwan Sediemnt, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan

SKCGW013

MG940965

MN513295

CMTCC KNU015

SKCGW014 SKCGW015

MG940966 MG940967

MN513296 MN513297

CMTCC KNU016 CMTCC KNU017

SKCGW016

MG940968

MN513298

CMTCC KNU018

harzianum Trichoderma aureoviride Hypocrea lixii Trichoderma koningiopsis Trichoderma koningii

Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan Sediment, Coastal wetland, Gangwan

Table 2. Interactions between Trichoderma derived compounds and pathogenicity related protein bcpmr1 of Botrytis cinerea and FgSwi6 of Fusarium graminearum. S.no

1 3 5

11.089 24.271

6-Pentyl-2H-pyran-2-one Propionamide

Docking Score (Kcal/mol) Molecular Weight (g/mol) FgSwi6 Bcpmr1 166.22 -8.677 -9.001 73.095 -5.639 -6.192

23.341

2-Aminooctane

129.247

Retention Compound Name time

6 25.554 9 10.382 10 16.287 11

12

17.610 33.302

Bicyclo[2.2.1]hept-5-ene2,3-dicarboxylic acid 182.175 2H-Pyran, 3-bromo-2butoxytetrahydro-, cis 237.13 2,4-Cyclopentadiene-1ethanamine. 109.17 1,3,3-Trimethyl-2(hydroxymethyl)-5-hydroxy1-cyclohexene 170.25 3-phenylpropinoic acid 150.17

-8.356

-9.075

-8.460

-8.344

-8.812

-9. 808

-7.17

-8.485

-8.393 -8.411

-7.683 -9.721

Fig.1. Soil samples collected from two different wetland locations. (i) Soil from wetland forest, Chuncheon si (37°51'19.84"N; 127°44'50.28"E), 2. Sediment from Coastal wetland, Gangwan do (37°24'33.64"N; 129°12'12.89"E) (Sourcehttps://www.google.com/maps/place/Chuncheonsi,+Gangwon-do)

Fig.2. Phylogenetic tree inferred by neighbor joining analysis performed on the ITS-Tef 1α concatenated sequences dataset of Trichoderma spp.

Fig.3. Antagonistic activity of newly isolated Trichoderma strains against three different plant pathogens on PDA (a), percentage of pathogens growth inhibition (b), MP- Macrophomina phaseolina, FG- Fusarium graminearum, BC- Botrytis cinerea.

Fig.4. Distribution of secondary metabolites profile from unbounded extract TAE (a), Antifungal activity of unbounded metabolites derived from Trichoderma sp. (b) and % of inhibition of FG at different concentration of Trichoderma extracts (c).

Fig.5. Chromatography of the potent antifungal compound isolated from T. aureoviride at retention time of 11.089, 24.271, 23.341, 25.554, 10.382, 16.287, 17.610, 33.302 min corresponding to 6-Pentyl-2H-pyran-2-one, Propionamide, 2-Aminooctane, Bicyclo[2.2.1]hept5-ene-2,3-dicarboxylic acid, 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis, 2,4-Cyclopentadiene1-ethanamine, 1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene, and 3phenylpropinoic acid (a), the potent antifungal compound structure of 2H-Pyran, 3-bromo-2butoxytetrahydro-, cis, (b)

Fig.6. 3D and 2D structure demonstrate the interaction between 2H-Pyran, 3-bromo-2butoxytetrahydro-, cis with FgSwi6 from filamentous fungus Fusarium graminearum (a, b) and Bcpmr1 from B. cinera (c,d).

Highlights • This work reported the potent biocontrol strain from wetland soil of Republic of Korea. • A total of nine Trichoderma species was isolated with potent biocontrol properties • T. auroviride (SKCGW013) inhibited the growth of various phytopathogens • Metabolites from the T. auroviride induced biocontrol activity synergistically

Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.