Physiological and Molecular Plant Pathology 109 (2020) 101458

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier.com/locate/pmpp

Isolation and molecular identification of Trichoderma species from wetland T

soil and their antagonistic activity against phytopathogens
Kandasamy Saravanakumar, Myeong-Hyeon Wang∗
Department of Medical Biotechnology, College of Biomedical Sciences, Kangwon National University, Chuncheon, Gangwon do, 24341, Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: Trichoderma species are known to protect the plants from pathogen infections through multifunctions, such as
Biocontrol secondary metabolism, mycoparasitism, hyperparasitism, nutrient competition, enzymes and induced systemic
Trichoderma resistance (ISR). Herein, we isolated a total of 18 Trichoderma strains divided to nine species such as T. atroviride, T.
Phytopathogens virens, T. velutinum, T. harzianum, T. asperellum, T. koningiopsis, T. aureoviride, H. lixii, and T. koningii from the soils
Enzymes
samples, collected from the wetland ecosystem of South Korea. These strains were screened against the pathogens-
Metabolites
Macrophomina phaseolina (MP), Fusarium graminearum (FG), and Botrytis cinerea (BC) - by in vitro antagonistic assay.
Amongst, T. aureoviride (SKCGW013) showed higher antagonistic activity against the targeted pathogens than
other isolates did. The strain SKCGW013 was further used for extraction, purification and analysis of the meta-
bolites by column chromatography (CC) and gas chromatography mass spectroscopy (GC-MS). The expression of
secondary metabolites regulatory genes of non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS)
were studied by RT-qPCR. The results showed the presence of eight dominant compounds in the ethyl acetate
fraction of the strain SKCGW013 and these compound were then screened by molecular modeling method against
phytopathogens. In addition, RT-qPCR study revealed the significant expression of metabolites related genes.
Further molecular docking study showed that the compounds from strain SKCGW13 synergistically inhibited the
targeted pathogens. Among the compounds - 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis - exhibited high docking
inhibitory energy against the targeted proteins, FgSwi6 and Bcpmr1 from FG and BC respectively. Overall, this
study concluded that T. aureoviride SKCGW013 was an excellent source for discovery of novel metabolites as bio-
control agents as evident by its metabolite profile with antifungal activity.

1. Introduction agricultural crops [11–14]. In addition, Trichoderma strains have recently

received a greater attention in bio-nanotechnology, specifically in the
The ubiquitous fungi Trichoderma species belong to the Ascomycota synthesis of various bioactive inorganic nanoparticles [15–20].
are present in a wide range of geographical locations. They can be isolated The plant diseases are caused by various pathogens including the
from various ecological sources including soil, water, plant parts and de- Macrophomina phaseolina (MP), Fusarium graminearum (FG), Botrytis cinerea
laying woody materials, etc., by applying the conventional microbiological (BC), Rhizoctonia, phythium (RP), phytophthora (P) and Curvularia lunata
methods of culture in laboratory or industrial scale production for the (CL) that lead to significant economic loss in various agricultural crops
generation of various bioactive metabolites and enzymes [1,2]. Tricho- [21]. Trichoderma strains are promising biocontrol against pathogens (MP,
derma strains are rich in the synthesis of various microbial molecules with FG, BC, RP, P and CL) and also stimulating plant growth [22]. They are
promising bioactivities [3]. The molecules reported from Trichoderma recognized as economically important fungal groups, involved in biocon-
species act as the elicitor to interact with the phytopathogens or plants to trol of various phytopathogens and nematodes through mycoparasitism,
induce the biocontrol activity through the molecular mechanism such as hyperparasitism, nutrient competition [23]. Being avirulent and en-
systemic acquired resistance (SAR), and induced systemic resistance (ISR) dophytic plant symbionts, Trichoderma strains penetrate in plants via roots
[4–7]. Moreover, the enzymes and metabolites derived from Trichoderma and trigger beneficial effects through activation of plant innate immunity
can synergistically induce the biocontrol activity against various patho- and nutrients uptake [13,24]. Remarkably, the antibiotic metabolites and
gens [8–10]. Universally, it is claimed that Trichoderma species are potent enzymes produced from Trichoderma species synergistically inhibit the
biofertilizer or bio-control agents to enhance the productivity of the plant disease incidents [25]. Apart from the agricultural applications,

∗
Corresponding author.
E-mail address: [email protected] (M.-H. Wang).

https://doi.org/10.1016/j.pmpp.2020.101458
Received 18 July 2019; Received in revised form 31 December 2019; Accepted 5 January 2020
Available online 06 January 2020
0885-5765/ © 2020 Elsevier Ltd. All rights reserved.
K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

Trichoderma strains are utilized in biotechnology as cell factory for pro- factor 1 alpha (tef1a) gene sequencing analysis according to the methods
duction of various enzymes with industrial importance [26,27]. Tricho- described elsewhere [32–34]. All the Trichoderma isolated were preserved
derma strains do produce a number of industrially important molecules in 20% glycerol stock in −80 °C.
(for the review see Ref. [28]), a few of which are available in the market,
such as cellulase from T. reesei, cellobiohydrolase from T. viride and T.
2.2. Screening of active biocontrol strain against phytopathogens
reesei, pectin lyases from T. reesei, xylanases from T. reesei and T. konignii
and hydrophobin from T. reesei [29]. Moreover, Trichoderma spp. are
The phytopathogens Macrophomina phaseolina (MP), Fusarium grami-
known producer of carbohydrate active enzymes (CAZymes), cellulase,
nearum (FG), and Botrytis cinerea (BC) were obtained from Korean culture
exoglucanase, endoglucanase, β-glucosidase, xylanase, pectinase, amylase,
center of microorganisms, Seoul, Republic of Korea. To select the potent
glucose isomerase, glucoamylase, protease, phytase, β-glucanase, lipase,
biocontrol strain, a total of 18 Trichoderma strains were screened against the
phospholipase, and lysophospholipase with extensive biotechnological
three phytopathogens by antagonist assay described earlier [35,36]. In brief,
applications; but, the level of enzyme production from the naturally oc-
the 5 mm of the growing edge of Trichoderma and phytopathogens were
curring strains is low for industrial application. Therefore, some of Tri-
placed on opposite direction of PDA plates and incubated at 27 ± 2 °C in
choderma strains are genetically modified to increase the production of
incubator for 5 days. Then the growth inhibition was measured using the
targeted molecules especially proteins in large scales [30]. The bio-
roller and percentage of the inhibition rate was calculated using the formula
technological and economical importance of Trichoderma has increased the
described elsewhere [8,36,37] as I = (Control-Test)/control x 100, where I-
interest of searching the novel strains from various ecological niche.
percent of inhibition, control-pathogens radial growth (cm), and test-pa-
However, only very few works are demonstrating the isolation and
thogens radial growth (cm) in dual culture plate. Followed by the cell wall
screening of biotechnologically important Trichoderma strains from the
degrading enzyme activity from the potent antagonist strain was analyzed
wetland soils of Republic of Korea. Hence, the present work was under-
using the methods reported elsewhere [38,39].
taken on isolation, molecular identification and screening of antagonistic
Trichoderma from wetland soil, collected from Republic of Korea against
various phytopathogens. 2.3. Extraction and GC-MS analysis of secondary metabolites

Among the tested Trichoderma strains, T. aureoviride (SKCGW013)

2. Materials and methods was selected as potent biocontrol strain and used for the extraction of
metabolites. The strain was cultured in Trichoderma biomass production
2.1. Collection of soil samples, isolation and Molecular identification medium described elsewhere [8] at 28 ± 2 °C in 180 rpm for 10 days in
Trichoderma shaking incubator. After the incubation period the extracellular products
and fungal mycelia were separated by filtration using the Whatman No. 4
A total of 92 soil samples were collected from two different locations, and then the extracellular products was extracted with 250 ml of ethyl
namely (i) wetland forest, Chuncheon si (37°51′19.84″N; 127°44′50.28″E), acetate for overnight at 180 rpm. The ethyl acetate phase and water
(ii) coastal wetland, Gangwan do (37°24′33.64″N; 129°12′12.89″E) phase were separated using a separating funnel. The ethyl acetate phase
(Fig. 1). The collected soil samples were kept in the ice box (4 °C) and containing metabolites was concentrated using a rotary evaporator at
transported to the laboratory of Kangwon National University, Chuncheon 40 °C. Finally the ethyl acetate extract was re-extracted and then sub-
for the isolation of Trichoderma strains. The strains were isolated using the jected to the gas chromatography and mass spectrophotometry (GS-MS;
selective medium modified potato dextrose agar according to the methods HP Agilent Technology, 7890A California, USA) analyses. Secondary
described in earlier studies [1,31]. The strains were purified by repetitive metabolites from the extract of T. aureoviride (SKCGW013) were identi-
colonies picking and culturing in potato dextrose agar (PDA). Then they fied by matching the GC-MS results with electronic searches of the Na-
were identified by applying the conventional morphological properties tional Institute of Standard and Technology (NIST) GC-MS chromato-
and molecular internal transcribed spacer (ITS) and translation elongation gram and mass electronic library W8N05ST.L.

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/Chuncheon-si,+Gangwon-do).

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K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

2.4. Antifungal activity of metabolites Software LLC). Finally the interactions between the receptor and ligands
were observed by BIOVIA Discovery Studio 2016 (Accelrys Software Inc.,
The antifungal activity of T. aureoviride extracts (TAE) was tested San Diego, CA, USA).
against the targeted fungal pathogen (FG) in PDA plates according to
the methods described earlier [8]. In brief, the different concentrations
3. Results and discussion
of TAE (50–500 μg mL−1) was sterilized by filtration and then in-
corporated into cooled PDA. After solidification, the FG was inoculated
3.1. Isolation and identification of Trichoderma
on the PDA medium, incorporated with TAE and the plates were in-
cubated in 27 ± 2 °C for 4 days and then the percentage of growth
Generally the identification of the Trichoderma spp. has been ac-
inhibition was measured using the standard formula by comparing the
cepted based on the two DNA gene sequence analysis, while the new
growth of the FG on PDA plates containing with or without TAE and the
Trichoderma spp. identification can be accepted by at least three DNA
results are presented as FG growth inhibition (%) calculated according
gene sequence analysis [44–46]. Thus present work a total of 18 isolates
to the formula described above.
divided into nine species of Trichoderma were isolated from two dif-
ferent sampling sites of Republic of Korea (Fig. 1) and identified based
2.5. Virtual screening of active metabolites on two DNA gene sequence analysis such as internal transcribed spacer
(ITS) and translation elongation factor 1 alpha (tef 1α) based NCBI blast
The protein FgSwi6 from FG is known to be involved in the growth, analysis (Fig. 2). However, a number of classification studies have
pathogenicity carbendazim sensitivity, cellulose utilization, lithium toler- shown that individual sequence (ITS, or tef- 1α gene sequences) based
ance, deoxynivalenol production and virulence of filamentous fungus FG phylogenic tree analysis was not able to distinguish all Trichoderma spp.
[40]. Another protein Bcpmr1 from BC is also known to be involved in the The combination of multi-loci sequences based phylogenic tree analysis
pathogenicity and growth of BC [41,42]. These two proteins were targeted is suggested for better distribution of Trichoderma spp. [47,48].
using the metabolites identified from TAE by applying the computational Therefore, in the present work was contracted the phylogenetic tree
modeling study. For the computational study, the 3D structure of the using the concatenated dataset of ITS- tef 1α inferred by maximum
proteins, FgSwi6 and Bcpmr1 was prepared by retrieving their sequences parsimony method [49]. The same or closely related species were
from the NCBI (https://www.ncbi.nlm.nih.gov/protein/). These protein clustered on a clade on the resulting tree (Fig. 2). The test and con-
sequences were used to generate the 3D structure using the SWISS MODEL ference taxa was compared clearly according to earlier report
(https://www.swissmodel.expasy.org/). The proteins were then pretreated [1,2,46,50,51]. The present results revealed the similarities between
according to the protocols described earlier [43]. The structure of the li- Trichoderma species while the out-group sequence of Nectria berolinensis
gands (compounds identified from TAE), such as 6-Pentyl-2H-pyran-2-one, formed the non-similarity clusters. Interesting T. harzianum was formed
Propionamide, 2-Aminooctane, Bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic a group cluster in association with reference sequence while another
acid, 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis, 2,4-Cyclopentadiene-1- group was formed for same clades association with other species such as
ethanamine, 1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene, T. virens, T. velutinum and Hypocrea lixii. Similar results were obtained
and 3-phenylpropinoic acid were generated using the ACD/ChemSketch for the Viride clades, which indicated the similarity between the species
using the canonical SMILES retrieved from the PubChem (https://www. within clades of Trichoderma species. Although, the concatenated da-
ncbi.nlm.nih.gov/pccompound). The ligand and receptor interactions taset of ITS- tef 1α of Trichoderma spp. formed the similar groups ac-
based on their docking energy score were measured by computational cording to their species but in case of clades were formed the two dif-
modelling using the ArgusLab 4.0.1 (Mark Thompson and Planaria ferent group that's indicated the difference within the clades (Fig. 2).

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

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K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

Table 1
Description of List of Trichoderma strains collected by this study and their sources.
Strain code NCBI accession Culture collection Identification Source

ITS1,ITS2 tef-α

KNUP001 MG552067 MN513281 CMTCC KNU001 Trichoderma atroviride Soil, Wetland forest, Chuncheon
KNUP002 MG552068 MN513282 CMTCC KNU002 Trichoderma virens Soil, Wetland forest, Chuncheon
SKCGW001 MG552069 MN513283 CMTCC KNU003 Trichoderma velutinum Sediemnt, Coastal wetland, Gangwan
SKCGW002 MG552070 MN513284 CMTCC KNU004 Trichoderma harzianum Sediemnt, Coastal wetland, Gangwan
SKCGW003 MG552071 MN513285 CMTCC KNU005 Trichoderma asperellum Sediemnt, Coastal wetland, Gangwan
SKCGW004 MG940956 MN513286 CMTCC KNU006 Trichoderma harzianum Sediemnt, Coastal wetland, Gangwan
SKCGW005 MG940957 MN513287 CMTCC KNU007 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW006 MG940958 MN513288 CMTCC KNU008 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW007 MG940959 MN513289 CMTCC KNU009 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW008 MG940960 MN513290 CMTCC KNU010 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW009 MG940961 MN513291 CMTCC KNU011 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW010 MG940962 MN513292 CMTCC KNU012 Trichoderma koningiopsis Sediment, Coastal wetland, Gangwan
SKCGW011 MG940963 MN513293 CMTCC KNU013 Trichoderma koningiopsis Sediment, Coastal wetland, Gangwan
SKCGW012 MG940964 MN513294 CMTCC KNU014 Trichoderma harzianum Sediment, Coastal wetland, Gangwan
SKCGW013 MG940965 MN513295 CMTCC KNU015 Trichoderma aureoviride Sediment, Coastal wetland, Gangwan
SKCGW014 MG940966 MN513296 CMTCC KNU016 Hypocrea lixii Sediment, Coastal wetland, Gangwan
SKCGW015 MG940967 MN513297 CMTCC KNU017 Trichoderma koningiopsis Sediment, Coastal wetland, Gangwan
SKCGW016 MG940968 MN513298 CMTCC KNU018 Trichoderma koningii Sediment, Coastal wetland, Gangwan

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.

For instance, the present study observed two different cluster for Viride antagonist activity against the targeted pathogens. Among the strains,
and Green/Harzianum from the concatenated dataset of ITS- tef 1α T. aureoviride (SKCGW013) showed a high inhibition activity against
inferred by maximum parsimony tree, which indicated the requirement MP (92.5%), FG (94.5%) and BC (89.32%) (Fig. 3). Similarly, earlier
of further depth molecular assessment for better understanding of the reports also evidenced the potent inhibitory effect of Trichoderma on
Trichoderma taxonomy using multiple gene sequence data. Among the pathogens such as Botrytis cinerea [52], Fusarium graminearum [25,53]
two sites of the coastal area, the site II showed high species diversity and Macrophomina phaseolina [54] through the production of antibiotic
and richness (Table .1). The dominant species recorded were T. har- metabolites and enzymes mediated competition for nutrients and space
zianum. T. atroviride, T. virens, T. velutinum, T.harzianum, T. asperellum, [55,56]. Moreover, the strain SKCGW013 was showed the higher en-
T. koningiopsis, T. aureoviride, and T. koningii (Table 1). zyme activity such as chitinase (71.21 ± 1.44%), cellulase
(68.45 ± 2.32%), protease (48.65 ± 0.12%) and β-(1–3) glucanase
3.2. Screening of active biocontrol strains (78.15 ± 1.84%) and it was higher than other strains tested in this
study. Further study analyzed the metabolites profile of the strain
Trichoderma isolates were tested against three phytopathogens (MP, SKCGW013 by applying the chromatography assay. The results showed
FG and BC) by antagonistic assay. All the strains showed the potential a total of the 185 secondary metabolites including the polyketides,

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K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

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]hept-5-ene-2,3-dicarboxylic acid, 2H-Pyran, 3-bromo-2-bu-
toxytetrahydro-, cis, 2,4-Cyclopentadiene-1-ethanamine, 1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene, and 3-phenylpropinoic acid (a), the potent
antifungal compound structure of 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis, (b).

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K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

Table 2
Interactions between Trichoderma derived compounds and pathogenicity related protein bcpmr1 of Botrytis cinerea and FgSwi6 of Fusarium graminearum.
S.no Retention time Compound Name Molecular Weight (g/mol) Docking Score (Kcal/mol)

FgSwi6 Bcpmr1

1 11.089 6-Pentyl-2H-pyran-2-one 166.22 −8.677 −9.001

3 24.271 Propionamide 73.095 −5.639 −6.192
5 23.341 2-Aminooctane 129.247 −8.356 −9.075
6 25.554 Bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid 182.175 −8.460 −8.344
9 10.382 2H-Pyran, 3-bromo-2-butoxytetrahydro-, cis 237.13 −8.812 −9. 808
10 16.287 2,4-Cyclopentadiene-1-ethanamine. 109.17 −7.17 −8.485
11 17.610 1,3,3-Trimethyl-2-(hydroxymethyl)-5-hydroxy-1-cyclohexene 170.25 −8.393 −7.683
12 33.302 3-phenylpropinoic acid 150.17 −8.411 −9.721

esters, nitriles, alkanes, benzenes, olefins, acids, alcohols and aldehydes potential and stability of unbounded compounds of Trichoderma against
in the ethyl acetate extract of SKCGW013 (Fig. 4a). Similarly, Tricho- phytopathogens.
derma strains are known to produce chemically diversified antifungal
metabolites as the biological weapon against various phytopathogens 3.3. GC-MS based identification of dominant compounds and
[57–60]. The antifungal activity of unbounded metabolites of Tricho- computational studies
derma was then tested against fungal pathogen FG. The results showed
significant inhibition of FG at the dose depended manner (Fig. 4b&c). Based on the chromatography, the dominant metabolites from TAE of
Similarly the crude extracts of Trichoderma strains are reported to in- SKCGW013 were identified as 6-Pentyl-2H-pyran-2-one, Propionamide, 2-
hibit the growth of F. graminearum and F. oxysporum f. sp. cucumerinum Aminooctane, Bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid, 2H-Pyran,
in PDA plates [8,25]. Thus the present results confirmed the biocontrol 3-bromo-2-butoxytetrahydro-, cis, 2,4-Cyclopentadiene-1-ethanamine,

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

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K. Saravanakumar and M.-H. Wang Physiological and Molecular Plant Pathology 109 (2020) 101458

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

nylpropinoic acid (Fig. 5a). These compounds were selected for the mo-
lecular inhibitory interaction towards the proteins - FgSwi6 from the FG This work was supported Korea Research Fellowship Program
[40] and Bcpmr1 from BC [41,42] by using the computation approach. through the National Research Foundation of Korea (NRF) funded by
The docking results showed that all the tested compounds showed good the Ministry of Science, ICT (2017H1D3A1A01052610).
docking score against the targeted proteins, evidencing the synergetic
antifungal activity of metabolites from TAE (Table 2). Among the com- References
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