Microbiological Research 246 (2021) 126721

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Belowground fungal volatiles perception in okra (Abelmoschus esculentus)

facilitates plant growth under biotic stress
Jyoti Singh a, b, Prachi Singh b, Anukool Vaishnav c, **, Shatrupa Ray b, Rahul Singh Rajput b,
Shiv Mohan Singh a, Harikesh Bahadur Singh b, c, d, *
a
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
b
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, 221005, India
c
Department of Biotechnology, Institute of Applied Sciences & Humanities, GLA University, Mathura, 281406, India
d
Somvanshi Research Foundation, 13/21, Vikas Nagar, Lucknow, 226022, India

A R T I C L E I N F O A B S T R A C T

Keywords: Microbial volatile organic compounds (mVOCs) have great potential in plant ecophysiology, yet the role of
Biocontrol belowground VOCs in plant stress management remains largely obscure. Analysis of biocontrol producing VOCs
Induced systemic resistance into the soil allow detailed insight into their interaction with soil borne pathogens for plant disease management.
Sclerotium rolfsii
A root interaction trial was set up to evaluate the effects of VOCs released from Trichoderma viride BHU-V2 on
Trichoderma
Volatiles
soil-inhabiting fungal pathogen and okra plant growth. VOCs released into soil by T. viride BHU-V2 inhibited the
growth of collar rot pathogen, Sclerotium rolfsii. Okra plants responded to VOCs by increasing the root growth
(lateral roots) and total biomass content. VOCs exposure increased defense mechanism in okra plants by inducing
different enzyme activities i.e. chitinase (0.89 fold), β-1,3-glucanase (0.42 fold), peroxidase (0.29 fold), poly­
phenol oxidase (0.33 fold) and phenylalanine lyase (0.7 fold) when inoculated with S. rolfsii. In addition, T. viride
BHU-V2 secreted VOCs reduced lipid peroxidation and cell death in okra plants under pathogen inoculated
condition. GC/MS analysis of VOCs blend revealed that T. viride BHU-V2 produced more number of antifungal
compounds in soil medium as compared to standard medium. Based on the above observations it is concluded
that okra plant roots perceive VOCs secreted by T. viride BHU-V2 into soil that involved in induction of plant
defense system against S. rolfsii. In an ecological context, the findings reveal that belowground microbial VOCs
may play an important role in stress signaling mechanism to interact with plants.

1. Introduction ACC-deaminase enzymes, exopolysaccharides, siderophores, volatiles

The soil inhabiting microorganisms are essential component of plant
ecophysiology (Dini-Andreote and van Elsas, 2013). An interest is
increasing among agricultural scientist to harness benefits of soil mi­
croorganisms for plant health and sustainable food production. Below­
ground interactions are crucial to a plant and are likely to be more
complicated, because soil microorganisms are potential interaction
partners both in mutualism and parasitism way (Werner et al., 2016). In
the belowground region, rhizosphere and root regions are key spots,
where plants are associated with vast number of microorganisms
(Raaijmakers et al., 2009). Recent studies suggest that plant associated
microbes are involved in plant stress tolerance to biotic and abiotic
conditions through different mechanisms. This includes secretion of
and antimicrobial compounds in the rhizosphere that directly or indi­
rectly affect plant defense mechanism (Vaishnav et al., 2019; Liu et al.,
2020a, 2020b). Although aboveground mechanisms of plant associated
microbes are well understood, belowground interactions of soil mi­
crobes are less explored. Recently, belowground volatile attracted more
attention since soil microorganism and plant roots were shown to pro­
duce and perceive volatiles as a signaling molecule (Werner et al., 2016;
Sharifi et al., 2018; Fincheira and Quiroz, 2018; Bouwmeester et al.,
2019).
Volatile organic compounds (VOCs) are signaling molecule with low
molecular weight and low boiling pressure. The lipophilic nature of
VOCs allows them to diffuse into soil, water and air and make them an
ideal infochemical for belowground interaction (Bouwmeester et al.,

* Corresponding author at: Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India.
** Corresponding author.
E-mail addresses: anukoolv7@gmail.com (A. Vaishnav), hbs1@rediffmail.com (H.B. Singh).

https://doi.org/10.1016/j.micres.2021.126721
Received 8 October 2020; Received in revised form 4 January 2021; Accepted 1 February 2021
Available online 5 February 2021
0944-5013/© 2021 Published by Elsevier GmbH. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
J. Singh et al.
2019). These VOCs belong to various chemical classes including fatty
acids derived molecules, terpenoids, phenolic compounds, sulfides,
benzenoids, nitriles etc (Werner et al., 2016). Through VOCs, plants are
in continuous communication with their holobiont. Plant secretes
different VOCs through root exudates during stress condition, which
may attract distinct beneficial microbial groups in the rhizosphere that
help plants to overcome stress and facilitate adaptation under changing
environment (Liu and Brettell, 2019). For instance, Schulz-Bohm et al.
(2018) reported that an infection of fungal pathogen Fusarium culmorum
in Carex arenaria modifies VOCs profile secreted in root exudates that
attract specific bacteria with antifungal activity in the rhizosphere. On
the contrary, VOCs produced by soil microorganisms can act as stimuli
to activate a series of signals that regulate physiological and metabolic
processes involved in plant growth and defense mechanism (Kan­
chiswamy et al., 2015; Sharifi and Ryu, 2018; Garbeva and Weisskopf,
2020). In an earlier study, Bacillus amyloliquefaciens GB03 producing
VOCs induced plant growth by triggering the iron acquisition machinery
in plants (Zhang et al., 2009). Similarly, VOCs produced by a symbiotic
bacteria Sinorhizobium meliloti increased rhizosphere acidification and
iron uptake in Medicago plant (Orozco-Mosqueda et al., 2013). Likewise,
later studies revealed that microbial VOCs provoke plant defense
mechanism through modulating defense genes expression, ion trans­
porters activity, osmolytes accumulation and antioxidant system during
biotic and abiotic stresses (Vaishnav et al., 2015, 2016; Ledger et al.,
2016; Jain et al., 2018; Kong et al., 2018).
Antagonistic fungi in soil are of high interest to explore their VOCs
interaction with soil-inhabiting organism and their consequences for
their targets as well as benefits for host plants (Sridharan et al., 2020).
Fungi cannot move by growth to distinct directions as bacteria can. The
main mechanism of antagonistic fungi to defend themselves from other
soil organisms is the secretion of soluble metabolites and VOCs
(Schenkel et al., 2018). Trichoderma spp. are well-established as antag­
onistic fungi for soil borne plant pathogens and also to promote plant
growth as well as health (Singh et al., 2013b; Kumar et al., 2017; Singh
et al., 2017). Numbers of soluble and volatile metabolites are produced
by different Trichoderma spp. which depicts them as potential compet­
itors among other biocontrol agents in the soil (Keswani et al., 2017;
Mukherjee et al., 2012). Trichoderma VOCs can aid various signaling in
plants by priming them against future pathogen attack (Boots et al.,
2014; Vinodkumar et al., 2017; Sunpapao et al., 2018). Recently, few
research groups made effort to study the diversity and biological roles of
Trichoderma VOCs in root development, physiological alterations, hor­
monal pathways, plant growth promotion and inhibition the growth of
plant pathogens including fungal, insect and aphids (Chen et al., 2016;
Li et al., 2018; Guo et al., 2019; Coppola et al., 2019). In all these studies,
VOCs experiment was performed in in-vitro condition, where Tricho­
derma was grown on a standard nutrient medium and physically sepa­
rated by plants. To investigate the effect of microbial VOCs at
belowground level, experiment should be performed in the soil condi­
tion, as nutrient substrate is the main factor to affect VOCs emission in
microbes (Wilkins et al., 2000; Vaishnav et al., 2017; Guo et al., 2019;
Srinivasan et al., 1992; Gokulan et al., 2014). Due to the variation in soil
physico-chemical characteristics, the quantity and diversity of microbial
volatiles (mVOCs) may change from in-vitro production.
Yet, to our knowledge, no reports are available on belowground
VOCs produced by Trichoderma spp. into soil and it also remains un­
known whether these volatiles involve in plant defense signaling against
soil borne pathogens. Additionally, the effect of different growth me­
dium on Trichoderma producing volatiles profile has not been addressed
till date. Therefore, here, we investigated the effects of belowground
volatiles produced by Trichoderma viride BHU-V2 on okra (Abelmoschus
esculentus) plant growth and defense to soil borne fungal pathogen
Sclerotium rolfsii (causative agent of color rot disease). Furthermore, we
performed a comparative study of Trichoderma producing VOCs profile
on Potato Dextrose Agar (PDA) and sterilized potting mixture through
Gas Chromatography–Mass Spectrometry (GC–MS) analysis.
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Microbiological Research 246 (2021) 126721
2. Materials and methods
2.1. Sample collection and isolation of Trichoderma spp
Trichoderma spp. were collected from rhizospheric soil of five
different eco-geographical locations of India such as Bilaspur (22.07 ̊ N,
82.14 ̊ E), Lalitpur (24.69 ̊ N, 78.41 ̊ E), Kanpur (26.45 ̊ N, 80.33 ̊ E),
Jaunpur (25.74 ̊ N, 82.68 ̊ E) and Varanasi (25.28 ̊ N, 82.95 ̊ E), followed
by serial dilution (up to 10− 7) and plating 10-5 to 10− 7dilutions on
Trichoderma selective medium (TSM) (Elad et al., 1981). The TSM plates
were further incubated at 28 ± 2 ◦ C for 72 h. Colonies of Trichoderma
spp. obtained on TSM plates were sub-cultured on potato dextrose agar
(PDA) and incubated at 28 ± 2 ◦ C to obtain pure colonies. The colonies
were further stored on PDA slants and as spore suspension in 20 %
glycerol at -80 ◦ C.
2.2. Re-isolation of Sclerotium rolfsii from diseased plant
The pure culture of Sclerotium rolfsii (NAIMCC-F-03053) was previ­
ously procured from IDA approved culture collection National Agricul­
turally Important Microorganism Culture Collection (NAIMCC), India.
This culture was used to infect collar region of okra plant through a
lesion. The sclerotia were re-isolated from the infected collar region of
okra plant. Sclerotia were subjected for sterilization with 3% sodium
hypochlorite solution followed by washing with sterilized distilled
water. The surface sterilized sclerotia were inoculated on PDA medium
followed by incubation at 28 ± 2 ◦ C. Colonies obtained were further
sub-cultured on fresh PDA medium for 4 days at 28 ± 2 ◦ C.
2.3. In-vitro study of VOCs for antagonistic activity of selected isolates
against Sclerotium rolfsii and on seed germination
Trichoderma isolates were screened on the basis of their antagonistic
activity (dual plate) against S. rolfsii (Fig. S1). Three isolates BHU-V2,
BHU-V3 and BHU-V5 were screened out from this study and further
subjected for VOCs assay through bipartite plate assay. Mycelium discs
from seven days old culture of Trichoderma isolates were placed in one
compartment and S. rolfsii were placed on another compartment of
bipartite plate containing PDA medium. Petri plates were completely
sealed by parafilm and kept in an incubator at 28 ± 2℃ for four days.
Control treatment was comprised of S. rolfsii placed alone in one
compartment. The experiment was conducted in triplicates. The inhi­
bition of growth of pathogen will indicate the production of bioactive
VOCs from the Trichoderma strains.
Okra seeds of cultivar “Kashi Krishna VRCAR 126” were used in this
study and surface sterilized with 1% sodium hypochlorite for 5 min and
rinsed thrice with sterilized distilled water followed by air-drying. Effect
of Trichoderma VOCs on seed germination was observed through
bipartite plate assay technique, wherein PDA medium was filled in one
section and other section contained Murashige and Skoog (MS) medium.
Mycelium disc (5 mm) from seven days old culture of Trichoderma iso­
lates was inoculated on PDA medium while surface sterilized Okra seeds
were placed on the MS medium compartment. The positive control
comprised of sterilized seeds on MS medium only. Bipartite plates were
sealed with parafilm and incubated at 23 ± 1 ◦ C and 45 % relative hu­
midity with 16 h photoperiod for two days. The experiment was con­
ducted in triplicates and effect of VOCs was measured by observing the
radical length of seedlings.
2.4. In-vitro assessment of Trichoderma viride BHU-V2 isolate on plant
growth promotion (PGP) activities
Based on antagonism activity and seed germination assay, T. viride
BHU-V2 isolate was screened out for further experiments. The qualita­
tive plant growth promotion activities i.e. indole-3 acetic acid (IAA)
production, phosphate solubilization and siderophore production were
J. Singh et al.
analyzed in T. viride BHU-V2 isolate. IAA production was tested by
Patten and Glick (1996) method. The quantification of IAA production
was performed by method of Kasotia et al. (2012). Phosphate solubili­
zation assay was performed on NBRIP medium according to the method
of Mishra and Nautiyal (2012). The quantification of Pi-solubilization
was performed by Diby et al. (2005). Siderophore production was
assayed according to the method described by Bano and Musarrat
(2003).
2.5. Analysis the efficiency of Trichoderma viride BHU-V2 VOCs in soil
conditioning experiment
The efficiency of T. viride BHU-V2 VOCs was further checked on soil
condition for seed germination and antagonism assay. Experimental
setup was similar to in vitro study with slight modification in nutrient
substrate for Trichoderma isolate. In the seed germination and antago­
nism assay, one compartment of bipartite plate was filled with sterilized
potting mixture (soil and sand in 2:1) and other compartment hold MS
and PDA medium respectively. In this study, soil was collected from
Banaras Hindu University agriculture farm with following characteris­
tics (pH- 7.3; EC- 0.24 dS m− 1; organic carbon- 2.1 g kg− 1 soil; available
N- 82 mg kg− 1 soil; available P- 4.89 mg kg− 1; available K- 52 mg kg− 1
soil). T. viride BHU-V2 isolate was grown on potting mixture for treat­
ment set whereas sterilized distilled water was applied in control plates.
Sterilized okra seeds and mycelial discs of S. rolfsii were placed indi­
vidually on other compartments of bipartite plate holding MS and PDA
medium respectively. Plates were sealed with parafilm and kept at
28 ± 2℃ for four days. The differences in observations between treated
and control sets suggested the emission of bioactive VOCs from T. viride
BHU-V2 into soil.
Microscopic analysis of S. rolfsii hyphal structure in the presence of
T. viride BHU-V2 secreted volatiles was performed according to Sand­
wiched Petri plates method described by Dennis and Webster (1971). A
5 mm diameter mycelial plug of S. rolfsii was placed at the center of PDA
plate with sterilized cover slips placed obliquely at a distance of
approximately 1 cm from the mycelial plug. Another Petri plate with
sterilized soil inoculated with Trichoderma isolates was placed beneath
the plate containing S. rolfsii. The entire experimental set up was sealed
with parafilm and incubated at 28 ± 2 ◦ C for 72 h (Fig. S2). PDA plate
with S. rolfsii alone served as the positive control. The morphological
changes occurred in S. rolfsii mycelium due to VOCs exposure was
examined under light microscope after staining with through
lactophenol-cotton blue (Tenorio-Salgado et al., 2013).
2.6. Molecular identification of Trichoderma viride BHU-V2 isolate
Identification of T. viride BHU-V2 was carried out by ITS sequencing
and the amplification of the gene was done by using universal primers,
ITS1-F (5̍-TCCGTAGGTGAACCTGCGG-3̍) and ITS4-R (5̍-
TCCTCCGCTTATTGATAT-3̍) (Rajput et al., 2020). The PCR reaction was
performed in a thermocycler (Techne, UK) with initial denaturation at
94 ◦ C for 1 min followed by 35 cycles of 4 min, annealing at 56 ◦ C for
1 min, elongation period at 72 ◦ C for 50 s and a final extension step of
7 min at 72 ◦ C. The PCR product was analyzed on 1.5 % (w/v) agarose
gel electrophoresis with ethidium bromide (0.5 μg/ML) and was further
subjected to sequencing. The gene sequence was subjected to BLAST for
identification and making phylogenetic tree.
2.7. Collection and characterization of VOCs profiling produced by
Trichoderma viride BHU-V2
Collection of T. viride BHU-V2 producing VOCs was performed in
bipartite Petri plate system with two different conditions i.e. (1) T. viride
BHU-V2 on soil + sterilized activated charcoal and (2) T. viride BHU-V2
on PDA medium + sterilized activated charcoal. Both conditions devoid
of T. viride BHU-V2 inoculation were used as control treatment.
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Microbiological Research 246 (2021) 126721
Activated charcoal used as an absorbent for VOCs (Fig. S3). Plates were
incubated at 28 ± 2 ◦ C for 72 h. Thereafter, the activated charcoal was
collected from all the treatments and extracted with 5 mL of ethyl ace­
tate to collect all trapped volatile compounds and used for GC–MS
analysis (Agilent GC–MS system). The generated data were analyzed by
two spectra libraries WIlEY8.LIB and NIST1.LIB. A Venn diagram was
created for identifying the shared compounds in both the PDA and soil
medium. The individual compound names were arranged and used as
inputs for the R package RAM (Chen et al., 2018). We also created two
chord diagrams with the help of Circos tool to analyze and present the
compounds and their respective properties (Krzywinski et al., 2009).
2.8. Evaluation of belowground VOCs effect on plant growth and disease
incidence
The effect of belowground VOCs produced by T. viride BHU-V2 was
conducted in glass- house conditions at Department of Mycology and
Plant Pathology, Banaras Hindu University, Varanasi, India. A system
was designed to evaluate Trichoderma VOCs effect on okra plant growth
and defense system against S. rolfsii. There were two boxes in this system
(polypropylene made), where one box is used for growing plants and
connected to other box containing Trichoderma culture plate (Fig. S4).
Plant growth and Trichoderma growth medium was mixture of soil and
sand in ratio of 2:1 respectively. Volatiles released from T. viride BHU-V2
isolate exposed in the belowground of growing plant through air pipe
(PVC made). There were four sets of treatments i.e.T1- Control (without
any treatment), T2- S. rolfsii inoculation, T3- T. viride BHU-V2 VOCs
treatment and T4- T. viride BHU-V2 VOCs + S. rolfsii inoculation. In the
VOCs treatments (T3 and T4), fresh culture of T. viride BHU-V2 was
inoculated in soil mixture and placed in the one box of system, while T1
and T2 treatments contained only soil mixture in the box. Surface ster­
ilized okra seeds were sown into one box and kept complete set ups in
glass house at 30/22 ◦ C day/night temperatures with 16/8 h light/dark
cycle photoperiod and ~70 % relative humidity. In T2 and T4 treat­
ments, plant soil was mixed with mass cultured S. rolfsii at the rate of
50 g per pot (Singh et al., 2013a). Plant growth parameters were
measured after 25 days of seed germination.
2.9. Assessment of plant growth parameters and total chlorophyll content
Plant samples from all the treatments were harvested and analyzed
for measuring plant growth parameters including shoot length (SL), root
length (RL), lateral roots, leaf area (LA) and fresh weight (FW) of shoot
and root tissues. The total biomass (DW) of samples from each treatment
was measured after oven drying at 80 ◦ C (Afzal et al., 2013). Total
chlorophyll content was measured by soaking 1 g of leaf tissue into
10 mL of 80 % acetone followed by incubation under dark conditions for
24 h. The amount of pigment released from the leaf tissue was deter­
mined by spectrophotometric analysis at 645 nm and 663 nm. The result
was expressed as mg chlorophyll/g FW (Wellburn, 1994).
2.10. Estimation of enzyme activities
The effect of VOCs exposure on plant defense response upon S. rolfsii
inoculation was analyzed post 7, 14 and 21 days after seed germination.
Three plants were randomly harvested from each treatment and ho­
mogenized in 2 mL of ice cold potassium phosphate buffer (0.1 M; pH 7).
The homogenate was centrifuged at 12,000 g for 10 min at 4 ◦ C and
supernatant used as crude enzyme extract. Phenylalanine ammonia
lyase (PAL) activity was estimated according to the method of Jain et al.
(2012) and expressed as μM trans-cinnamic acid (TCA) g− 1 fresh weight
(FW). Peroxidase (PO) activity was determined through Hammersch­
midt et al. (1982) method and expressed as U min-1 g-1 FW. Polyphenol
oxidase (PPO) enzyme assay was performed according to the method of
Singh et al. (2013a) and expressed as min-1 mg− 1FW. Chitinase and β-1,
3-glucanase assays was conducted according to the method of Chairin
J. Singh et al. Microbiological Research 246 (2021) 126721

Fig. 1. Antifungal activity of VOCs produced by Trichoderma isolates on S. rolfsii growth. (A) Condition-I: all Trichoderma isolates and S. rolfsii were inoculated on
PDA medium in separate compartment of bipartite Petri plates; (B) Graphical representation of growth inhibition in S. rolfsii (Percent inhibition was compared with
Control. Results are expressed as mean of three replication and vertical bars indicate standard deviation of the mean. Different letters above error bar indicate
significant difference among treatment results taken at same time interval according to Duncan’s multiple range test at p ≤ 0.05); (C) Condition-II: S. rolfsii was
inoculated on PDA medium, while T. viride BHU-V2 (most prominent under condition-I) was grown on soil medium.

Fig. 2. Seed germination ability of VOCs produced by Trichoderma isolates. (A) Condition- I: Bipartite plate assay in which Trichoderma isolates were grown on PDA
medium and seeds were sown on MS medium in different compartments; (B) Graphical representation of radical length after seed germination under condition-I
(results are expressed as mean of three replication and vertical bars indicate standard deviation of the mean. Different letters above error bar indicate significant
difference among treatment results taken at same time interval according to Duncan’s multiple range test at p ≤ 0.05.); and (C) Condition- II: where T. viride BHU-V2
was grown on soil medium in one compartment and seeds were sown in another compartment on MS medium.

4
J. Singh et al. Microbiological Research 246 (2021) 126721

Fig. 3. Microscopic analysis of S. rolfsii hyphae after exposed under T. viride BHU-V2 producing VOCs. (A) Control condition: no exposure of VOCs, shows intact
hyphae of S. rolfsii; and (B) under VOCs exposure, shows degradation and thinning of S. rolfsii hyphae. Photographs were taken on 10μM bar scale.

and Petcharat (2017). SDS in 50 % (v/v) methanol by incubating at 50 ◦ C for 2 h on water

bath. Thereafter, absorbance was taken at 600 nm. The data is repre­
2.11. In-situ detection of lipid peroxidation and cell death sented in cell death (%).

Histochemical analysis of lipid peroxidation was performed in the
leaf tissues through staining with Schiff’s reagent for 1 h followed by
destaining in ethanol: acetic acid (3:1) solution (hot) for 30 min. The
development of pink color in the leaf tissues was indicated as lipid
peroxidation of membrane proteins (Airakiet al., 2012). Cell death was
determined according to the method of Iannone et al. (2015). Leaves
were immersed in 0.1 % Evan’s blue solution for 15 min, thereafter
decolourization was performed in 95 % boiling ethanol for 30 min. The
necrotic lesions were developed as indigo blue spots on the leaves. For
quantitative estimation in both experiments, dye was eluted in 1% (w/v)
2.12. Statistical analysis
All experiments were designed and repeated in Randomized Block
Design (RBD) with three replications. Data were subjected in one-way
ANOVA analysis through SPSS package version 20. Means were
compared by Duncan’s multiple range tests at p ≤ 0.05 significant. Re­
sults were discussed in terms of percentage and fold changes with
respect to control plants.

5
J. Singh et al.
Fig. 4. Plant growth promoting properties and molecular characterization of T. viride BHU-V2 isolate. (A) IAA production; (B) Phosphate solubilization; (C) Side­
rophore production; (D) Quantitative estimation of all above three properties (Results are expressed as mean of three replications ± SD); and (E) Phylogenetic
analysis of BHU-V2 based on ITS sequence (The red asterisk represents sequence generated from this study and submitted in NCBI GenBank with their respective
accession number- MW081914).
3. Results
3.1. Isolation and characterization of Trichoderma isolates
A total of five different isolates were obtained on TSM. All the iso­
lates were further screened out on the basis of their antagonism activity
against S. rolfsii through dual culture assay (Fig. S1). Three isolates BHU-
V2, BHU-V3 and BHU-V5 showed biocontrol activity were further
selected for VOCs study. In VOCs assay, BHU-V2 producing VOCs
showed higher antagonism (83 %) at 72 h as compared to other isolates
(Fig. 1). In addition, the effect of VOCs from BHU-V2, BHU-V3 and BHU-
V5 on seed germination percentage was recorded after two days of
experiment initiation. The radical length of Okra seeds was higher
(1.3 cm) in the presence of BHU-V2 VOCs exposure as compared to other
isolates (Fig. 2). On the basis of antagonism and seed germination ac­
tivity, BHU-V2 isolate was selected for further studies. BHU-V2 was also
tested for similar effects during in situ setup. The artificial nutrient
medium i.e. PDA was replaced by sterilized soil inoculated with BHU-V2
strain in bipartite plate method and was analyzed for S. rolfsii inhibition
and enhanced seed germination. Microscopic analysis of change in hy­
phal morphology in sandwiched plate method revealed hyphal distor­
tion, thinning, node formation, deformed cells and cytoplasm
aggregation under VOCs exposure in comparison to compact and spindle
mycelia of control set (Fig. 3).
During the analysis of plant growth promoting activities, BHU-V2
isolate was found positive for the production of IAA (13.5 μg/mL) as
the color of media changed after the utilization of L- tryptophan and
variation in pH. The strain also solubilized tri calcium phosphate
(0.14 g/mL) after the reduction caused by stannous chloride and
changed the decolorized the medium. Siderophore activity was also
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Microbiological Research 246 (2021) 126721
reported in the selected BHU-V2 strain (2.1 cm) as the orange color of
media was observed around the inoculated culture (Fig. 4A–D). Further,
BHU-V2 was identified by ITS gene sequencing. The phylogenetic
analysis of BHU-V2 ITS sequence showed 99.67 % homology with Tri­
choderma viride (Fig. 4E). The sequence was submitted to GenBank, NCBI
database (accession number MW081914).
3.2. GC/MS profiling of VOCs emitted from Trichoderma viride BHU-V2
GC/MS profiling of VOCs produced by T. viride BHU-V2 was identi­
fied by NITS library. Final list of compounds was summarized after
devoid of control treatment compounds. Different nutrient medium
conditions affected VOCs pattern released from T. viride BHU-V2.
T. viride BHU-V2 released more number of compounds on PDA me­
dium as compared to soil medium. A total of 46 compounds were
summarized (>0.5 % peak area) in PDA medium condition, while 30
compounds were recorded in soil condition (Tables 1 & 2 ). These
compounds belong to different functional groups such as alcohol, alde­
hyde, acid, alkane, fatty acids and many more (Fig. 5A & B). Nine
common compounds were released on the both mediums with slight
quantitative variations. On the PDA medium, Benzestrol was exhibited
with highest peak area (10.96 %), while Tetradecanoic acid was present
on the soil medium with highest 9.27 % peak area. During literature
survey for listed compounds, it was observed that most of the com­
pounds released on soil medium were identified as antifungal and plant
growth promoting in nature. On the other hand, varied range of com­
pounds with different biological activities were emitted from PDA me­
dium were reported as antioxidants, nematicidal, pest repellent, etc. in
different organisms.
J. Singh et al. Microbiological Research 246 (2021) 126721

Table 1
GC/MS profiling of volatile organic compounds released by T. viride BHU-V2 on PDA medium along with their properties as reported in previous literatures.
S.No. Name of compounda RTb Area%c Propertiesd Referencese

1 Cymol 5.03 0.97 Biocontrol activity Rabari et al., 2018

2 3-Trifluoroacetoxy- pentadecane 5.47 1.19 NOT REPORTED
3 Benzyl alcohol 5.85 0.95 Antimicrobial agent Karabit et al., 1986
4 Nonane, 4-ethyl-5-methyl- 6.60 0.84
5 Vanillin 7.30 0.91 Antioxidant property Dalmolin et al., 2016
6 Cyclopentane, 1-methyl-3-(1-methylethyl)- 7.56 0.85 NOT REPORTED
7 6-Methyl-bicyclooctan-7-ol 8.75 1.29 Antifungal activity Awad et al., 2018
8 3-Methyl-2-butenoic acid, hexadecyl ester 9.38 0.91 NOT REPORTED
9 3-methyl butanal 9.41 1.12 Plant growth promoter Farag et al., 2006
10 Cubenene 10.88 0.82 NOT REPORTED
11 1,2-Ethanediol, 1-(2-furanyl)- 11.69 1.97 Antibacterial activity Hai-bin et al., 2008
12 2,4-Di-tert-butylphenol 12.08 1.09 NOT REPORTED
13 1-Hexadecanol 12.61 1.12 NOT REPORTED
14 Phosphonoacetic Acid 12.96 2.35 Antifungal activity Banfi et al., 2006; Yang et al., 2008
15 Benzeneethanol, 4-hydroxy- 14.75 7.97 Antioxidant property Ashwathanarayana and Naika, 2017
16 2,5-Furandione, 3-dodecyl- 15.77 0.94 NOT REPORTED
17 7-Methyl-Z-tetradecen-1-ol acetate 16.16 0.87 NOT REPORTED
18 1-Heptatriacotanol 16.48 1.15 NOT REPORTED
19 Benzestrol 16.68 10.96 Fungistatic property Heinemann, 1947
20 1-Hexadecanol, 2-methyl- 17.07 1.75 NOT REPORTED
21 Decadienal 17.31 0.98 Nematicidal activity
22 17-Pentatriacontene 17.40 1.77 NOT REPORTED
23 Hydrazine 17.63 1.15 Pest repellent Horvitz et al., 2007
24 Cis-jasmone 17.90 2.74 Induce plant defense Moraes et al., 2008
25 Phenol,2-methyl 18.02 1.39 Antimicrobial activity Hussein et al., 2016
26 1-Decanol, 2-octyl- 18.28 0.84 NOT REPORTED
27 Globulol 18.44 9.49 Plant growth regulator Aghaei et al., 2013
28 Isopentyl alcohol 18.54 1.87 Plant growth regulator Nieto and Frankenberger, 1991
29 Octatriacontylpentafluoropropionate 19.06 0.90 NOT REPORTED
30 Isoaromadendrene epoxide 19.56 1.39 Antioxidant property Hameed et al., 2015
31 Eicosylheptyl ether 19.90 1.42 NOT REPORTED
32 n-Tetracosanol-1 19.98 4.57 Antibacterial activity Kumari et al., 2019
33 (2,3-Diphenylcyclopropyl) methyl phenyl sulfoxide, trans- 21.09 1.11 NOT REPORTED
34 Ethyl iso-allocholate 21.17 1.69 Antifungal compound Abubacker and Deepalakshmi, 2013
35 9-Hexacosene 21.5 4.61 NOT REPORTED
36 2,4-Dodecadienal 21.61 1.15 Plant growth promoter Awad et al., 2018; El-Leel et al., 2019
37 α-humulene 22.18 1.18 Antifungal & antibacterial Hossain et al., 2008; Pichette et al., 2006
38 Heptyltetracosyl ether 21.78 4.34 Insecticidal activity Howard et al., 1960
39 Benzamide 22.26 0.91 Plant growth regulator Domínguez et al., 2016
40 Oleic acid 22.46 1.13 NOT REPORTED
41 1-Methyl cycloundecanol 22.89 3.86 Biocontrol activity Awad et al., 2018
42 2-butyl-1- octanol 24.16 2.19 Antifungal activity Mannaa and Kim, 2018
43 Dodecanoic acid 25.34 2.67 Antimicrobial activity Jasim et al., 2013
44 α-Copaene 29.60 4.23 Antioxidant property Costa et al., 2011
45 Guaiol 34.91 1.32 Antimicrobial activity Santos et al., 2013
46 Cedrane-813-diol 39.52 1.46 Antimicrobial activity Ugur et al., 2009
a
Compounds were identified by through mass spectral data with NIST and WIlEY8 library after GC–MS analysis.
b
RT- Retention time.
c
Peak area percentage.
d
Properties reported in available literatures.
e
Reference of literatures.

3.3. Belowground VOCs produced by Trichoderma viride BHU-V2 3.4. Belowground VOCs augmented defense enzyme activities in okra
promoted plant growth plants

In this study, belowground VOCs secreted by T. viride BHU-V2 were
found to interact with okra plants and significantly effect on root and
leaves (Fig. 6A & B). Plant growth promotion parameters were higher in
treatments T3 and T4 as compared their respective control. S. rolfsii
inoculation into soil was found to reduce okra plant growth in terms of
length, lateral root formation, fresh weight and biomass content. How­
ever, VOCs exposed plants (T4) were exhibited significantly higher
shoot length (21.79 %), root length (24.94 %), lateral root number
(121.17 %), fresh weight (37.89 %) and biomass content (42.5 %) as
compared to respective control plants (T2). Total chlorophyll content
was also reduced under pathogen inoculation condition. VOCs exposed
plants (T4) were retained (34 %) higher chlorophyll content as
compared to T2 treatment plants (Table 3).
All defense enzymes activities were induced in okra plants during
S. rolfsii inoculation. However, VOCs exposed plants (T4) were exhibited
higher activity as compared to respective control plants (T2). PAL and
PPO activities were found higher (0.7 & 0.33 fold respectively) in T4
treated plants as compared to T2 plants at 14th day (Fig. 7A & B). PO
activity was exhibited higher at 7th day in T4 plants (0.29 fold) as
compared to T2 plants (Fig. 7C). Activities of cell wall degrading en­
zymes chitinase and β-1, 3-glucanase were also measured in okra plants
upon S. rolfsii pathogen inoculation. Chitinase enzyme was found
highest at 7th day in T4 plants (0.89 fold) as compared to T2 plants
(Fig. 8A). β-1, 3-glucanase was observed highest at 14th day in T4 plants
(0.42 fold) as compared to T2 plants (Fig. 8B).

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J. Singh et al. Microbiological Research 246 (2021) 126721

Table 2
GC/MS profiling of volatile organic compounds released by T. viride BHU-V2 on soil medium along with their properties as reported in previous literatures.
S.No. Name of compounda RTb Area%c Propertiesd Referencese

1 3,7-Diacetamido-7H-s-triazolo 13.41 1.97 Insecticidal activity Hussein et al., 2016

2 1,3-Benzenediol 13.82 0.91 Antioxidant property Mathew et al., 2015
3 Anethole 14.47 0.98 Antioxidant property Freire et al., 2005
4 Oleic acid 15.32 1.97 Antifungal activity Abubacker and Devi., 2014
5 Bisabolol 16.43 1.29 Antioxidant property Braga et al., 2009
6 Isolongifolol 16.82 1.13 Antioxidant property Adibe et al., 2019
7 Benzestrol 17.02 1.24 Fungistatic activity Heinemann, 1947
8 Cis-jasmone 17.23 0.91 Induce plant defense Moraes et al., 2008
9 Cyclopropanepentanoic acid 17.86 1.03 Antioxidant property Aihetasham et al., 2015
10 Ledene oxide-(II) 18.08 1.81 Antioxidant property Al-Qudah et al., 2012
11 trans-Z-Bisabolene epoxide 18.23 4.27 Antioxidant property Yuvarajan et al., 2014
12 10-Heneicosene 18.33 4.85 Antifungal and antibacterial activity Mahamuni, 2015; Gurnani et al., 2016
13 Globulol 18.66 2.03 Plant growth regulator Aghaei et al., 2013
14 11-Octadecenoic acid 19.11 4.12 Antimicrobial activity Aihetasham et al., 2017
15 Oleic Acid 19.14 4.92 Antifungal activity Priyanka and Nakkeeran, 2019; Mahamuni, 2015
16 Tetradecanoic acid 19.19 9.27 Antifungal activity Salem et al., 2019
17 1-Decanol, 2-hexyl- 19.47 7.18 Antibacterial and antifungal activity Abdu, 2019; Elbanhawy et al., 2019
18 13-Docosenoic acid, methyl ester 19.84 5.91 Antifungal and antibacterial activity Chandrasekaran et al., 2011; Abubacker andDevi, 2014
19 Hexadecanoic acid 20.2 6.84 Plant growth regulator Salem et al., 2014
20 Ascaridole 20.48 4.01 Insectidal activity Kong et al., 2004
21 Heptacos-1-ene 20.53 4.71 Antimicrobial activity dos Reis et al., 2019
22 Ethyl iso-allocholate 20.84 2.01 Antifungal and antibacterial activity Abubacker and Deepalakshmi, 2013
23 9-Tricosene 21.21 2.07 Antifungal activity Pandey et al., 2013
24 Benzamide 21.32 4.91 Plant growth regulator Domínguez et al., 2016
25 Heptyltetracosyl ether 21.67 3.47 Insecticidal activity Howard et al., 1960
26 2,4-Dodecadienal 21.93 1.51 Plant growth promoter Awad et al., 2018; El-Leel et al., 2019
27 α-humulene 22.26 3.35 Antifungal and antibacterial activity Hossain et al., 2008; Pichette et al., 2006
28 Androstane-11,17-dione 22.87 1.03 Antioxidant property Awad et al., 2018
29 β-bisabolene 23.28 0.97 Bactericidal activity Nascimento et al., 2007
30 2-butyl-1-octanol 23.48 2.84 Antifungal activity Mannaa and Kim, 2018
a
Compounds were identified by through mass spectral data with NIST and WIlEY8 library after GC–MS analysis.
b
RT- Retention time.
c
Peak area percentage.
d
Properties reported in available literatures.
e
Reference of literatures.

3.5. Belowground VOCs reduced cell death and lipid peroxidation in okra
plant against S. rolfsii inoculation
In situ cell death was analyzed through the amount of blue spots
owing to hypersensitive reactions (HR), which was found maximum in
T2 plants (75 %) (S. rolfsii inoculation only). However, VOCs produced
by T. viride BHU-V2 (T4) were able to mitigate the cell death resulting in
spot reduction on leaf tissues (43 %) (Fig. 9A). Intensity of pinkish color
developed on leaves surface is directly proportional to the rate of lipid
peroxidation leading to malondialdehyde (MDA) from lipid peroxides.
Maximum amount of color development were observed on the leaves of
T2 plants (88 %), whereas spots on T4 plants were reduced substantially
(35 %) (Fig. 9B).
4. Discussion
Plant holobiont interactions are essential for plant growth and health
asthe associated microbes get involved in plant stress tolerance mech­
anism to cope with abiotic and biotic stresses (Trivedi et al., 2020). To
protect plant from soil borne pathogens, the rhizospheric microbes
developed various sophisticated mechanisms including the production
of antimicrobial secondary metabolites and emission of bioactive vola­
tile compounds to activate plant defense mechanism (Liu and Brettell,
2019; Moisan et al., 2020). In the present study, we found that below­
ground volatiles emitted by T. viride BHU-V2 affected okra plant growth
and resistance to soil-borne fungal pathogen S. rolfsii. Despite an overall
reduction in okra plant growth and biomass content upon inoculation
with S. rolfsii, volatile exposed plants (T. viride BHU-V2 inoculated)
remained tolerant and healthy. Belowground exposure to T. viride
BHU-V2 producing VOCs accelerated root growth, which resulted in­
crease in overall plant growth and biomass. In addition, VOCs exposure
affected plant defense mechanism by inducing different defense enzyme
activities, resulting in reduced lipid peroxidation and cell death. T. viride
BHU-V2 released volatile compounds were variable and dependent on
the nutrient medium.
Trichoderma species are widely reported as potent biofertilizer and
biocontrol agents owing to their properties of mycoparasitism, antibi­
osis, plant growth promotion, induction for systemic resistance, colo­
nization of roots and inhibition of phytopathogens (Hansen et al., 2010;
Kamal et al., 2018; Liu et al., 2020a, 2020b; Swain et al., 2018). In the
present study, T. viride BHU-V2 was found able to inhibit the growth of
S. rolfsii in a partite Petri plate system through producing VOCs. In this
experiment, there was no physical contact between both fungal strains.
Hence, at preliminary level, it hypothesized that T. viride BHU-V2
secreted such volatile compounds that are responsible for inhibition of
S. rolfsii growth. This assumption was further confirmed by microscopic
analysis of S. rolfsii mycelium. Microscopic analysis exposed the reduc­
tion and thinning of S. rolfsii hyphae in the presence of T. viride BHU-V2
producing VOCs which confirmed the presence of antifungal compounds
in VOCs blend. The similar findings have been observed by Amin et al.
(2010) and Hosen et al. (2016), where T. viride producing VOCs
inhibited mycelial growth and sclerotia formation in S. rolfsii. Further­
more, T. viride BHU-V2 producing VOCs significantly affected seed
germination of okra that suggests the presence of plant growth pro­
moting compounds in VOCs blend. Similar results have been observed in
which T. viride producing VOCs promoted growth of Arabidopsis thaliana
and tomato plants (Hung et al., 2013; Lee et al., 2016). In the present
study, T. viride BHU-V2 isolate was exhibited auxin production, phos­
phate solublization and siderophore production activity that assures the
proliferation of plants in different manner (Harman et al., 2004; Con­
treras-Cornejo et al., 2009; Mastouri et al., 2010; Lee et al., 2016).
The results of GC–MS analysis revealed the presence of fungistatic,

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J. Singh et al. Microbiological Research 246 (2021) 126721

Fig. 5. Multivariate analysis of metabolites obtained on

PDA and soil medium after GC–MS analysis. (A) Venn
diagram represents a total of 9 compounds identified as
common between PDA and soil medium datasets. The
Venn diagram shows the common as well as the mutually
exclusive compound names for each medium along with
the counts. The 9 common compounds are 2,4-Dodecadie­
nal, α-humulene, Benzamide, Benzestrol, Cis-jasmone,
Ethyl iso-allocholate, Globulol, Heptyltetracosyl ether
and Oleic acid; and (B) Chord diagram presents the prop­
erties of compounds identified on both PDA and soil me­
dium and their networks. The nodes represent both
compounds and properties and edges represent their as­
sociation. There were total of 11 properties associated in
PDA medium whereas in case of soil medium, the number
of properties is 9. In case of both mediums, the highest
association was obtained for the Antifungal property;
however compounds obtained on soil share a major
portion in this group.

9
J. Singh et al. Microbiological Research 246 (2021) 126721

Fig. 6. Effect of VOCs exposure on (A) root morphology and (B) leaf number and area under different treatments. The quantification analyses of these parameters are
represented in Table 3. Where, T1- Control (without any treatment), T2- S. rolfsii inoculation, T3- T. viride BHU-V2 VOCs treatment and T4- T. viride BHU-V2 VOCs +
S. rolfsii inoculation.

Table 3
Effect of T. viride BHU-V2 producing VOCs on okra plant growth parameters under different treatment conditions. Results are expressed as mean of three repli­
cations ± SD. Significant difference among the treatments taken at same time interval are indicated by different letters according to Duncan’s multiple range test at
p ≤ 0.05.T1- Control (without any treatment), T2- S. rolfsii inoculation, T3- T. viride BHU-V2 VOCs treatment and T4- T. viride BHU-V2 VOCs + S. rolfsii inoculation.
Treatments Shoot length Root length Fresh weight Dry weight No. of leaves Leaf area No. of lateral roots Total chlorophyll content
(cm) (cm) (g) (g) (number) (cm2) (number) (mg chlorophyll/g FW)

T1 22.43 ± 0.46c 6.86 ± 0.03c 3.12 ± 0.26c 1.52 ± 0.05b 5.66 ± 0.57b 22.52 ± 1.02c 33.66 ± 2.51c 4.9 ± 0.06c
T2 16.06 ± 0.25d 4.93 ± 0.25d 2.19 ± 0.10d 0.8 ± 0.01c 3.33 ± 0.57c 13.74 ± 1.51d 23.66 ± 2.01d 2.85 ± 0.07d
T3 25.23 ± 0.61a 9.66 ± 0.77a 4.40 ± 0.36a 2.40 ± 0.35a 8.33 ± 0.57a 28.42 ± 1.25a 61.66 ± 3.05a 5.16 ± 0.15a
T4 19.56 ± 0.98b 6.16 ± 0.65b 3.02 ± 0.23b 1.14 ± 0.04b 4.33 ± 0.57c 18.22 ± 1.87b 52.33 ± 3.05b 3.84 ± 0.11b

antibacterial, antioxidant and plant growth regulating compounds in
T. viride BHU-V2 producing VOCs blend i.e. 2-butyl-1-octanol, methyl-
bicyclooctan-7-ol, 1-methyl cycloudecanol and Tetradecanoic acid,
benzestrol, Oleic acid, bezenediol, 9-Tricosene, Hexadecanoic acid,
Ascaridole and oleic acid. The production of these compounds varied
with growth medium condition of T. viride BHU-V2. Our GC–MS results
explored that T. viride BHU-V2 produce more numbers of volatile com­
pounds on PDA medium as compared to soil medium. However, an
interesting finding observed in the VOCs blend produced on the soil
medium is that most of the compounds have been reported as fungicides
in the previous studies. Several other studies also reported production of
different functional group compounds such as alcohols, pyrans, alde­
hydes, ketones, fatty acids, etc. from Trichoderma spp. (Vinodkumar
et al., 2017; Sunpapao et al., 2018; Nagamani et al., 2017; Kumari et al.,
2019). The production of a wide array of volatile compounds are depend
on species, strain, substrate concentration, presence of pathogen and
various other abiotic factors (Wheatley, 2002; Insam and Seewald,
2010). The secretion of majority of fungicidal and plant growth pro­
moting compounds on soil medium as compared to PDA suggests the
effect of soil complexity on metabolic activity of Trichoderma sp. VOCs
profiling produced on PDA medium possessed compounds with various
biological activities such as antioxidants, pest repellents, nematicidal
compounds along with limited amount of antifungal and antibacterial
compounds. The confirmation of the activity of different VOCs com­
pounds were done by literature survey (Mannaa and Kim, 2018; Awad
et al., 2018; Salem et al., 2019; Pandey et al., 2013; Salem et al., 2014;
Huang et al., 2012; Priyanka and Nakkeeran, 2019).
Exposure of okra roots to T. viride BHU-V2 volatiles significantly
improved the growth of S. rolfsii inoculated plants. Although S. rolfsii
inoculation resulted in intense wilting and finally resulted in reduced
growth of control plants, fungal volatile-exposed plants remained
tolerant and compensated for the loss of plant weight and height. The
VOCs exposure significantly modified root morphology of okra plants
along with plant height, number and size of leaves, plant biomass and
chlorophyll content. Microbial VOCs have already been reported as a
modifier for root architecture of host plants to increase colonization of
microbes (Werner et al., 2016). Other studies also confirmed the role of
Trichoderma secreted VOCs in plant growth promotion (Garnica-Vergara
et al., 2015; Lee et al., 2016; Wonglom et al., 2020). The induction in
plant biomass and chlorophyll content upon VOCs exposure suggest
effect of VOCs on phytohormone pathways (Contreras-Cornejo et al.,
2009). VOCs profiling of T. viride BHU-V2 also exhibited the presence of
plant growth promoting compounds including globulol, benzamide,
cis-jasmone, isopentyl alcohol and 2, 4-dodecadienal which can be

10
J. Singh et al.
Fig. 7. Effect of VOCs exposure on defense enzymes activity in okra plants
under different treatments. (A) Phenyl ammonia lyase activity (PAL); (B)
Polyphenol oxidase activity (PPO); and (C) Peroxidase activity (PO). Where, T1-
Control (without any treatment), T2- S. rolfsii inoculation, T3- T. viride BHU-V2
VOCs treatment and T4- T. viride BHU-V2 VOCs + S. rolfsii inoculation. Results
are expressed as mean of three replication and vertical bars indicate standard
deviation of the mean. Different letters above error bar indicate significant
difference among treatment results taken at same time interval according to
Duncan’s multiple range test at p ≤ 0.05.
asserted for their involvement in enhanced growth of okra (Aghaei et al.,
2013; Domínguez et al., 2016). In addition, 2, 4-dodecadienal and
cis-jasmone has been reputed to confer defense activities in host plant
(Moraes et al., 2008; Awad et al., 2018; El-Leel et al., 2019). Although
VOCs exposure induced okra plant growth, it is difficult to correlate one
compound for this contribution. More detailed study is required to un­
derstand the contribution of a single compound.
In the current study, VOCs of T. viride BHU-V2 not only promoted the
plant growth but also systematically stimulated the defense mechanisms
of okra plant during plant-pathogen interaction. Belowground VOCs
have been affirmed for their potential in induction of resistance in plants
against soil-borne pathogens by activating defense mechanism (Werner
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Microbiological Research 246 (2021) 126721
Fig. 8. Effect of VOCs exposure on defense enzymes activity in okra plants
under different treatments. (A) Chitinase activity and (B) β 1-3 Glucanase ac­
tivity. Where, T1- Control (without any treatment), T2- S. rolfsii inoculation, T3-
T. viride BHU-V2 VOCs treatment and T4- T. viride BHU-V2 VOCs + S. rolfsii
inoculation. Results are expressed as mean of three replication and vertical bars
indicate standard deviation of the mean. Different letters above error bar
indicate significant difference among treatment results taken at same time in­
terval according to Duncan’s multiple range test at p ≤ 0.05.
et al., 2016). The induced systemic defense system has been associated
with increased activity of fungal cell wall degrading enzymes and
up-regulation of defense enzymatic activities. Previous studies have
mentioned intensified antioxidant effects of volatiles produced by fresh
culture of T. viride against different pathogens (Vos et al., 2015; Naznin
et al., 2014; Gajera et al., 2016; Awad et al., 2018). In support to above
findings, some volatile compounds including benzeneethanol,
α-copaene, diepoxyhexadecane, isoaromadendrene epoxide,
trans-Z-bisabolene epoxide, ledene oxide, bisabolol, Cyclo­
propanepentanoic acid, isolongifolol,androstane-11 and 17-dionefrom
were detected in the VOCs blend produced by T. viride BHU-V2 and
reported to induce antioxidant properties for better immunity of host
plant (Hameed et al., 2015; Shareef et al., 2016; Ashwathanarayana and
Naika, 2017; Adibe et al., 2019).
In our experiment, VOCs exposure induced PAL, PO and PPO activity
in okra plants during their interaction with S. rolfsii inoculated in soil.
The maximum induction of PAL was observed during progressive days of
plant growth that ensure higher production of lignin and phenol com­
pounds for defense as it is the initial enzyme of phenylpropanoid
metabolism (Tonelli et al., 2011). The higher activity of PO and PPO
provides an insight for reduction in reactive oxygen species level that
was accumulated due to pathogen infection. Similar findings have been
reported by other studies, in which bacterial emitted VOCs induced PAL,
phenol content and other disease resistance enzymes to alleviate toxic
effect of ROS in host plants (del Rosario Cappellari et al., 2017; Zhao
et al., 2019). Our results also showed a higher level of cell wall
degrading enzymes β-1,3-glucanase and chitinase in the VOCs exposed
okra plants as compared to control plants during pathogen inoculation.
The induction of these enzymes can also be linked with the growth in­
hibition of S. rolfsii in the soil through the changes in hyphal morphology
that includes thinning of hyphae, node formation, movement of plasma,
distortion in structures and partial degradation of fungal cell wall
J. Singh et al.
Fig. 9. Histochemical analysis of (A) hypersensitive cell death and (B) lipid peroxidation in okra leaves under different treatments. Where, T1- Control (without any
treatment), T2- S. rolfsii inoculation, T3- T. viride BHU-V2 VOCs treatment and T4- T. viride BHU-V2 VOCs + S. rolfsii inoculation. In the graphs, results are expressed
as mean of three replication and vertical bars indicate standard deviation of the mean.
observed during microscopic analysis of hyphae from control and VOCs
exposed S. rolfsii (Tenorio-Salgado et al., 2013; Wonglom et al., 2020). In
situ histochemical staining of leaves for lipid peroxidation and cell death
cast further confirmatory information to the existing analysis regarding
plant defense mechanism. In this experiment, reduced H2O2 production
in volatile exposed okra plants in comparison to control plants may be
correlated with increased level of PO enzyme. Augmented level of H2O2
along with enhanced lipid peroxidation is a parameter for extent of
membrane damage that ultimately leads to cell death (Airaki et al.,
2012; Jain et al., 2015; Ray et al., 2016). In the similar pattern, higher
amount of lipid peroxidation within tissues has been observed in control
plants that ultimately lead to augmented cell death was observed in
Evan’s blue staining.
The results from our study revealed that VOCs released by T. viride
BHU-V2 in belowground mediated (i) antifungal activity against collar
rot pathogen of okra (S. rolfsii), (ii) defense responses including
enhanced cell-wall degrading and defense enzyme activities supported
by histological and microscopic analysis and (iii) improved plant
growth. Hence, T. viride BHU-V2 can be used as a potent biocontrol
agent for soil-inhabiting pathogens and plant growth promoter for sus­
tainable crop production. Furthermore, this study provides an impetus
to validate the role of specific VOCs and their pathways for plant growth
promotion and utilize volatile compounds in form of suitable bio­
formulation for field application. Formulations of individual VOCs or
VOCs blend can be directly apply through spraying or gel based tech­
nology in the soil at the time of seed sowing for management of soil
borne diseases.
12
Microbiological Research 246 (2021) 126721
Author contributions
JS and PS performed the experiments and drafted the manuscript.
RSR helped in compilation of data and interpretation of the results. AV
and SR edited entire manuscript. SMS and HBS coordinated the work
and gave valuable comments.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgment
J. Singh is thankful to CSIR-JRF fellowship. P. Singh and R. S. Rajput
are grateful to UGC-RET fellowship for providing financial assistance. A.
Vaishnav wants to acknowledge SERB-NPDF (PDF/2017/000689) for
providing financial assistance. Authors would like to thank Dr. Jagajjit
Sahu and Roshan Kumar for performing multivariate analysis of GC–MS
metabolites.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.micres.2021.126721.
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