Impact of Seed Treatments with Fungal Biocontrol Agents
on Enzymatic Activities and Phenolic Content of Soybean under Greenhouse and
Field Conditions
Anum Intisar1*,
Zafar Iqbal1 and Shahbaz Talib Sahi2
1Department of Plant Pathology, College of Agriculture,
University of Sargodha, Pakistan
2Department of Plant Pathology, University of
Agriculture, Faisalabad, Pakistan
*For correspondence: anum.intisar2009uaf@gmail.com
Received 26 March
2021; Accepted 27 May 2021; Published 10 July 2021
Abstract
Fungi in the genus Trichoderma are widely used as
biological control agents because they can suppress plant pathogens and
activate plant defense systems. In the present study, efficacy of microbial
antagonists viz., T.
harzianum and T. viride or their combination was evaluated
against the pathogenic fungus Macrophomina phaseolina and their effect
on enzymatic activities and phenol content of soybean [Glycine max (L.)
Merr.] plants. Soybean seeds were inoculated with T. harzianum and T.
viride separately or in combination, and sown in pots under green house
and under field conditions. Host enzymatic activities and phenol levels were
measured at 14, 28 and 42 days after sowing (DAS) in both field and greenhouse
experiments. Seed treatments with T. harzianum, T. viride or
their combination increased peroxidase, polyphenol oxidase and β-1, 3-glucanase activities, and
also the total phenol content in soybean leaves as compared to a non-treated
control treatment. Concentration of peroxidase and β-1, 3-glucanasepeaked at 14 DAS and decreased thereafter in
all the treatments under greenhouse and field conditions. All the treatments
showed the highest levels of total phenols and polyphenol-oxidase at 28 DAS
under both greenhouse and field conditions. At 14 DAS in both trials, the
combination of T. viride + T. harzianum resulted in
the highest level of peroxidase and β-1,
3-glucanase activities. This combination also resulted in the highest levels of
total phenols and polyphenol oxidase content at 28 DAS. Our findings
demonstrated that application of Trichoderma species as seed treatment
has potential to trigger key mechanisms of systemically acquired resistance in
soybean, and thereby enhanced efficacy of disease management tactics. © 2021
Friends Science Publishers
Keywords: Macrophomina
phaseolina; Peroxidase; Phenolics; Seed inoculation; Soybean;
Trichoderma
Introduction
Soybean is an important leguminous crop and a major
source of vegetable oil and proteins worldwide. The crop is prone to many
economically important fungal diseases of which charcoal rot caused by Macrophomina
phaseolina is the major constraint causing significant losses every year
(Marquez et al. 2021). This pathogen also causes diseases in other
legumes such as chickpea, mungbean and mashbean (Banaras et al. 2021;
Javed et al. 2021). Different management methods such as physical,
chemical (fungicides), regulatory, cultural and biological have been used to
control Macrophomina phaseolina (Khan and Javaid 2020a; Khan et al.
2021; Um-e-Aiman et al. 2021). However, these methods are helpful only
when used well in advance as precautionary measures (Ganeshamoorthi et al.
2010; Marquez et al. 2021). In addition, conventional chemical
fungicides for M. phaseolina infections may be less helpful due to
soil-borne nature of the pathogen, and may interrupt the balance of beneficial
microbes in the soils (Anis et al. 2010). Furthermore, the
indiscriminate use of chemical pesticides and fungicides may develop resistance
to pathogenic strains and can cause harmful environmental risks and health
hazards (Afouda et al. 2012). Efforts to manage the disease in soybean
through crop rotation has also been suggested (Mengistu et al. 2007),
but it may be inadequate for control of soil-borne fungal diseases with
long-surviving propagules such as charcoal rot. Therefore, alternative methods
of disease control are need of the time.
During the past few
decades, different biocontrol agents have been identified, characterized and
commercialized (Javaid et al. 2021; Sharf et al. 2021).
Biocontrol organisms have gained more attention as component of integrated
disease management programs (Shahid and Khan 2019; Ali et al. 2020). Biological control is an effective way to
enhance resistance in plants against pathogens, and this technique may play a
significant role in sustainability of agricultural systems. Biocontrol
organisms are helpful against seed- and soil-borne fungal diseases of several
crops (Akhtar and Javaid 2018; Javaid et al. 2018; Yasmin et al.
2020). The fungus Trichoderma harzianum has been documented to suppress
many soil-borne fungal pathogens including M. phaseolina
(Mukhopadhyay and Kumar 2020; Khan and
Javaid 2020b). Aly et al. (2007) enlisted different antagonists
of Trichoderma spp. against M. phaseolina. Sreedevi et al.
(2011) depicted that T. viride and T. harzianum isolates had
antifungal activity against M. phaseolina. Trichoderma spp. act
as biocontrol organisms and also stimulate the plant resistance and growth
resulting in overall improvement in yield (Javaid et al. 2017; Shoaib et
al. 2018). The biocontrol activity related to antibiotics and
mycoparasitism also improves defense response or systemic resistance in plants
(Naher et al. 2014). The germination percentage of melon was 96.7% when
seeds were treated with commercial T. harzianum + M. phaseolina as
compared to M. phaseolina alone (46.7%) and showed excellent results
against charcoal stem rot of water melon (Etebarian 2006). The antagonistic
characteristics of the biocontrol species depend on multiple mechanisms that
are involved in activation of specific properties (Khan and Javaid 2020b).
The most important
mechanism of Trichoderma spp. is the induction of plant defense response
to specific pathogens (Harman 2006; Inayati et al. 2020). Other than chemical and
physical obstructions, plants have immune systems. The system is able to
identify motifs that contain common structural features of all microbes but not
present in their host plants. The defense response of plants is rapid,
transitory and generalized. During biotic stress, host plant shows various
cellular and physiological changes such as ion influx across the plasma
membranes; activation of nitric oxide, defense-related genes; high production
of ROS (reactive oxygen species), different phytohormones; biosynthesis of
specific stress related proteins and production of antimicrobial chemicals such
as phenolics (Wu et al. 2014; Nishad et al. 2020). Different
biocontrol organisms may cause distinct molecular and cellular transformations
in plants that enhance the resistance to biotic and abiotic stress (Brotman et
al. 2013; Kumar 2013). The activity of defense-related enzymes such as
phenylalanine ammonia lyase, polyphenol oxidase and peroxidase was documented to be progressively enhanced in plants of
green gram (Vigna radiata) when inoculated with T. viride alone
or in combination with Pseudomonas fluorescens against M. phaseolina
(Thilagavathi et al. 2007). Tomato plants treated with T.
arundinaceum showed early expression of defense-related genes against Rhizoctonia
solani and Botrytis cinerea (Malmierca et al. 2012). Although
there are reports on role of Trichoderma spp. as a biological control
agent and induced defense-related enzymatic changes in plants, however, there
is little information available on combined effect of T. harzianum and T.
viride to induce defense-related enzymes in soybean plants. Therefore, the
main objective of the present investigation was to determine a suitable
combination of Trichoderma spp. in improving the enzymatic and phenolic
contents of soybean under greenhouse and field conditions.
Materials and Methods
Collection of fungal isolates
Soybean plants infected with M. phaseolina were collected from
soybean growing areas of Punjab, Pakistan. These infected samples were kept in
polythene bags and brought to plant pathology laboratory for isolation and
further processing. Potato dextrose agar (PDA) medium was used to culture M.
phaseolina. For this purpose, 200 g peeled and sliced potatoes, 20 g agar
and 20 g dextrose were used. The potatoes were sliced, boiled in 400 mL
distilled water and their extract was used after filtration with muslin cloth.
Likewise, agar was boiled in distilled water (400 mL); after boiling, 20 g of
melted agar and 20 g of dextrose were mixed with potato extract. After
preparation, the medium was autoclaved at 121°C for 30 min. Symptomatic
portions of stems were chopped into 5- to 7-mm long pieces. The chopped pieces
were disinfested with mercuric chloride (0.1%), washed with sterilized
distilled water and then placed on PDA plates with the help of sterilized
forceps. These PDA plates were incubated at 27 ± 1°C for 4 days to get suitable
growth of M. phaseolina. Characteristics of M. phaseolina were
identified on the basis of formation of sclerotia and morphology of colony by
following guidelines of Mahdizadeh et al. (2011). To maintain fungal culture in a viable condition, the PDA plates were
placed in a refrigerator at 4°C
until used.
For mass culturing of M. phaseolina, rice seeds
were washed with distilled water, placed in narrow glass flasks of 250 mL, and
soaked with enough water to cover the seeds. The flasks were plugged with
cotton and wrapped with aluminum foil. After 12 h seeds were autoclaved at
121°C for 30 min. After cooling, 5 mm mycelial discs were taken from 7 days old
culture of M. phaseolina, which had been prepared in PDA medium. These
discs of M. phaseolina were placed in flasks containing rice seeds and
incubated at 27 ± 1°C for 15 days in dark. From 3rd day on, flasks
were stirred daily to avoid aggregate formation. After 15 days, the seeds were
completely colonized showing black color and became ready for use. After
incubation, the inoculum was kept at 4°C till further utilization in the
experiments.
Application of fungal antagonists
Greenhouse experiment: Plastic pots (17 ×
20 × 20 cm3) were filled with a mixture of clay, sand and peat
(1:1:1). Soil was autoclaved at 121°C for 30 min for 2 successive days prior to
use. For fungal bio-control agents, treatments were: T. harzianum alone,
T. viridealone and T. harzianum + T. viride in
combination at three levels of concentration of conidia (2×104, 2×106
and 2×107 spores mL-1) (Karthikeyan et al. 2015).
Equal concentration of each species was used in the combined treatment. The
spore concentration was determined using hemocytometer.
Field experiment: Fungal bio-control agents or
their combination i.e., T.
harzianum, T. viride and T. harzianum + T. viride
were used at the same concentrations as for greenhouse. Seeds of soybean
variety NARC-3 (80 kg ha-1) were treated with fungal bio-control
agents using gum Arabicas sticky material. Seeds were coated with 1% gum Arabic
(10 g for 1 kg of soybean seed) as an adhesive and suspended in the conidial
suspension and kept at 25 ± 2°C in a rotary shaker for 6 h to
ensure uniform coating. After coating, seeds were dried in shade, and then used
for sowing. Both the experiments (greenhouse and field) were conducted in
research area of University of Agriculture Faisalabad, Pakistan using RCBD with
factorial arrangement and three replications. The net plot size for each
treatment unit was 3 × 3 meter. The inoculum of the pathogen M. phaseolina
developed on rice grains was added along the length of the lines @ 6 g m-1
along with sowing seeds. Crop was sown with the help of hands in rows in first
week of February, 2017 and 2018. The distance between rows was 25 cm, while
between plants was 5 cm. Fertilizers such as nitrogen, phosphorus and potassium
were used @ 25, 60, 50 kg ha-1, respectively. When the crop needed
water, it was irrigated and weeds were controlled manually during growing
season.
Observations
Peroxidase activity, total phenol content (TPC),
polyphenol-oxidase (PPO) and β-1,
3-glucanase activity were determined in leaves of NARC-3 14, 28 and 42 days
after sowing (DAS) in the field studies.
Peroxidase (PO) activity
The procedure for determining the activity of peroxidase
was adopted from Fehrmann and Dimond (1967). Approximately 0.5 g fresh leaves
of treated or non-treated (control) soybean leaves ground in a pre-chilled
mortar with 0.1 M ice cold phosphate
buffer (20 mL) at pH 7.1. Later on, it was kept for centrifugation (3000 rpm)
for 15 min. The supernatant (25 mL) was used for assay. Freshly prepared
pyrogallol, reagent, enzyme extract and phosphate buffer were mixed in a
cuvette tube and the blend was tuned to zero absorbance on a spectrophotometer.
The activity of enzyme was measured as the alteration in absorbance per minute
(ΔA/min) at 430 nm.
Total phenol content (TPC)
TPC was estimated by the Folin-Ciocalteu reagent method
(Bray and Thorpe 1954). Folin-Ciocalteu reagent (1 mL) and 20% sodium carbonate
(2 mL) were added together with ethanol extract (1 mL) in a test tube and then
heated for 1 min in a boiling water bath. After cooling, distilled water was
added and final volume was made up to 25 mL. The absorbance of the blue color
was determined with Spectronic-20 colorimeters at 725 nm. Total phenol content
was noted from the standard curve used for catechol.
Polyphenol oxidase (PPO)
Enzyme extract (0.5 mL) and 0.1 M phosphate buffer (2.3 mL) were added to a cuvette, which was
adjusted to zero absorbance on a spectrophotometer (Mahadevan and Sridhar
1982). A 0.2 mL aliquot of 0.1 M
catechol was added and then reactants were rapidly mixed. The activity of
enzyme was noted as variation in absorbance instantaneously after adding 0.1 M catechol (0.2 mL).
β-1, 3-glucanase activity
Approximately 1 g soybean leaves from each treatment
were homogenized separately in a mortar containing 0.1 M. sodium phosphate buffer at pH 7.1 at the rate of 2 mL g-1
fresh weight leaves for 1 min. This preparation was then passed through cheese
cloth and filterate was centrifuged at 3000 rpm for 15 min at 6oC.
The clear supernatant was collected and considered to be a crude extract for
enzymes assay. The supernatant was stored in the refrigerator at -20°C until
determination of β-1, 3-glucanase
activity by following the procedure of El-Gamal et al. (2016).
Statistical analysis
Data were statistically analyzed using Statistix 8.1
software and means were compared by least significant difference test (LSD) at
5% probability level.
Results
β-1, 3-glucanase activity of soybean plants
Fungal bio-control agents significantly increased β-1, 3-glucanase activity in
soybean compared to the control under both greenhouse and field conditions.
Among fungal bio-control agents, T. harzianum + T. viride greatly
increased β-1, 3-glucanase
activity (6.07 and 2.98 µg g-1 under
greenhouse and field conditions, respectively) followed by T. harzianum,
whereas plants treated with T. viride expressed the least β-1, 3-glucanase activity (3.75 and
1.37 µg g-1 under
greenhouse and field conditions, respectively). At 14 DAG, plants exhibited
maximum β-1, 3-glucanase
activity where T. harzianum + T. viride was applied (9.88 and
3.16 µg g-1 under
greenhouse and field conditions, respectively) which had not changed at 28 DAG.
At 42 DAG, plants expressed the lowest β-1,
3-glucanase activity where T. viride was applied (Fig. 1‒4).
Fig. 1: Effect of fungal biocontrol agents on β-1, 3-glucanase activity of soybean under greenhouse
conditions
Fig. 2: Impact of interaction between fungal biocontrol agents and days on β-1, 3-glucanase activity of
soybean under greenhouse conditions
Fig.
4: Impact of interaction between fungal biocontrol agents
and days on β-1, 3-glucanase
activity of soybean under field conditions
Fig.
5: Effect of fungal biocontrol agents on peroxidase
activity of soybean under greenhouse conditions
Fig.
6: Impact of interaction between fungal biocontrol agents
and days on peroxidase activity of soybean under greenhouse conditions
Fig. 3: Effect of fungal biocontrol agents on β-1, 3-glucanase activity of soybean under field conditions
Fig. 7: Effect
of fungal biocontrol agents on peroxidase activity of soybean under field
conditions
Fig. 8: Impact
of interaction between fungal biocontrol agents and days on peroxidase activity
of soybean under field conditions
Fig. 9:
Effect of fungal biocontrol agents on polyphenol-oxidase of soybean under
greenhouse conditions
Control plants had less peroxidase activity than
bio-control agents (Fig. 5‒8). Among fungal bio-control agents, T. harzianum +
T. viride progressively improved peroxidase activity in soybean (3.05
and 1.98 µg g-1 under
greenhouse and field conditions, respectively), while soil application of T.
viride expressed minimum peroxidase activity (2.32 µg g-1). At 28 DAG, plants with T. harzianum + T.
viride showed maximum peroxidase activity (3.10 and 2.16 µg g-1 under greenhouse and
field conditions, respectively). Peroxidase activity decreased with passage of
time to a minimum at 42 DAG in the T. viride treatment.
Polyphenol oxidase (PPO) activity of soybean plants
Concentration of PPO in leaves of soybean was consider
ably higher when seeds were inoculated with fungal antagonists before sowing
(Fig. 9‒12) than in the non-inoculated controls. Fungal bio-control agents also
increased the PPO concentration in soybean leaves compared to control. Among
fungal bio-control agents, T. harzianum + T. viride greatly
enhanced PPO activity (1.26 and 2.90 µg
g-1), whereas T. harzianum showed the least activity (0.63
and 1.27 µg g-1) under greenhouse and field conditions,
respectively. At 42 DAG, PPO activity was the highest when T. harzianum +
T. viride was applied (1.50 µg
g-1). Polyphenol-oxidase activity was the lowest (0.50 and 3.10 µg g-1)
at 42 DAG for the T. harzianum treatment under greenhouse and field
conditions, respectively.
Total phenol content (TPC)
Fungal bio-control agents significantly enhanced total
phenol content in soybean leaves compared to control (Fig. 13‒16). The combination
of T. harzianum + T. viride significantly improved total phenol
content (3.41 and 4.20 µg g-1 under greenhouse and field conditions,
respectively), whereas T. viride exhibited the least total phenol. At 28
DAG, total phenol content was increased substantially when T. harzianum +
T. viride was applied (3.83 and 4.66 µg g-1 under greenhouse
and field conditions, respectively), which was equivalent to the values at 14
DAG. Total phenol content was the least at 42 DAG in the T. viride treatment
(2.23 µg g-1).
Discussion
In
the present study, higher β-1, 3-glucanase,
peroxidase, polyphenol oxidase activities and maximum total phenol content were
observed in the soybean plants when seeds were sown after treatment with T. harzianum + T.
viride,
while the minimum values of these parameters were observed in the plants sown
with untreated seed under both greenhouse and field conditions. These
results indicate that combinations of T. harzianum + T. viride
triggers stronger soybean defense signals than alone T. harzianum and T.
viride. Increased activity of these host enzymes during plant-fungus
interactions have been reported previously by several researchers (Khaledi and
Taheri 2016; Yusnawan
et al. 2019; Inayati et al. 2020). Peroxidase and β-1, 3-glucanase play a
significant role to initiate the plant defense response against various
pathogens through production of highly toxic phenolic compounds and higher
production of reactive oxygen species or establishment of structural barriers
such as lignin accumulation (Yusnawan et al. 2019; Inayati et al. 2020).
β-1,3-glucanase degrades the
cell wall polysaccharides of fungal pathogens and kills the pathogens (Ueki et
al. 2020). Khaledi and Taheri (2016) reported
Fig. 10: Impact
of interaction between fungal biocontrol agents and days on polyphenol-oxidase
of soybean under greenhouse conditions
Fig. 11:
Effect of fungal biocontrol agents on polyphenol-oxidase of soybean under field
conditions
Fig. 12: Impact
of interaction between fungal biocontrol agents and days on polyphenol-oxidase
of soybean under field conditions
Fig. 13:
Effect of fungal biocontrol agents on total phenol contents of soybean under
greenhouse conditions
Fig. 14:
Impact of interaction between fungal biocontrol agents and days on total phenol
contents of soybean under greenhouse conditions
Fig. 15:
Effect of fungal biocontrol agents on total phenol contents of soybean under
field conditions
significant increase in peroxidase activity and phenolics
in soybean roots when seeds were sown after inoculation with T. harzianum
isolates. Similarly, Rajeswari (2019) observed that leaves of Arachis
hypogaea sprayed with combinations of T. viride and T. harzianum
significantly increased phenols concentration. The study of Yusnawan et al.
(2019) showed that the activity of peroxidase increased in soybean
plants treated with T. virens. Phenolics are one of the largest and most
diverse groups of plant active substances involved in the plant growth
regulation, and also play important role in defense responses during pathogen
infection and abiotic stress (Kubalt 2016). Phenolic
compounds are produced by plant when the plant recognizes harmful pathogens or
beneficial microbes. Polyphenol oxidase is involved in synthesis of phytoalexin
and
Fig. 16:
Impact of interaction between fungal biocontrol agents and days on total phenol
contents of soybean under field conditions
phenolic compounds, and the studies show that activity of
polyphenol oxidase increases in legumes when treated with T. viride
(Surekha et al. 2014). Seed treatment with T. virens increased
the accumulation of total phenols in legumes (Inayati et al. 2020).
PPO has been suggested to play important role in disease resistance due to its
ability to catalyze oxidation of phenolic compounds into quinones and lignin
biosynthesis (Kavitha and Umesha 2008; Inayati et al. 2020). According to
the reported study, it is suggested that the induction of plant resistance in
different hosts may require different signaling, and the induction is
represented in different manner (Martínez-Medina et al. 2014). An
increase of phenolic contents was also observed in soybean when seeds were
treated with T. virens (Yusnawan et al. 2019). Trichoderma
species have been studied for decades as effective bio-control agents against
many pathogens through various modes of action (Inayati et al. 2020)
and these Trichoderma spp., can induce systemic resistance in various
plant species and pathogens (Angel et al. 2016; Małolepsza et al.
2017). The study of Dubey et al. (2018) showed that there were up-regulated expression of some defense-related genes
and catalase in response to the presence of T. virens and R. solani. Numerous
studies indicate the ability of Trichoderma spp. to reprogram plant
genes expression that changes plant proteome and metabolome which alleviates
physiological and biochemical change, and improve plant resistance to biotic
and abiotic stresses (Mazzei et al. 2016). In plants, Trichoderma
is able to activate plant defense mechanisms mostly for induced systemic
resistance. Studies show that Trichoderma colonization triggers the
plant defense systems (Pieterse et al. 2014; Inayati et al. 2020).
Conclusion
The present study showed that combining of T.
harzianum with T. viride significantly increased the peroxidase,
polyphenol oxidase, phenolics, polyphenol and β-1, 3-glucanase concentration in soybean compared to T.
harzianum or T. viride alone. The increase in peroxidase, polyphenol
oxidase, phenolics, polyphenol and β-1,3-glucanaseactivity
and phenols concentration demonstrates that T. harzianum + T. viride are
synergistic and have beneficial impact on growth of soybean plants.
Acknowledgements
Authors acknowledge the Dr. Usman Ghazanfer from
department of Plant Pathology, College of Agriculture, University of Sargodha
and Dr. Muhammad Atiq from Department of Plant Pathology, University of
Agriculture, Faisalabad, for their support in data analysis.
Author Contributions
ZI and STS planned
the experiments and AI interpreted the results and wrote the
manuscript.
Conflicts of Interest
All authors declare no conflicts
of interest.
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author.
Ethics Approval
Not applicable in this paper
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