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Abstract

The current research based on greenhouse experiment evaluates the impact of the Lactobacillus strains (Lactobacillus plantarum, Lactobacillus paralimentaris, Lactobacillus fermentum, Lactobacillus pentosus, and Lactobacillus buchneri) previously isolated from maize silage on the Fusarium verticillioides-infected maize plants. The growth parameters as well as catalase, superoxide dismutase, ascorbate peroxidase, and peroxidase antioxidant enzymes activity were investigated in one-month old seedlings, after inoculations with Fusarium or co-inoculations with Fusarium and the Lactobacillus strains. Application of Lactobacillus strains in maize seedlings significantly enhanced the plant growth and biomass. The best effect was observed when the L. buchneri was applied. It was revealed that inoculation with Fusarium stimulated antioxidant enzyme activity and co-inoculation with Lactobacillus strains reduced the enzyme activity, compared to Fusarium treatment alone. This is the first report that revealed the bioprotective role of Lactobacillus strains against F. verticillioides.
Journal of Horticultural Research 2017, vol. 25(2): 67-74
DOI: 10.1515/johr-2017-0015
_______________________________________________________________________________________________________
*Corresponding author:
e-mail: m.aghdasi@gu.ac.ir
EFFECTS OF FUSARIUM VERTICILLIOIDES AND LACTOBACILLUS
STRAINS INOCULATION ON GROWTH AND ANTIOXIDANT ENZYMES
ACTIVITY OF ZEA MAYS PLANTS
Zohreh Akhavan KHARAZIAN1, Mahnaz AGHDASI1*, Gholamreza Salehi JOUZAN2,
Majid ZAMANI3
1Golestan University, Shahid Beheshti street, 159 Gorgan, Iran
2Agricultural Biotechnology Research Institute of Iran (ABRII)
3Maize & Forage Crops Research Department, Seed & Plant Improvement Institute (SPII)
Received: May 2017; Accepted: October 2017
Abstract
The current research based on greenhouse experiment evaluates the impact of the Lactobacillus strains
(Lactobacillus plantarum, Lactobacillus paralimentaris, Lactobacillus fermentum, Lactobacillus pentosus,
and Lactobacillus buchneri) previously isolated from maize silage on the Fusarium verticillioides-infected
maize plants. The growth parameters as well as catalase, superoxide dismutase, ascorbate peroxidase, and
peroxidase antioxidant enzymes activity were investigated in one-month old seedlings, after inoculations
with Fusarium or co-inoculations with Fusarium and the Lactobacillus strains. Application of Lactobacil-
lus strains in maize seedlings significantly enhanced the plant growth and biomass. The best effect was
observed when the L. buchneri was applied. It was revealed that inoculation with Fusarium stimulated
antioxidant enzyme activity and co-inoculation with Lactobacillus strains reduced the enzyme activity,
compared to Fusarium treatment alone. This is the first report that revealed the bioprotective role of Lac-
tobacillus strains against F. verticillioides.
Key words: biocontrol, Fusarium disease, lactic acid bacteria, maize, plant growth promotion
INTRODUCTION
Maize (Zea mays L.) is one of the most im-
portant and the third most traded cereal grain in the
world (Pereira et al. 2011b). Fusarium verticillioides
(Sacc.) Nirenberg (syn. Fusarium moniliforme) is
known as one of the most frequent fungal pathogens
in maize worldwide. In the suitable conditions, the
pathogen induces root, stalk, ear, kernel, and seed-
ling rot, which causes serious production losses. F.
verticillioides secretes several toxins that are poten-
tially toxic for humans and farm animals. The most
important of these toxins produced by F. verticil-
lioides are mycotoxins, the fumonisins (Oren et al.
2003), possessing carcinogenic effects (Pereira et
al. 2011b). This species, in association with maize,
can appear as both a pathogen or a symptomless in-
tercellular endophyte, depending on diverse factors
such as plant and fungal genotypes, environmental
conditions, fungal inoculum size, and the presence
of antagonists (Bacon et al. 2001; Pereira et al.
2011a). The contamination of maize and wheat
fields with Fusarium strains, particularly F. verticil-
lioides and F. proliferatum, is commonly reported
(Mohammadi-Gholami et al. 2013). This contami-
nation is a serious public health hazard because of
the food spoilage and the presence of carcinogenic
fumonisin B1 in high levels. Biological control of
crops̕ disease and pets using microbial inoculants is
being increasingly noticed as a feasible, ecofriendly
alternative that limits the enormous use of the syn-
thetic chemical pesticides (Gajbhiye & Kapadnis
2016; Oliveira et al. 2014; Pereira et al. 2011a).
Lactic acid bacteria (LAB) are a family of
gram-positive, non-spore forming, cocci- or rod-
shaped, catalase (CAT)-negative organisms (Patil et
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68 Z.A. Kharazian et al.
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al. 2010). LAB have been widely and safely used in
the food and feed industries as probiotics or starters
during the past decades (Franz et al. 2010; Oliveira
et al. 2014). Recently, some studies reported the an-
tifungal activities of these bacteria against some
plant pathogenic fungi (Gajbhiye & Kapadnis 2016;
Gupta & Srivastava 2014; Kharazian et al. 2017;
Kıvanc et al. 2014; Oliveira et al. 2014; Tropcheva
et al. 2014; Varsha et al. 2014).
The resistance of plants to fungal colonization
is often manifested by the hypersensitive reaction
(HR) of challenged plant cells and the reactive oxy-
gen species (ROS) production. It is the evidence of
successful recognition of infection and activation of
plant defenses. The excess of ROS causes damage
to proteins, lipids, carbohydrates, DNA and finally
results in cell death (Torres 2010). The role of the
ROS family is that of a double-edged sword; while
they act as secondary messengers in various key
physiological phenomena, they also induce oxida-
tive damages under several environmental stress
conditions (Das & Roychoudhury 2014).
The induction of ROS-scavenging enzymes,
such as superoxide dismutase (SOD), peroxidases
(PODs), and CAT, are the most important and com-
mon mechanism for detoxifying ROS, synthesized
during stress responses. These enzymes act by either
the partial suppression of ROS production or the scav-
enging of the ROS already produced (Torres 2010).
Many references report the impact of
Fusarium maize pathogens on antioxidative re-
sponses of the plants (GarcíaLimones et al. 2009;
Gherbawy et al. 2012; Sorahinobar et al. 2015), but
there is no report on the effects of LABs as biocon-
trol agents on the antioxidant enzymes in the
Fusarium-infected plants. So the objective of the
present study was to evaluate the impact of the Lac-
tobacillus strains previously isolated from maize si-
lage (Kharazian et al. 2017) on the physiological re-
sponses and growth parameters of Fusarium-in-
fected maize plants.
MATERIAL AND METHODS
Microbial strains
The F. verticillioides was kindly provided by the
Maize & Forage Crops Research Department, Seed
and Plant Improvement Institute (SPII), Karaj, Iran.
This strain was previously isolated from diseased
maize plants in the fields. For spore production, the
fungus was grown in the Potato Dextrose Broth me-
dium at 28 °C, and the spores were collected by fil-
tration.
The Lactobacillus strains used in the present
study were isolated from Iranian maize silages, and
their high antifungal activities against some plant
pathogenic fungi, including F. verticillioides, Peni-
cillium sp., Pythium aphanidermatum, and Verticil-
lium dahliae have been confirmed (Kharazian et al.
2017). The Lactobacillus strains used in the present
work were Lactobacillus plantarum E2, Lactobacil-
lus pentosus E4, Lactobacillus paralimentaris Q2,
Lactobacillus fermentum Q4, and Lactobacillus
buchneri (sunkii) Q6 with NCBI nucleotide se-
quence databases (https://www.ncbi.nlm.nih.gov)
accession numbers KJ736725, KJ736733,
KJ736727, KJ736732, and KJ736735, respectively
(Kharazian et al. 2017).
The Lactobacillus strains were inoculated into
De Man, Rogosa, and Sharpe (MRS) broth and cul-
tivated overnight at 37 °C. Bacterial suspensions
were centrifuged at 10,000 g for 20 min to remove
the nutritional medium, and then they were washed
twice with sterile water. The bacterial pellets were
suspended in sterile water to the volume of about
108 (CFU/ml) and immediately used for inoculation
of the seedlings.
In the treatment with the combination of five
Lactobacillus strains, equal amount of each strain
was given to the final concentration of about 108
(CFU/ml).
Plant material
The seeds of F. verticillioides susceptible maize
line K74/1 were kindly provided by the Maize &
Forage Crops Research Department of SPII. The
seeds were surface sterilized by bleach (5.25% so-
dium hypochlorite) for 10 min and then were rinsed
several times in sterile water. The kernels beaker
was placed in 60 °C water bath for 3 min, then the
water was removed, and the kernels were trans-
ferred to a Petri dish and covered with water. For
germination, the kernels were incubated in the dark
for two days at 25 °C and then for two days at 4 °C
(Bacon et al. 1994).
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Lactobacillus strains as a biocontrol against Fusarium verticillioides 69
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Table 1. Design of the greenhouse experiment
Treat-
ment
Strains
Fungus
(Pathogen)
Bacteria (Antagonists)
T1
-
-
T2
F. verticil-
lioides
L. plantarum E2,
L. pentosus E4,
L. paralimentaris Q2,
L. fermentum Q4,
L. buchneri (sunkii) Q6
T3
-
L. plantarum (E2),
L. pentosus E4,
L. paralimentaris Q2,
L. fermentum Q4,
L. buchneri (sunkii) Q6
T4
-
L. buchneri (sunkii) Q6
T5
F. verticil-
lioides
L. buchneri (sunkii) Q6
T6
F. verticil-
lioides
Benomyl (Fungicide)
T7
-
Benomyl (Fungicide)
T8
F. verticil-
lioides
-
Plant microbe interactions and growth parame-
ters analysis
Eight different combinations of the Lactobacillus
strains were used for inoculation of the maize seed-
lings under greenhouse conditions (Table 1). For the
treatments 6 and 7, a solution of the fungicide beno-
myl (commercial powder Benlate) with a concentra-
tion of 100 mg·ml-1 was added to the soil substrate
for controlling Fusarium.
Maize seedlings with aerial parts of 2.03.5 cm
in length were placed in Petri dishes together with
Lactobacillus strains suspensions and left for 4 h at
25 °C and then 4 h with F. verticillioides spore sus-
pensions and then transferred to pots in the green-
house. In the control samples, the seedlings were
soaked in water instead of bacterial suspensions.
The greenhouse experiment was conducted in pots
containing a mixture of 40% peat, 30% loam, 20%
vermiculite, and 10% compost. There were three
seedlings per pot and three pots for each treatment.
The greenhouse temperature was 2527 °C with 12-
h photoperiod. The plants were irrigated twice in
a week. After one month, the maize plants were har-
vested. Then different growth parameters, including
shoot and root length and fresh and dry weights of
plants were measured.
Measurement of enzymes activity
To prepare crude enzyme extracts, fresh leaves
(0.05 g) were ground with 2 ml of 0.1 M cool phos-
phate buffer (pH 6.8) as described by Kar and Mishra
(1976). The obtained homogenate was then centri-
fuged at 15,000 g for 15 min at 4 °C. The clear super-
natant was used for assaying the enzyme activities.
CAT activity was determined by monitoring
the destruction of H2O2 at 240 nm. The reaction
mixture in a final volume of 3 ml contained 50 mM
phosphate buffer (pH 6.8), 100 μl enzyme extract,
and 15 mM H2O2. The decrease in absorbance at
240 nm was recorded with a spectrophotometer
(Shimadzu UV-160) (Aebi 1984). The POD reac-
tion mixture in a final volume of 3 ml contained
20 mM guaiacol, 25 mM phosphate buffer (pH 6.8),
40 mM H2O2, and 10 μl of the crude enzyme extract.
The increase in absorbance at 470 nm because of
tetra-guaiacol formation was recorded spectropho-
tometrically (Chance & Maehly 1955). Superoxide
dismutase (SOD) activity was measured by using
the photochemical nitro blue tetrazolium (NBT)
method (Beauchamp & Fridovich 1971). The SOD
reaction mixture in a final volume of 1 ml con-
tained 50 mM potassium phosphate buffer (pH
7.8), 0.1 mM ethylenediaminetetraacetic acid
(EDTA), 20 µl of the extract, 75 µM NBT, 13 mM
methionine, and 4 µM riboflavin. One unit of SOD
was defined as the quantity of enzyme required to
inhibit the reduction of NBT by 50%. Total ascor-
bate peroxidase (APX) activity was measured
spectrophotometrically by detecting the absorb-
ance at 290 nm during oxidation of ascorbic acid,
using the method described by Nakano and Asada
(1981). One milliliter of the reaction mixture con-
tained 50 mM potassium phosphate buffer (pH
7.0), 0.45 mM l-ascorbic acid, 0.3 mM H2O2, and
30 µl of the extract. One unit of APX was defined
as the quantity of enzyme required to consume
1 µM of substrate.
Statistical analysis
The experiment was carried out in three replications.
Analysis of variance, average comparing, and treatment
groups score were obtained by using SAS (version 9.1)
and the Duncan’s Multiple range tests (P < 0.05).
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70 Z.A. Kharazian et al.
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RESULTS AND DISCUSSION
In the current study, we have made the green-
house experiment to evaluate the impact of inocula-
tions of the F. verticillioides with or without Lacto-
bacillus strains (as biocontrol agent) on maize seed-
lings growth and antioxidant enzymes activity. Our
previous data from in vitro experiments showed that
all selected Lactobacillus strains can inhibit growth
of F. verticillioides (Kharazian et al. 2017).
Root lengths increased in the seedlings that
were soaked in suspensions of Lactobacillus
strains (Figs. 1 and 2A). The treatments 2
(Fusarium + all 5 Lactobacillus strains) and 5
(Fusarium + L. buchneri) caused the longest roots
(24 cm) compared to the control (T1) (11 cm). The
treatment 6 (fungicide + Fusarium) resulted in the
shortest roots, which was similar to that of the con-
trol with Fusarium. The root weight increased in
all Lactobacillus-containing treatments (with or
without Fusarium inoculation) compared to the
control (Fig. 2B). The maximum root weight,
1.854 and 1.729 mg, belonged to the treatments 3
(all 5 Lactobacillus) and 4, respectively. The low-
est root weight was recorded in the Fusarium treat-
ment (81 mg fresh weight). The treatments 3 and 5
caused significant increase in the shoot length and
weight, compared to the control (Fig. 3A and 3B).
However, other treatments did not show any sig-
nificant differences in shoot lengths compared to
the control. Fresh weight of the shoots in all Lac-
tobacillus treatments (also in the co-inoculation of
Lactobacillus with Fusarium) was significantly
bigger than that of the control, while dry weight of
the shoots was higher only in treatments 3 and 5.
The effect of Lactobacillus strains on plant
growth was previously described by Hamed et al.
(2011), Limanska et al. (2013), and Narasimha
Murthy et al. (2012). The positive effect of Lacto-
bacillus strains inoculation on shoot growth and lat-
eral root number was reported by Hamed et al.
(2011). According to Limanska et al. (2013), the
physiological response of seedlings for inoculation
with suspensions of Lactobacillus depends on the
tested strain.
Fusarium caused a significant increase in the
activity of all enzymes (Fig. 4). All the treatments
with microorganisms have increased POD and SOD
activities compared to the control (Fig. 4A and 4B).
The activity of APX and CAT was higher after in-
oculations with the mixture of bacteria and with
L. buchneri (Fig. 4C and 4D).
The above results are in agreement with
Gherbawy et al. (2012), who demonstrated that
F. moniliforme inoculation resulted in enhanced
activity of antioxidant enzymes (SOD, CAT, and
APX) in the wheat shoots. Meanwhile, Pereira et
al. (2011a) demonstrated that inoculation of maize
seeds with F. verticillioides, either alone or co-in-
oculated with the Bacillus, resulted in enhanced
SOD activity. The chances of oxidative burst and
programmed cell death are minimized because of
the enhanced antioxidant enzymes activity. As
a result, F. verticillioides can be protected from the
oxidative damage during colonization (Kumar et
al. 2009). Another interesting result of this study
was that the antioxidant enzymes activity are de-
creased in plants that were co-inoculated with
F. verticillioides and Lactobacillus strains as com-
pared to plants inoculated with F. verticillioides
only. Previously, two characteristics, including an-
tagonistic effects against plant pathogenic fungi
(Yan et al. 2017; Russo et al. 2017; Guo et al.
2012) and also high antioxidant activity, ROS
scavenging and inhibition of the production of free
radicals have been reported for different Lactoba-
cillus species (Virtanen et al. 2007; Xing et al.
2015).
Our experiments confirmed the high antifun-
gal activity of the selected Lactobacillus strains
against F. verticillioides, which may be caused by
the secretion of antifungal substances by Lactoba-
cillus strains. Some of the known secreted sub-
stances by Lactobacillus strains are cyclic dipep-
tides, proteinaceous compounds, organic acids,
fatty acids, nisin, and reuterin (Crowley et al. 2013;
Gajbhiye & Kapadnis 2016; Limanska et al. 2013).
Further experiments are needed to determine how
Lactobacillus strains prevent F. verticillioides in-
fection.
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Lactobacillus strains as a biocontrol against Fusarium verticillioides 71
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Fig. 1. The effect of Lactobacillus strains and Fusarium verticillioides inoculation on maize seedlings growth in the
greenhouse for 4 weeks. T1: control, T2: F. verticillioides + L. plantarum + L. pentosus + L. paralimentaris + L. fer-
mentum + L. buchneri, T3: L. plantarum + L. pentosus + L. paralimentaris + L. fermentum + L. buchneri, T4: L. buch-
neri, T5: F. verticillioides + L. buchneri, T6: F. verticillioides + fungicide, T7: fungicide, T8: F. verticillioides
Fig. 2. A) Root length and B) root fresh and dry weight
of maize seedlings inoculated with Lactobacillus strains
and F. verticillioides after 4 weeks of growth in the
greenhouse. The results are the means of three replicates
of experiment ± SE. Different letters above the columns
indicate significant differences between treatments
(P 0.05) according to Duncan’s multiple range tests. For
treatments see Table 1 and Fig. 1.
Fig. 3. A) Shoot length and B) shoot fresh and dry weight
of maize seedlings inoculated with Lactobacillus strains
and F. verticillioides after 4 weeks of growth in the
greenhouse. The results are the means of three replicates
of experiment ± SE. Different letters above the columns
indicate significant differences between treatments
(P 0.05) according to Duncan’s multiple range tests. or
treatments see Table 1 and Fig. 1.
0
5
10
15
20
25
30
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Root length (cm)
Treatment
d
0
500
1000
1500
2000
2500
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Root weigt (mg)
Treatment
Root Fresh Weight
Root Dry Weight
d
0
5
10
15
20
25
1 2 3 4 5 6 7 8
Shoot length (cm)
Treatment
c
0
200
400
600
800
1000
1200
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Shoot weight (mg)
Treatment
Shoot Fresh Weight
Shoot Dry weight
d
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72 Z.A. Kharazian et al.
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Fig. 4. A) Peroxidase (POD), B) superoxide dismutase
(SOD), C) ascorbate peroxidase (APX) and D) catalase
(CAT) activities in shoots of maize seedlings inoculated
with Lactobacillus strains and F. verticillioides after
4 weeks of growth in the greenhouse. The results are the
means of three replicates of experiment ± SE. Different
letters above the columns indicate significant differences
between treatments (P 0.05) according to Duncan’s mul-
tiple range tests. For treatments see Table 1 and Fig. 1.
CONCLUSION
The current results of the greenhouse experi-
ment on maize seedlings suggest that studied Lacto-
bacillus strains may have potential to be used as bi-
ocontrol agents. Field experiments are needed to
propose using of these strains in crop farming.
Acknowledgment
We thank the Golestan University Deputy of Research
and Office of Higher Education for the financial support
to Zohreh Akhavan Kharazian in the form of grants for
PhD research project.
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... The highest POD activity was recorded for the combined effect of F. oxysporum and methanol extract of P. boergesenii (206.2%) on plant shoots. In this connection, Kharazian et al. [50] demonstrated that F. verticillioides inoculation resulted in an enhancement in the activity of the antioxidant enzymes (CAT, APX, and POD) in the shoot of maize. Anand et al. [51] mentioned that an increase of CAT activity in the inoculated fruits was due to an increased H 2 O 2 in the host tissues. ...
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