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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:
Zohreh Akhavan KHARAZIAN1, Mahnaz AGHDASI1*, Gholamreza Salehi JOUZAN2,
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
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
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
Download Date | 1/24/18 1:45 AM
68 Z.A. Kharazian et al.
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.
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-
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 (
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
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
Table 1. Design of the greenhouse experiment
Bacteria (Antagonists)
F. verticil-
L. plantarum E2,
L. pentosus E4,
L. paralimentaris Q2,
L. fermentum Q4,
L. buchneri (sunkii) Q6
L. plantarum (E2),
L. pentosus E4,
L. paralimentaris Q2,
L. fermentum Q4,
L. buchneri (sunkii) Q6
L. buchneri (sunkii) Q6
F. verticil-
L. buchneri (sunkii) Q6
F. verticil-
Benomyl (Fungicide)
Benomyl (Fungicide)
F. verticil-
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).
Download Date | 1/24/18 1:45 AM
70 Z.A. Kharazian et al.
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.
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-
Download Date | 1/24/18 1:45 AM
Lactobacillus strains as a biocontrol against Fusarium verticillioides 71
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.
Root length (cm)
Root weigt (mg)
Root Fresh Weight
Root Dry Weight
1 2 3 4 5 6 7 8
Shoot length (cm)
Shoot weight (mg)
Shoot Fresh Weight
Shoot Dry weight
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72 Z.A. Kharazian et al.
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.
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.
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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The present research was conducted to evaluate the marine algal extracts effectiveness on tomato Fusarium wilt disease. The organic extracts of macroalgae exhibited antagonistic activity against Fusarium oxysporum. The highest antifungal activity obtained from the methanolic extract of Cystoseira myrica followed by methanol extract of Sargassum cinereum. GC–mass analysis of some seaweed extracts was used to identify the presence of main compounds as dimethylocta-1,6-dien-3-ol,1-methoxy-4-(2-propenyl), hexadecanoic acid, methyl ester, and octadecanoic acid, and methyl ester. The infection of tomato plants with F. oxysporum induced a significant decrease in shoot and root dry weights as well as the photosynthetic pigments. There was a marked increase in soluble contents of saccharides and protein for infected tomato plant shoots and roots. On the other hand, pathogenicity stress induced a significant decrease in total contents of saccharides and protein of tomato shoots and roots. The results indicated a significant increase in total free amino acid content and antioxidant enzymes (CAT, POD, and APX) activities of inoculated tomato shoots and roots. The plant fresh and dry weights increased significantly by increasing its pigments content as a result of marine algal extracts application. On the other hand, algal extracts pretreatment decreased soluble saccharides and protein contents of plants, whereas increased significantly amino acid content in shoots of the inoculated plants. The increase in the activity of antioxidant enzymes played an essential role in increasing plant resistance against F. oxysporum. Finally, the marine macroalgae could serve as a new bioagent source for biological control of soil fungi.
This study describes the effects of lactic acid bacteria (LAB) on photosynthetic performance and antioxidant metabolism in Solanum lycopersicum L. (tomato) plants. Two strains of previously isolated and characterized LAB, Enterococcus sp. BB3 and Lactobacillus sp. BB6, were used alone or in combination as inoculants of tomato plants under controlled conditions in a 30-day assay. At the end of the assay, the physiological parameters of the plants were studied, including chlorophyll fluorescence, net photosynthesis, lipid peroxidation, antioxidant activity, and the content of photosynthetic pigments, total phenols, and flavonoids. The results show that, both individually and combined, these strains improve all physiological parameters in S. lycopersicum plants, where BB3 and BB6 alone increase photochemical features such as Fv′/Fm′, ФPSII, and ETR. Pn, gs, and E were higher in all three treatments with LAB. Meanwhile, oxidative damage, measured as lipid peroxidation, significantly decreased (P<0.05) in all bacterial treatments, especially in the combined assay (BB3+BB6) in comparison with the control assays. Finally, a significant increase (P<0.05) in antioxidant activity was observed but not in total phenol and flavonoid content in tomato plants treated with bacteria. Conclusion. Our data suggest positive effects on physiological features in S. lycopersicum L. mainly related to the BB3 and BB6 strains of LAB and a reduction of oxidative damage and antioxidant metabolism, generally associated with combined treatments.
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The incidence of Fusarium head blight (FHB) in cereal grains such as barley and wheat is of growing concern due to climate change threatening food safety. Further processing of cereals by malting provides an ideal environment for the growth of Fusarium, leading to food safety concerns due to the production of mycotoxins, production challenges with the negative effects to malt and beer qualities, and economic loss owing to the field yield reduction. To improve food safety and product quality, different methods of fungal control have been investigated and reported in the literature. Traditional methods to control fungal growth and mycotoxin production have included chemical and physical methods, but these treatments led to worsened malt properties, limiting their applicability to the brewing industry. Biological control methods have, therefore, attracted wide interest as alternative treatments due to their ability to limit Fusarium growth and mycotoxin production in malting cereals without toxic by-products, thus exhibiting promise for improving food safety. Various biological agents have been investigated and applied in malting and have shown the potential to suppress Fusarium spp. growth and mycotoxin production. These agents include several lactic acid bacterial (LAB) species and Geotrichum candidum. Another promising biocontrol agent for malting control is Pythium oligandrum, which has successfully limited Fusarium infection in other agricultural crops. The review outlines the Fusarium-control methods reported referenced for the brewing industry and the present prospects in biological control applications on the promise of P. oligandrum as a novel agent for malting.
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Lactic acid bacteria (LAB), isolated from milk and yoghurt, were tested for their efficacy against some phytopathogenic fungi under in vitro and in vivo tests. Fusarium oxysporum, one of most important pathogenic fungi invade tomato plants, was chosen to evaluate the effectiveness of LAB as a biocontrol agent under in vivo tests. Culture broth of LAB was applied as seed treatment or soil drench. The protective effect of LAB significantly increased after challenging inoculation by F. oxysporem, especially when LAB were applied as seed treatment; the number of roots increased by 216, 311, and 358% over control with LB-1, LB-4, and LB-5, respectively, whereas the increment was 169, 163, and 181% for soil drench. Interestingly, when LAB were applied as seed treatment, in soil infested with F.oxysporum, the total fresh weight of tomato plants increased by 348, 260, and 390% with LB-1, LB-3, LB-5, respectively, whereas the increment was 268, 427, and 393% with LB-1, LB-4, and LB-5, respectively, for soil drench. Overall, while previous reports of antifungal activity by LAB under in vitro tests are scarce, we have demonstrated for the first time the capability of LAB to act as plant growth promoting bacteria and biocontrol agent against some phytopathogenic fungi under in vivo tests.
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The antifungal activity of Lactobacillus plantarum was evaluated in vitro using Penicillium roqueforti as the indicator fungus and investigated the preservative effect of Chinese steamed bread. The screening of antifungal activity was assayed by double-layer plate point inoculation, mycelial growth rate inhibition, conidial germination assays and agar diffusion, L. plantarum CCFM259 (CCFM259) exhibited good inhibition of P. roqueforti mycelial growth and conidial germination, with inhibition rates were 45.3 and 18.0%, respectively. The water/salt-soluble extract of CCFM259 was partially purified and exhibited the properties of strong antifungal activity and good thermal stability, and kinds of organic acids were identified. Agar diffusion assays results illustrated that acetic and phenllactic acids showed the most significant inhibition, and the mixture of acids also exhibited a synergistic effect. The Chinese steamed bread manufactured with CCFM259 possessed desirable sensorial characteristics and did not show any contamination by fungi until 7 days of storage, a similar level of inhibition comparable to that afforded by 0.25% (w/w) calcium propionate. Interest in the application of sourdough for the steamed bread is growing constantly in China, mainly due to the improvement of quality requirements. Type II sourdough as an industrial culture starter is generally not suitable for achieving dough leavening but are used for dough acidification, and as dough improvers. These sourdoughs are generally fermented by the selection of microbial strains with a long fermentation time, high dough yield and temperature of fermentation in order to shorten the fermentation process. The main objectives of this study were to evaluate the antifungal activity of Lactobacillus plantarum CCFM259 in vitro using Penicillium roqueforti as the indicator fungus, to develop a natural improver of dough and to extend the shelf life and sensory characteristics of steamed bread.
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Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.
Nitro blue tetrazolium has been used to intercept O2⁻ generated enzymically or photochemically. The reduction of NBT by O2⁻ has been utilized as the basis of assays for superoxide dismutase, which exposes its presence by inhibiting the reduction of NBT. Superoxide dismutase could thus be assayed either in crude extracts or in purified protein fractions. The assays described are sensitive to ng/ml levels of super-oxide dismutase and were applicable in free solution or on polyacrylamide gels. The staining procedure for localizing superoxide dismutase on polyacrylamide electrophoretograms has been applied to extracts obtained from a variety of sources. E. coli has been found to contain two superoxide dismutases whereas bovine heart, brain, lung, and erthrocytes contain only one.
Fungal infection represents a severe problem that decreases the yield and market value of fruit crops. The use of fungicides is a conventional method to control infections but it is associated with disadvantages such as hazardous impact on public health, environmental contamination, resistance development among pathogens and high cost of agrochemicals. Biological control is an alternative approach for the treatment of fungal infections. The species of Bacillus, Pseudomonas, Enterobacter, Pantoea, Burkholderia, Lysobacter and Serratia have been successfully used in the control of fungal infections. The mechanisms involved in biocontrol are hyperparasitism or predation, production of antibiotics, lytic enzymes and induction of host resistance. Lactic acid bacteria have been used as biopreservative organisms in food and feed systems. They are a cluster of Gram-positive bacteria and include, for example, species of the genera Enterococcus, Lactobacillus, Leuconostoc, Lactococcus and Pediococcus. The ability to produce several antibacterial and antifungal substances confers biopreservation potential to lactic acid bacteria. Many have ‘generally regarded as safe’ (GRAS) status and are considered as safe both from human and environmental point of view. Their isolation is reported from vegetables, aerial plant surfaces, pickled cabbage, grass silage, malted cereals and also from soil. They produce antifungal substances such as cyclic dipeptides, proteinaceous compounds, organic acids, fatty acids and reuterin. The biocontrol potential of lactic acid bacteria is demonstrated in the prevention of fungal infections of fruits such as apples and grapes. Thus, living cells or product formulations of antifungal lactic acid bacteria may be prepared and used as an alternative biocontrol technology.
Cereal-based fermented products are worldwide diffused staple food resources and cereal-based beverages represent a promising innovative field in the food market. Contamination and development of spoilage filamentous fungi can result in loss of cereal-based food products and it is a critical safety concern due to their potential ability to produce mycotoxins. Lactic Acid Bacteria (LAB) have been proposed as green strategy for the control of the moulds in the food industry due to their ability to produce antifungal metabolites. In this work, eighty-eight Lactobacillus plantarum strains were screened for their antifungal activity against Aspergillus niger, Aspergillus flavus, Fusarium culmorum, Penicillium roqueforti, Penicillium expansum, Penicillium chrysogenum, and Cladosporium spp. The overlayed method was used for a preliminary discrimination of the strains as no, mild and strong inhibitors. L. plantarum isolates that displayed broad antifungal spectrum activity were further screened based on the antifungal properties of their cell-free supernatant (CFS). CFSs from L. plantarum UFG 108 and L. plantarum UFG 121, in reason of their antifungal potential, were characterized and analyzed by HPLC. Results indicated that lactic acid was produced at high concentration during the growth phase, suggesting that this metabolic aptitude, associated with the low pH, contributed to explain the highlighted antifungal phenotype. Production of phenyllactic acid was also observed. Finally, a new oat-based beverage was obtained by fermentation with the strongest antifungal strain L. plantarum UFG 121. This product was submitted or not to a thermal stabilization and artificially contaminated with F. culmorum. Samples containingL. plantarum UFG121 showed the best biopreservative effects, since that no differences were observed in terms of some qualitative features between not or contaminated samples with F. culmorum. Here we demonstrate, for the first time, the suitability of LAB strains for the fermentation and antifungal biopreservation of cereal products.
ABSTRACT: Fusarium graminearum is an important disease globally that causes serious damages on crops particularly on wheat. Currently, the mechanisms underlying resistance to Fusarium head blight (FHB) are still unknown. To understand the host response to challenge by F.graminearum, we examined enzymatic activities in the wheat spikelet following inoculation with F.graminearum macroconidia. Greenhouse grown of two wheat cultivars Falat (susceptible) and Sumai3 (resistant) were inoculated by F.graminearum macronidia at the anthesis. Spikelets were harvested at 3, 5 and 7 days post inoculation (dpi). According to our results FHB infection in Sumi3 lead to significant increase of H2O2 and MDA contents as Falat but this induction was earlier in Sumi3. It seemed that increase in hydrogen peroxide in resistant genotypes was due to rapid induction of SOD (Superoxide dismutase) and decrease of CAT (Catalase) activity. Our results also indicated that POX (Peroxidase) and PPO (Polyphenol oxidase) activities were significantly increased in the resistant cultivar at 3 dpi, whereas increased activity of these enzymes was later in Falat at 5 and 7dpi. According to these results we suggest that rapid and more significant induction of antioxidative systems is the critical trait of wheat genotypes to resist FHB infection.