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AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
80
Original scientific paper
10.7251/AGRENG1803080A
UDC 582.28:579.264
ISOLATION, CHARACTERIZATION AND FORMULATION OF
ANTAGONISTIC BACTERIA AGAINST FUNGAL PLANT
PATHOGENS
Natalija ATANASOVA-PANCEVSKA*, Dzoko KUNGULOVSKI
Department of Microbiology and Microbial Biotechnology, Institute of Biology, Faculty of
Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Skopje,
Macedonia
*Corresponding author: natalijaap@gmail.com
ABSTRACT
Concerns regarding food safety and the environment have led to reduced use of
agrochemicals and the development of sustainable agriculture. In this context,
biological control of fungal plant pathogens can improve global food availability,
one of the three pillars of food security, by reducing crop losses, particularly for
low-income farmers. Antagonistic bacteria are common soil inhabitants with
potential to be developed into biofungicides for the management of fungal plant
pathogens. In this study, antagonistic bacterium was isolated from the commercial
compost from a Resen factory for compost and screened for its growth inhibition of
fungal pathogens in laboratory tests. The zone of inhibition (mm) was recorded by
measuring the distance between the edges of the growing mycelium and the
antagonistic bacterium. Five replications were maintained for each isolate. Based
on phenotypic characteristics, biochemical tests, and sequence analysis of 16S
rRNA, the antagonistic bacterium was identified as Paenibacillus alvei (strain DZ-
3). The bacterium suppressed the growth of all five tested fungal plant pathogens
(Fusarium oxysporum, Rhizoctonia solani,Alternaria alternata,Botrytis cinerea
and Plasmopara viticola) in in vitro conditions over. The survival of antagonistic
bacterium in peat and talc formulations decreased time at room temperature, but
the populations remained above 108CFU/g during the 180-day storage period. This
study suggests that this bacterium can be developed and formulated as
biofungicides for minimizing the crop losses caused by fungal plant pathogens and
diseases caused by them.
Keywords: biocontrol, fungal plant pathogens, biofungicides, antagonistic
bacteria.
INTRODUCTION
As agriculture struggles to support the rapidly growing global population, plant
disease reduces the production and quality of food, fibre and biofuel crops. Farmers
spend billions of dollars on disease management, often without adequate technical
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
81
support, resulting in poor disease control, pollution and harmful results. In
addition, plant disease can devastate natural ecosystems, compounding
environmental problems caused by habitat loss and poor land management. Disease
losses can mean that communities become dependent on imported foods, often
replacing a balanced diet with processed foods that create further health problems.
A variety of fungi are known to cause important plant diseases, resulting in a
significant lost in agricultural crops. Fungal plant diseases are considered the most
important microbial agents causing serious losses in the agriculture annually
(Agrios, 1988). Plant diseases caused by a variety of fungi may cause significant
losses on agricultural crops. All plants are attacked by several pathogenic fungi.
Each pathogenic fungi can attack one or more plants. More than 10,000 species of
fungi can cause disease in plants (Agrios, 2005).
The plant diseases need to be controlled to maintain the level of yield both
quantitatively and qualitatively. Farmers often rely heavily on the use of synthetic
fungicides to control the plant diseases. However, the environmental problems
caused by excessive use and misuse of synthetic fungicide have led to considerable
changes in people’s attitudes towards the use of synthetic pesticides in agriculture.
Today, there is an increased awareness about the healthy food and healthy
environment. In response to this, some researchers have focused their efforts on the
development of plant disease control methods alternative to the use of synthetic
chemicals, such as biological control using microbial antagonists. Many microbial
antagonists have been reported to possess antagonistic activities against plant
fungal pathogens, such as Pseudomonas fluorescens,Agrobacterium radiobacter,
Bacillus subtilis, B. cereus,B. amyloliquefaciens,Trichoderma virens,
Burkholderia cepacia,Saccharomyces sp., Gliocladium sp. (Suprapta, 2012; Pal
and Garderner, 2006). Biological control of plant diseases has been considered a
viable alternative method to manage plant diseases (Cook, 1993). Biocontrol is
environmentally safe and in some cases the only available option to protect plants
against pathogens (Cook, 1993). Biological control employs natural antagonists of
pathogens to eradicate or control their population. In broad terms, biological
control is the suppression of damaging activities of one organism by one or more
other organisms, often referred to as natural antagonists.
In recent years, research has lead to the development of a small commercial sector
which produces a number of biocontrol products. The market share of biopesticides
of the total pesticide market is less than three percent. However, significant
expansion is expected the upcoming decades due to the increased demand for
organic food, and safer pesticides in agriculture and forestry.
Biological control agents are generally formulated as wetable powders, dusts,
granules and aqueous or oil-based liquid products using different mineral, organic
or inert carriers (Ardakani et al., 2009). Despite of a lot of research on biological
control of plant diseases, the number of available products is limited and their
market share is marginal. The market for biological control products is not only
determined by agricultural aspects such as the number of diseases controlled by
one biocontrol product in different crops but also by economic aspects as cost-
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
82
effective mass production, easy registration and the availability of competitive
means of control including fungicides. The future development of low-chemical
input sustainable agriculture and organic farming will determine the eventual role
of biological control in agriculture.
The paper describes the method of isolation, characterization, biocontrol potential
and formulation of antagonistic bacteria against several fungal plant pathogens.
MATERIAL AND METHODS
Origin of the bacteria- Isolation of potentially antagonistic microorganisms
Fifty grams of compost from the composting plant in Resen, Macedonia, were
taken and added to 250 ml sterile distilled water in a 500 ml Erlenmeyer flask. The
flask was shaken on an orbital shaker for 30 min at 27°C and serial dilutions from
10-1 to 10-6 were performed. From each dilution, 0.5 ml of sample was taken and
placed on Muller Hinton agar (MHA) medium along with antimycotic
cycloheximide (5 g mL-1) using pour plate technique and incubated at 27°C for 1
week. After the incubation period, the plates were observed for microbial colonies
which had formed a clear zone of inhibition. The colony with the greatest zone of
inhibition was selected and picked up by a sterilized wire loop and sub-cultured on
MHA to obtain pure bacterial colonies. The pure culture was preserved on agar
slants of Muller Hinton medium for further studies.
Molecular characterization of antagonistic agents
The phenotypic properties of the selected strain wase determined using the
methods described in Bergey’s Manual of Determinative Bacteriology (Holt et al.,
1994). The selected antimicrobial strain wase identified by sequencing of the 16S
rRNA gene. First, DNA from each strain was isolated. Pure colony was grown
overnight in the appropriate medium, cells were harvested by centrifugation (14000
rpm, 10 min), washed twice with 1xPBS buffer (140 mM NaCl, 2.7 mM KCl, 100
mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) and kept at -20oC until further
processing. DNA extraction was done using PrepManUltra reagent (Applied
Biosystems), following the protocol for culture broth samples. The concentration of
DNA was determined spectrophotometrically. DNA working solution of 2.7 – 3.1
ng/µl was prepared by diluting the stock DNA. The sequence of the 16S ribosomal
RNA gene (rDNA) of bacterial strains was determined using MicroSeq Full Gene
Kit (Applied Biosystems), composed of two parts: MicroSeq® Full Gene 16S
rDNA Bacterial Identification PCR Kit and MicroSeq® Full Gene 16S rDNA
Bacterial Identification Sequencing Kit. Amplification of the three fragments of the
16S ribosomal RNA gene was done using 7.5 µl DNA working solution in a
reaction volume of 15 µl on 2720 Thermal Cycler (Applied Biosystems).
Purification of the amplified products was done using ExoSAP-IT® reagent (USB)
according to the manufacturer’s instructions prior to sequencing. The cycle
sequencing was performed with forward and reverse primers for each amplified
product according to the instructions provided by the kit with one exception: the
final volume of the sequencing reactions was 10 µl. After cycle sequencing, excess
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
83
dye terminators and primers were removed from the cycle sequencing reactions by
precipitation in separate tubes with 2 µl 5M Na-acetate and 50 µl ethanol. After
incubation at room temperature for 30 min, the tubes were centrifuged at 14000
rpm for 30 min, the supernatant was discarded, the precipitate was dried for 5 min
at room temperature and re-suspended in 20 µl of Hi-Di™ Formamide. Sequence
analyses were performed on a 3500 Genetic Analyzer (Applied Biosystems).
Plant pathogens
Phytopathogenic strains (Botrytis cinerea FNS- FCC 23, Fusarium oxysporum
FNS- FCC 103, Plasmopara viticola FNS- FCC 65, Alternaria alternata FNS-
FCC 624, Rhizoctonia solani FNS- FCC 218) were supplied by the Culture
Collection of the Department of Microbiology and Microbial Biotechnology,
Faculty of Natural Sciences and Mathematics, Skopje, Macedonia. Fungal cultures
of phytopathogenic strains were kept on Sabouraud Dextrose Agar (SDA) at 4°C.
Disc diffusion method
Disc diffusion method was used to check the antifungal properties of the isolated
bacterial strain against selected fungal pathogens. Petri plates containing equal
volumes of MHA and SDA (7.5+7.5 ml) medium were inoculated with a
standardized bacterial isolate. A filter disc containing 20 μl of bacterial suspension
was placed on a Petri plate pre-seeded with the fungal pathogen. The plates were
initially kept at 4°C for 2h to allow the diffusion of the isolate, and later incubated
at 28 ± 1°C. The zones of inhibition were measured after five days of incubation
and the mean values were calculated. Five replications were maintained for each
isolate. The zone of inhibition between pathogen and the bacterial isolate was rated
as significant (+++) if the inhibition zone was >10mm wide, moderate (++) if the
zone of inhibition was 2 to 10 mm wide, and poor (+) if it was <2 mm wide.
Antagonistic activity of isolated bacterium against phytopathogenic fungi
The suppressive effect and antagonistic activity of isolated bacterium against
phytopathogenic fungi was demonstrated using the technique of Landa et al.
(1997). Growth inhibition was expressed as the ratio of the radius of hyphal growth
relative to the radius of growth on a control plate without antagonist. Values were
conveniently corrected so they could be expressed in a scale from 0 (no inhibition)
to 1 (maximum inhibition).
Development of talc and peat formulations of antagonistic bacteria
The formulations of selected bacterial isolate DZ-3 was prepared in talc powder
and irradiated peat. The MHB broth was inoculated with a loopful of bacterium,
and the flask was incubated on a rotary shaker at 150 r/min for 72 h at room
temperature (24±2°C). The broth containing 8×108colony-forming units
(CFU)/mL, determined spectrophotometrically and by dilution plating on MHB
plates, was used for the preparation of talc and peat formulation. The talc
formulation was prepared with sterilized talc powder following the method
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
84
described by Vidhyasekaran and Muthamilan (1995). To 400 mL of MHB broth,
the following were added under sterile conditions: 1 kg of talc powder sterilized at
105°C for 12 h, 15 g of calcium carbonate to adjust the pH to neutral, and 10 g
carboxymethyl cellulose (CMC) as adhesive. The peat formulation was prepared
with sterile irradiated peat. To 70 mL of MHB broth, 120 g of irradiated peat and 5
mL of bacterial culture were added under sterile conditions. The formulated
products were air-dried in a laminar flow hood to a workable (15%–20%) moisture
level and kept in polyethylene bags and used for the treatments immediately or as
needed. The population of bacteria was around 2.5×108CFU/g in both talc and peat
formulations at the time of application.
Shelf life of formulated antagonistic bacteria
The shelf life of the products stored at room temperature (24±2°C) for 6 months
was studied by monitoring the viability of antagonistic bacterium in peat and talc
formulations by a serial dilution technique. One gram of the sample drawn from
each formulation periodically at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
150, and 180 days of storage period was mixed with 9 mL of sterile distilled water
(SDW). From this, serial dilutions were made. A 1 mL aliquot of each dilution was
pipetted out into sterilized Petri plates, and 15 mL of MHB was added and
incubated at room temperature. The bacterial colonies were counted 3 days after
plating and expressed as the number of CFU per gram of peat or talc formulation
(Vidhyasekaran and Muthamilan 1995).
RESULTS AND DISCUSSION
Biological control of soil borne pathogens by introduced microorganisms has been
studied over 80 years, but most of the time it has not been considered commercially
feasible. However interest and research in this area increased steadily. There is a
shift toward the important role of biological control in agriculture in the future.
Several companies now have programs to develop biocontrol agents as commercial
products. Morphological studies showed that the isolate with the greatest zone of
inhibition was Gram-positive, sporulating, rod shaped bacterium. Alignment of the
16S rRNA sequences of the bacterial species revealed identity of 99% to the genus
Bacillus. Isolate DZ-3 was identified as Paenibacillus alvei. Inoculated on MHA,
P. alvei produced large, circular, rough, white-yellowish colonies with irregular
margins. The spores of P. alvei are smooth, spherical and green in color using the
Schaeffer and Fulton staining method. Paenibacillus alvei are Gram-positive, rod-
shaped, motile, spore-forming and catalase-positive bacteria (Najafi et al., 2011).
The first report of antimicrobial peptide production by these bacteria was by
Anandaraj et al., 2009, who isolated a strain from fermented tomato fruit and
detected two antimicrobial peptides, Paenibacillin P and Paenibacillin N. The
isolated bacterial strain from compost was screened for secondary metabolites with
antimicrobial activity by diffusion agar method. Paenibacillus alvei DZ-3 showed
potential antifungal activity against all tested fungi, with the highest zones of
Alternaria alternata FNS- FCC 624 (Table 1).
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
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Table 1. Growth inhibition of Paenibacillus alvei DZ-3 on tested phytopathogenic
fungi with disc diffusion method.
(Five replications were maintained for each fungus; the mean values were
calculated).
phytopathogenic
fungus
inhibition zone (mm)
Paenibacillus
alvei DZ-3
+ positive
control (0.5
gL-1
nystatine)
+ positive
control (0.5 gL-
1
cycloheximide)
- negative
control
(sd H2О)
Botrytis cinerea FNS-
FCC 23
11.3
5.3
7.3
0
Fusarium oxysporum
FNS- FCC 103
12.1
5.4
8.7
0
Plasmopara viticola
FNS- FCC 65
14.6
6.5
6.1
0
Alternaria alternata
FNS- FCC 624
22.7
6.9
7.6
0
Rhizoctonia solani
FNS- FCC 218
19.2
5.4
5.6
0
significant activity (+++) (inhibition zone > 10 mm)
moderate activity (++) ( inhibition zone 2–10 mm)
poor activity (+) (inhibition zone <2 mm)
There are many different types of Gram positive and Gram negative bacteria (such
as Bacillus spp. and Pseudomonas spp.) exhibiting antifungal activities especially
toward different phytopathogenic fungi (Kobayashi et al., 2000; Gupta et al.,
2001). In this group we can add our antifungal isolate Paenibacillus alvei DZ-3,
who showed a wide range of antifungal activities toward phytopathogenic fungi.
Isolates of Pseudomonas were evaluated for antifungal activity against five fungal
plant pathogens, i.e. Fusarium oxysporum,Aspergillus niger,Aspergillus flavus,
Alternaria alternata and Erysiphe cruciferarum (Singh et al., 2011). All tested
fungal strains showed significant reduction in terms of radial diameter after the
treatment with Pseudomonas cultures, in comparison with the controls. Out of the
five fungal pathogens studied, Fusarium oxysporum showed maximum extent of
inhibition (% control inhibition = 51.76%) followed by Aspergillus niger (50.14%),
and least by Erysiphe cruciferarum (22.27%). The antagonistic effect of
Pseudomonas might be explained on the basis of its antifungal secondary
metabolites that are capable of lysing chitin which is the most important
component of fungal cell wall (Singh et al., 2011).
Biological control of plant diseases is a result of many different types of interaction
among microorganisms and can occur through different mechanisms, which are
generally classified as: parasitism/predation, antibiosis, competition, lytic enzymes,
and induced resistance (Pal and Gardener, 2006). The most effective biocontrol
active microorganisms studied appear to antagonize plant pathogen employing
several modes of actions. For example, Pseudomonas known to produce the
antibiotic 2,4-diacetylphloroglucinol (DAPG) may also induce host defenses.
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Since inhibition indexes directly obtained from antagonist-phytopathogen
confrontations came in different scales, they were appropriately corrected so they
ranged from 0 (no pathogen inhibition) to 1 (maximum pathogen inhibition) in
order to facilitate comparisons. Inhibition indexes varied widely and showed
indexes from 0.12458, for Botrytis cinerea FNS- FCC 23 to 0.94513 for Alternaria
alternata FNS- FCC 624 (Table 2). According to these we can conclude that the
results corresponds with that from disc diffusion method.
Table 2. Inhibition indexes obtained from antagonist-phytopathogen
confrontations.
(Five replications were maintained for each fungus; the mean values were
calculated).
phytopathogenic fungus
Paenibacillus alvei DZ-3
inhibition index*
corrected inhibition
index
Botrytis cinerea FNS- FCC 23
0.12458
0.2
Fusarium oxysporum FNS- FCC
103
0.26936
0.4
Plasmopara viticola FNS- FCC 65
0.48378
0.8
Alternaria alternata FNS- FCC 624
0.94513
1.0
Rhizoctonia solani FNS- FCC 218
0.86923
0.6
* ratio of the radius of hyphal growth relative to the radius of growth on a control plate
without antagonist
0- no pathogen inhibition
1- maximum pathogen inhibition
The initial bacterial isolate Paenibacillus alvei DZ-3 were higher in irradiated peat
than in talc powder formulation, although both products were inoculated with the
same bacterial concentrations. These population densities declined over time in
both formulations during the 180 days of storage at room temperature but remained
above 108CFU/g (Figure 1). In peat formulation, the viability of the bacterial
isolate during the first 60 days of storage did not decline significantly compared
with their respective initial populations of 9.4 log CFU/g at day 0 (Figure 1). The
populations of antagonistic bacterium dropped to 8.72 log CFU/g after 60 days (in
talk powder formulation) and to 9.1 log CFU/g (in peat formulation).
AGROFOR International Journal, Vol. 3, Issue No. 3, 2018
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Figure 1. Population densities of antagonistic bacteria, Paenibacillus alvei DZ-3 in
irradiated peat formulation and talc powder formulation during 180 days of storage
at room temperature (24±2°C).
The main focus of the study was the performance of antifungal activity and storage
stability of Paenibacillus alvei DZ-3, which were superior in in vitro conditions. In
terms of the formulation for showing the storage stability it was clear that both,
irradiated peat formulation and talc powder, formulation were suitable. The
survival of antagonistic bacterium in peat and talc formulations decreased over
time at room temperature, but the populations remained still above 108CFU/g
during the 180-day storage period on room temperature.
Today, the market share of biocontrol formulations is increasing and it occupies
1% of the overall pesticide sales. Montesinos (2003) and Fravel (2005) have drawn
up lists of biocontrol products and strains registered by the United States
Environmental Protection Agency (USEPA) and the European Protection Agency
(EPA). These strains mainly belong to Bacillus and Pseudomonas bacterial genera
and Aspergillus and Trichoderma fungal genera. Microbial pesticides are seen as a
tool for developing a more rational pesticide use strategy and future products
should have improved balance between efficiency and cost (El-Said, 2005; Rao et
al., 2007; Glare et al., 2012; Khater, 2012).
Additional studies on the mechanism(s) of action of newly discovered antagonist
against the tested phytopathogenic fungi are necessary to fully understand the
potential beneficial role of Paenibacillus alvei DZ-3. In addition, field
experiments are needed, particularly in regard to season long control of
phytopathogenic fungi. Generally, the cost and complexity of studies for the
registration of microbial pesticides is a barrier to the transfer of laboratory
knowledge to the commercialization of these substances.
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CONCLUSION
In order to have more effective biological control strategies in the future, it is
crucial to carry out further research on certain less developed aspects of biocontrol,
including development of novel formulations, understanding the impact of
environmental factors on biocontrol agents, mass production of biocontrol
microorganisms and the use of biotechnology and nano-technology in
improvement of biocontrol mechanisms and strategies. Biocontrol of plant diseases
has a bright and promising future, due to the growing demand for biocontrol
products by the farmers. In addition, it is possible to use biological control as an
effective strategy to manage plant diseases, increase yield, protect the environment
and biological resources, and establish a sustainable agricultural system.
The study suggests that Paenibacillus alvei can be developed and formulated as
biofungicide for minimizing crop losses and diseases caused by fungal plant
pathogens .
The challenge is to develop a formulation and application method which can be
implemented on a commercial scale. It must be effective, reliable, consistent,
economically feasible, and with a wider spectrum. Continuous laboratory research
followed by field experiments are needed to develop excellent biocontrol agents,
particularly against plant fungal pathogens.
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