Open Agriculture.
2018; 3: 264–272
Research Article
Irda Safni*, Widya Antastia
In vitro antagonism of five rhizobacterial species
against Athelia rolfsii collar rot disease in soybean
https://doi.org/10.1515/opag-2018-0028
received September 26, 2017; accepted June 26, 2018
Abstract: Plant Growth Promoting Rhizobacteria
(PGPR) influence plant growth by a number of direct
(producing plant growth promoting substances) and
indirect (through prevention of deleterious effects of
phytopathogenic microorganisms) mechanisms. Five
species of bacteria were isolated from rhizospheric soils of
soybean and peanut fields from several locations in North
Sumatra. On the basis of morphological and biochemical
characteristics, the bacteria were identified as Aeromonas
hydrophila, Burkholderia cepacia, Serratia ficaria, Pantoea
spp. 2, and Vibrio alginolyticus. These species were tested
in vitro against the causal pathogen of collar rot disease
of soybean, Athelia rolfsii, which is an important soybean
disease in Indonesia. The five species of bacteria were
subjected to screening of antagonistic activities against
A. rolfsii in vitro with a dual culture-technique. Of the
five species, B. cepacia, S. ficaria and V. alginolyticus
were the most effective antagonistic bacteria to control A.
rolfsii. B. cepacia, S. ficaria and V. algynolitycus produced
inhibiting zones against A. rolfsii of 98.35%, 97.83% and
96.97% respectively. All bacterial species showed their
antagonistic activity significantly with the inhibiting
zone percentage being more than 60%. The experimental
results suggested that all bacterial species have a future
potency as a biocontrol agent to reduce A. rolfsii collar rot
disease of soybean.
Keywords: plant growth promoting rhizobacteria,
Aeromonas hydrophila, Burkholderia cepacia, Serratia
ficaria, Pantoea sp 2, Vibrio alginolyticus
*Corresponding author: Irda Safni, Department of Agrotechnology,
Faculty of Agriculture, Universitas Sumatera Utara, Medan, 20155,
Indonesia, E-mail: irda@usu.ac.id
Widya Antastia, Department of Agrotechnology, Faculty of
Agriculture, Universitas Sumatera Utara, Medan, 20155, Indonesia
Open Access. © 2018 Irda Safni, Widya Antastia, published by De Gruyter.
NonCommercial-NoDerivs 4.0 License.
1 Introduction
Soybean (Glycine max (L.) Merril) is one of the important
legume crops worldwide including in Indonesia. National
demand for soybean increases 18% annually accordance
with domestic population growth, however the soybean
production is quite low (Direktorat Pangan dan pertanian
2015, Ratulangi 2004).
Soybean suffers considerable damage from collar
rot disease incited by Athelia rolfsii (Curzi) (formerly
Sclerotium rolfsii). A. rolfsii is a soil borne fungal pathogen
which has a wide host range including rice, mungbean,
peanut, soybean, sweet potato, banana, wheat and potato
(Agrios 1997). A. rolfsii was first reported as a tomato
blight pathogen in Florida, USA by Rolfs (1892). Saccardo
(1911) named the fungal pathogen as Sclerotium rolfsii,
which has had taxonomic revision and transferred into A.
rolfsii (Tu and Kimbrought 1978).
In Indonesia the yield losses due to collar rot disease
affecting soybean and other legumes reach 5-55% in dry
land or wet land (Nautiyal 2002, Wahyuningsih 2005).
The disease intensity in the field is mostly more than 5%,
which has an impact on the yields economically because it
causes yield reduction or failure to harvest (Wahyuningsih
2005). The infection occurs in the early plant growth,
showing seedling rot or damping-off of seedlings. In
the older plant (2-3 weeks old), rot symptom occurs in
the collar part of the plant with brown spots and white
mycelia on the infected area (Semangun 2000).
A. rolfsii can be managed by fungicide application,
soil solarization, crop rotation and the use of antagonistic
microorganisms (Punja 1988). To avoid the negative impact
of fungicide residue, more enviromental friendly methods
such as biological controls have been applied recently
(Hardaningsih 2011, Rahayu 2008). These include, the use
of beneficial fungi and bacteria (Suryanto 2009). Plant
Growth Promoting Rhizobacteria (PGPR), which are a
group of beneficial bacteria, colonizes rhizosphere, have
been shown to have potential to improve plant growth as
well as to give plant protection against pathogens direct
or indirect effects (Saharan and Nehra 2011). This research
This work is licensed under the Creative Commons Attribution-
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 In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean
was aimed to test the antagonistic effect of rhizobacteria
isolated from legumes in North Sumatra against A. rolfsii
in vitro.
2 Methods
2.1 Sample collection
Soil samples of soybean’s rhizosphere were collected from
districts of Tanjung Selamat, Lubuk Pakam and Binjai,
North Sumatera, Indonesia, while soil samples of peanut’s
rhizosphere were collected from districts of Tanjung
Selamat, Medan Johor and Binjai, North Sumatera. Soil
samples were collected on the basis of random sampling
from 5 points from a depth of 5-25 cm between healthy
plants and infected plants. The soils were composited in
one plastic bag before being brought to the laboratory.
2.2 Bacterial isolation
Ten grams of soil was mixed with 90 ml of sterile water for
15 min. A dilution series of 10-5 was conducted. The soil
suspension was streaked onto potato dextrose agar (PDA)
media, which was used to grow both phytopathogenic
fungi A. rolfsii and the bacteria, and incubated for 48
hours at 28°C. The bacteria grew very well on the PDA
media. The homogenous bacterial colonies based on the
colony morphology were separated and re-cultured until
pure cultures were obtained.
2.3 Bacterial identification
The bacterial strains were identified on the basis of
morphological and biochemical assays for several general
assays to observe the morphological characteristics and
biochemical reactions of the bacteria and PGPR screening
assays for determining which strains had potential as
PGPR.
The morphological assays included colony and cell
morphologies, while the biochemical assays included
Gram staining, catalase, oxidase (Schaad et al. 2001), and
physiological assays comprised of growth on different pH
values (5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) (Woyesa and Assefa
2011), and utilizing an API® 20E Microbial Identification
kit (Biomerieux). The growth at different pH values was
conducted for the five selected strains.
The PGPR screening assays included indole acetate
acid (IAA) production (Widayanti 2007), phosphate
 265
solubilization (Widayanti 2007), Hydrogen Cyanide (HCN)
production (Agustiansyah et al. 2013) and siderophore
production where the absorbance values were measured
by spectrophotometer (Dirmawati 2003).
The biochemical assays and the PGPR screening
assays were replicated three times.
2.4 In vitro screening for antagonism
Five selected bbacterial isolates, which were screened
based on a PGPR assay in vitro for growth inhibition of
the phytopathogenic fungus A. rolfsii. Briefly, 5 μl drops
of each bacterial culture (108 CFU/ml) were streaked
equidistantly on the margins of PDA plates. A mycelial
agar plug of 5 mm diameter from a 7-day-old culture of A.
rolfsii was grown on PDA plates next to the streak of the
test bacterium. Control plates not inoculated with bacteria
were also prepared. Two independent experiments with
each bacterial isolate were performed and each experiment
was replicated three times. Plates were incubated at 28°C
for 2 days. Antagonistic activity was assessed by relating
mycelia diameter on plates inoculated with bacteria to
mycelia diameter on control plates and computing the
percentage of Growth Inhibition (GI %). The inhibition of
the mycelial growth of A. rolfsii was measured by using the
following formula:
where I = inhibition of mycelial growth of the pathogen
(%), C = growth of A. rolfsii in the control plate (mm) and T
= growth of the pathogen in plates challenged with PGPR
(mm) (Kumar et al., 2011)
2.5 Statistical Analysis
Statistical analysis was performed using SPSS Statistics
version 21.0.0 (International Business Machines
Corporation, USA). For multiple-group comparisons
among the antagonistic bacterial strains and
phytopathogenic fungi A. rolfsii, two-way ANOVA at P <
0.05 was performed. All data were expressed as means ±
SD (standard deviations).
Ethical approval: The conducted research is not related
to either human or animals use.
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266   I. Safni, W. Antastia

3 Results 3.2 Bacterial identification

Colony and cell Morphological characteristics of seventeen

3.1 Bacterial isolation
bacterial strains isolated from the rhizospheres of soybeans
and peanuts are displayed in Table 2. Colony shapes of the
A total of seventeen bacterial strains were isolated from
strains were mostly circular with the exception of strain
the rhizhosphere of soybeans and peanuts from five
PJ23 and PJ22, which had a filamentous shape. The colony
locations in North Sumatera, Indonesia (Table 1). Ten
colour, margin and elevation as well as cell shape did not
strains and seven strains were isolated from soybeans and
have much variation (Table 2). Most rhizospheric bacterial
peanuts respectively.

Table 1: Bacteria that were isolated from the rhizosphere of soybean and peanut
No Bacterial strains Source of Plant rhizhosphere Location

1 SYT51 Soybean Tanjung Selamat

2 SYT22 Soybean Tanjung Selamat
3 SYT21 Soybean Tanjung Selamat
4 SYP21 Soybean Lubuk Pakam
5 SYP42 Soybean Lubuk Pakam
6 SYP43 Soybean Lubuk Pakam
7 SYP31 Soybean Lubuk Pakam
8 SYB43 Soybean Binjai
9 SYB43 Soybean Binjai
10 SYB51 Soybean Binjai
11 PT5 Peanut Tanjung Selamat
12 PT22 Peanut Tanjung Selamat
13 PJ23 Peanut Medan Johor
14 PJ51 Peanut Medan Johor
15 PJ22 Peanut Medan Johor
16 PB3 Peanut Binjai
17 PB42 Peanut Binjai

Table 2: Morphological characteristics of bacterial strains isolated from the rhizosphere of soybean and peanut in North Sumatera,
Indonesia
Colony morphology Cell morphology

Strains Shape Colour Margin Elevation Cell shape

SYT51 Circular White Undulate Flat Short rods

SYT22 Circular White Entire Flat Short rods
SYT21 Circular Cream Entire Flat Coccus
SYP21 Circular White Undulate Flat Short rods
SYP42 Circular White Entire Flat Short rods
SYP43 Circular White Undulate Flat Short rods
SYP31 Circular White Entire Flat Long rods, in chain
SYB43 Circular Cream Entire Flat Short rods
SYB42 Circular White Entire Raised Short rods
SYB51 Circular Cream Entire Flat Coccus
PT5 Circular White Entire Flat Short rods
PT22 Circular White Entire Flat Short rods
PJ23 Filamentous White Filiform Raised Coccus
PJ51 Circular White Undulate Flat Coccus
PJ22 Filamentous White Filiform Flat Short rods
PB3 Circular White Entire Flat Short rods
PB42 Circular White Entire Raised Short rods

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 In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean  267

strains were rods and only four strains were cocci (strain
SYT21, SYB51, PJ23 and PJ51). Among the strains, strain
PJ22 isolated from peanut and strain SYP21 isolated from
soybean were Gram positive and five selected strains
could grow well on media with different pHs (Table 3).
The results of the PGPR screening assays are displayed
in Table 4. On the basis of a PGPR screening assay, 47%
of the isolates were able to produce IAA, 58.8% could
solubilize phosphate, none produced HCN and 100%
produced siderophores. Isolate SYP31 was able to produce
IAA in the media without tryptophan addition, while
other isolates did not produce IAA in the media without
tryptophan (Table 4).
On the basis of PGPR screening assay, 5 best isolates
were selected for further test, i.e. in vitro antagonism
against phytopathogenic A. rolfsii:
SYP31
SYB43
SYT51
PB3
PB42
3.3 In vitro screening for antagonism
In vitro screening antagonism of five PGPR species against
A. rolfsii showed that Burkholderia cepacia had the highest
growth inhibition against A. rolfsii on the second day after
inoculation (98.35%) (Table 5). Serratia ficari and Vibrio
alginolyticus also inhibited the growth of A. rolfsii with
high percentage values (97.83% and 96.97% respectively).
Aeromonas hydrophyla and Pantoea sp. 2 just inhibited
the growth of A. rolfsii by 64.83% and 63.13% respectively.
Figure 1 showed the inhibition of five PGPR isolates
against the growth of A. rolfsii on PDA media compared
to the control without the bacterial treatments. All
bacterial isolates interfered with the growth of A. rolfsii
because the fungal mycelium could not fill the petri dish.
On microscopic observation, various impacts of PGPR
isolates on hyphae of A. rolfsii including crooked hyphae
(A. hydrophyla and V. alginolyticus), shorten hyphae (B.
cepacia), and lysed hyphae (S. ficaria, Pantoea sp.2, and
V. alginolyticus) (Figure 2) were observed.

These five bacterial isolates were identified using an

4 Discussion and Conclusions
API® test kit, and the results were the following:
On the basis of production of indole acetic acid (IAA),
SYP31 : Aeromonas hydrophila
siderophore and solubilization of phosphate, the five
SYB43: Burkholderia cepacia
selected bacterial strains might have potential to be used
SYT51 : Serratia ficaria
as PGPR in the field. Bashan and de-Bashan (2005) stated
PB3 : Pantoea sp. 2
that mechanisms including N2 fixation, solubilization of
PB42: Vibrio alginolyticus

Table 3: Biochemical and physiological assays of bacterial strains isolated from the rhizosphere of soybean and peanut in North Sumatra
Strains Gram Oxidase Catalase Growth on different pH

5.0 6.0 7.0 8.0 9.0 10.0

SYT51 Negative + + + + + + + +
SYT22 Negative + + NA1 NA NA NA NA NA
SYT21 Negative + + NA NA NA NA NA NA
SYP21 Positive + + NA NA NA NA NA NA
SYP42 Negative + + NA NA NA NA NA NA
SYP43 Negative + + NA NA NA NA NA NA
SYP31 Negative + + + + + + + +
SYB43 Negative + + + + + + + +
SYB42 Negative + + NA NA NA NA NA NA
SYB51 Negative + + NA NA NA NA NA NA
PT5 Negative + + NA NA NA NA NA NA
PT22 Negative + + NA NA NA NA NA NA
PJ23 Negative + + NA NA NA NA NA NA
PJ51 Negative + + NA NA NA NA NA NA
PJ22 Positive + + NA NA NA NA NA NA
PB3 Negative + + + + + + + +
PB42 Negative + + + + + +

Note: 1NA=Not Available

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268   I. Safni, W. Antastia

Table 4: Production of indole acetate acid (IAA), HCN, siderophore and phosphate solubilization of rhizospheric bacterial strains
Strains IAA1 production Phosphat HCN2 production Siderophore production
Solubilization

LB3 + tryptophan LB without Phosphate Media + glisin Media without Media + FeCl3 Media without
tryptophan glisin FeCl3

SYT51 + - + - - 1.14 1.65

SYT22 - - + - - 1.99 2.17
SYT21 - - - - - 2.31 2.39
SYP21 - - + - - 2.26 2.31
SYP42 - - - - - 1.50 2.34
SYP43 - - - - - 0.88 1.19
SYP31 + + - - - 1.63 2.20
SYB43 + - + - - 0.61 2.28
SYB42 + - + - - 2.21 2.59
SYB51 - - - - - 1.17 1.17
PT5 - - - - - 0.51 1.78
PT22 + - + - - 0.51 2.04
PJ23 - - + - - 1.88 1.21
PJ51 - - + - - 0.86 2.27
PJ22 + - - - - 2.12 2.63
PB3 + - + - - 2.42 2.47
PB42 + - + - - 1.14 1.77
Note: 1IAA=Indole Acetic Acid; 2HCN=Hydrogen Cyanide; 3LB=Luria-Bertani

Table 5: In vitro screening antagonism of five PGPR species against Athelia rolfsii
Treatments Growth inhibition (%) after 2 days inoculation

I II III Total Mean

Aeromonas hydrophila 97.70 96.80 0.00 194.50 64.83ab

Burkholderia cepacia 98.10 99.2 98.60 197.70 98.35a

Serratia ficaria 97.70 98.40 97.40 293.50 97.83b

Pantoea sp. 2 91.00 98.40 0.00 185.40 63.13ab

Vibrio alginolyticus 97.70 98.40 94.80 290.90 96.97b

phosphate and zinc, sequestration of iron by siderophore
production, production of phytohormones such as Auxins,
cytokinins and gibberellins, production of the enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase
promote direct plant growth. However, none of the isolates
produced hydrogen cyanide. Hydrogen cyanide is toxic
to some bacteria, fungi, protozoa, mammals and plants,
but some bacteria utilize hydrogen cyanide as a source of
nitrogen for growth (Knowles 1988).
Bacteria Burkholderia cepacia, Serratia ficaria and
Vibrio alginolyticus are the best PGPR isolates based on in
vitro screening antagonisms, therefore they are considered
to have potential to control phytopathogen A.rolfsii in the
field.
Since the first decription of the genus Burkholderia
and species of B. cepacia, it has been revealed that this
species has great potential in biotechnology as it produces
a large number of commercially hydrolytic enzymes and
bioactive substances which are beneficial for plant growth
and health (Eberl and Vandamme 2016). B. cepacia is a
ubiquitous soil organism which has been effectively used
as a biocontrol agent against many plant pathogenic
fungi such as Colletotrichum gloeosporioides (de Los
Santos-Villalobos et al. 2012), Pythium-induced damping-
off, Aphanomyces-induced root rot of pea, Rhizoctonia-
induced root rot of Poinsettia and other fungal diseases
(Parke et al. 1991; King and Parke 1993; Cartwright
and Benson 1994; Fridlender et al. 1993). In the USA,
several strains of B. cepacia have been registered by The
United States Enviromental Protection Agency (EPA) as
biocontrol agents against plant pathogenic fungi (Eberl
and Vandamme 2016; Rai 2006). The ability of B. cepacia
to act as a biocontrol agent is due to its production of
various compounds with antifungal activity (Vial et al.

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 Vibrio alginolyticus 97.70 98.40 94.80 290.90 96.97b

In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean  269

a b c d e

Aeromonas hydrophila Burkholderia cepacia Serratia ficaria vs Pantoea sp2 vs Vibrio alginolyticus
vs A. rolfsii vs A.rolfsii A.rolfsii A.rolfsii vs A.rolfsii

A.rolfsii control

Figure 1: In vitro antagonism of Plant Growth Promoting Rhizobacteria isolates against Athelia rolfsii

2007; Schmidt et al. 2009). Additionally, B. cepacia is also chitinases which were evidenced as crucial factors which
known to produce antibiotics. Many studies confirmed degraded the cell wall of fungal hyphae (Ordentlich et al.
that the inhibitory activity against plant pathogens was 1988). Serratia marcescens also effective killed root-knot
associated with the production of several secondary nematode (Meloidogyne sp.) in vitro (Safni et al 2018)
metabolites including altericidins, cepacin, and others This study identified Serratia ficaria as the selected PGPR
(Kirinuki et al. 1977; Parker et al. 1984; EI-Banna and strains which inhibited growth of A. rolfsii in vitro. There
Winkelmann 1998). Although B. cepacia has been proven is no report finding S. ficaria and Vibrio alginolyticus as a
as a good biocontrol agent, some strains may pose a biocontrol agent. Grimont et al. (1979) isolated S. ficaria
risk to human health which cause severe infections in from fig trees, caprifigs and fig wasps in California and
cystic fibriosis (CF) and immunocompromised patients Tunisia and from a small black ant in France. Grimont
(Mahenthiralingam et al. 2005; Vandamme and Peeters and Grimont (2006) added that four percent of S. ficaria
2014; Peeters et al. 2013). Because of this reason, the US isolates was isolated from plants. Vibrio species are
withdrew the biopesticide product of B. cepacia from the usually found in aquatic environment and sometimes
market (https://www.gpo.gov/fdsys/pkg/FR-2004-09-29/ 15a small amount are found in the wood substrates where
pdf/04-21695.pdf). However, B. cepacia have been used in earthworms live (Magdoff and Weil 2004). Nursyam (2017)
Asia because the bacterial strains of B. cepacia might have studied the primary metabolites of V. alginolitycus isolated
different characteristics and function. from sponge Haliclona sp. found they could inhibit the
Similarly, the genus Serratia has been widely used growth of the clinical bacterium Staphylococcus aereus in
in agriculture as a biocontrol agent against many plant vitro. The results of this study showed that S. ficaria and
pathogens. Several selected strains of Serratia plymuthica, V. alginolitycus might have potency as a biocontrol agent,
Serratia marcescens and Serratia liquefaciens have been particularly for A. rolfsii. These results suggested both
able to reduce plant disease severity using specific PGPR isolates produced IAA and siderophores as well
application strategies (Saha et al. 2017). Serratia species as they could solubilized phosphate which help plant
produce the red pigments, prodiogiosin and pyrrolnitrin growth and inhibited growth of the phytopathogenic
as well as producing chitinase and a siderophore which fungi A. rolfsii.
inhibit fungal growth (Saha et al. 2017). Bacteria with This in vitro study revealed that the effectiveness of
chitinolytic activity have shown potency as biocontrol A.hydrophila and Pantoea sp. 2 to inhibit the growth of A.
agents against plant pathogenic fungi by degrading the rolfsii was not as good as the other three bacterial isolates.
cell walls, which are mainly composed of chitin (Someya One replication showed that the bacterial isolates could not
et al. 2011). S. marcescens was an effective biocontrol agent inhibit the growth of A. rolfsii. This might occurs because
against Sclerotium rolfsii affecting beans and extracellular the bacterial isolates could not compete with the fungal

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270   I. Safni, W. Antastia

a b

Aeromonas hydrophila vs Athelia rolfsii Burkholderia cepacia vs Athelia rolfsii

c d

Serratia ficaria vs Athelia rolfsii Pantoea sp.2 vs Athelia rolfsii

e f

16

Vibrio algynolyticus vs Athelia rolfsii normal hyphae of Athelia rolfsii without bacterial treatment

Figure 2: Microscopic observation of the in vitro antagonism of Plant Growth Promoting Rhizobacteria against Athelia rolfsii, a. crooked
hyphae, b. shorten hyphae, c. lysed hyphae, d. lysed hyphae, e. lysed and crooked hyphae, f. normal hyphae

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 In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean
pathogen in obtaining sites and nutritients. The bacteria
might also lose their virulence due to incompatible abiotic
conditions. On the contrary, A. hydrophyla was studied
as a PGPR to increase nodulation and nitrogen fixation
of soybean (Zhang et al. 1997). Similarly, Pantoea sp. was
utilised as an effective stress mitigator in the saline soil
of mungbeans (Vigna radiata L.) and improved seed yield
(Panwar et al. 2016).
On the basis of morphological changes of fungal
hyphae after treatment with the five bacterial isolates, all
bacterial isolates showed an effect on the fungal hyphae,
such as crooking, shortening and lysing. Morphological
deformities of pathogenic fungal hyphae caused by PGPR
affects have been reported in early studies (Govindasamy
et al. 2017; Kumar et al. 2011; Sharma and Dubey 2017).
PGPR isolates produces enzymes that degrade fungal
cellular components including chitinase, β-1,3 glucanase,
and are cellulolytic (Heydari and Pessarakli 2010; Whipps
2001) which can cause the loss of structural integrity,
lysis, fragmentation and perforations of hyphae, and
sclerotial degradation (Sharma and Dubey 2017).
In summary, five bacterial species, which were
isolated from rhizospheric soils in several locations in
North Sumatra, were selected as potential biological
control agents against collar rot pathogen of soybean, A.
rolfsii, on the basis of in vitro antagonistic assay.
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