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Research Article
Siderophore Producing Pseudomonas cf. monteilii 9 for
Assured Biological Control of Sclerotium rolfsii Causing Stem
Rot of Groundnut
Dalvi S M1 and Rakh R R*
Department of Microbiology, Shri Guru Buddhiswami Mahavidyalaya, Purna (Jn.) Dist. Parbhani (MS)
India 1Department of Botany, Shri Guru Buddhiswami Mahavidyalaya, Purna (Jn.) Dist. Parbhani (MS) India
Article Info
Abstract
Received: 26-05-2017,
Revised: 21-06-2017,
Accepted: 22-06-2017
To search for the effective microbial control agent, specially Pseudomonas spp.
for management of stem rot disease caused by Sclerotium rolfsii in groundnut,
21 Pseudomonas spp. were isolated from rhizospheric soil of different plants.
These isolates were screened for their antagonistic activity against Sclerotium
rolfsii (a phytopathogen causing stem rot) by using dual culture technique. A
Pseudomonas isolate, identified as Pseudomonas cf. monteilii 9, showed highest
antagonistic activity against Sclerotium rolfsii. In dual cultures, the
Pseudomonas cf. monteilii 9 inhibited the Sclerotium rolfsii (94 %), in terms of
dry weight. Pseudomonas cf. monteilii 9 produce siderophore which was
responsible for suppression of Sclerotium rolfsii in vitro. In vitro results were
confirmed by undertaking pot assay for biocontrol of Sclerotium rolfsii.
Groundnut seeds treated with Pseudomonas showed decrease in incidence of
disease (45.45 to 66.67%) when challenged with Sclerotium rolfsii as compared
to untreated seeds.
Keywords:
Groundnut, Sclerotium
rolfsii Sacc, Pseudomonas
cf. monteilii 9, Siderophore.
INTRODUCTION:
Sclerotium rolfsii Sacc. [Athelia rolfsii
(Curzi) Tu and Kimbrough] is known to cause
southern blight in a wide variety of crops.
Sclerotium rolfsii forms brownish sclerotia that can
survive in soil for long periods, frequently
tolerating biological and chemical degradation due
to the presence of melanin in the outer membrane
(Chet, 1975). Variety of methods are employed to
manage S. rolfsii namely fungicide applications,
solarization, use of antagonistic microorganisms,
deep plowing, crop rotation, and incorporation of
organic and inorganic residues (Punja, 1985).
Chemical fungicides are extensively used in
current agriculture. However, excessive use of
chemical fungicides in agriculture has led to
deteriorating human health, environmental
pollution, and development of resistance in
pathogen to fungicide. Because of the worsening
problems in fungal disease control, a serious search
is needed to identify alternative methods for plant
protection, which are less dependent on chemicals
and are more environmental friendly. Microbial
antagonists are widely used for the biocontrol of
fungal plant diseases.
Rhizospheric bacteria have proved as
excellent agents to control soil-borne plant
pathogens. Bacterial species like Bacillus,
Pseudomonas, have been proved in controlling the
fungal diseases.
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Dalvi and Rakh
Bacteria identified as plant growth
promoting rhizobacteria and biocontrol strains often
belong to the following genera (i) Bacillus (Nair et
al., 2002; Shadi Selseleh-zakeri et al., 2015 and
Ray et al., 2012), (ii) Pseudomonas (Mark et al.,
2006). B. aryabhattai has produced lipopeptide
antibiotic iturin to inhibit F. graminearum isolated
from infected wheat seeds (Shadi Selseleh-zakeri et
al., 2015).
Pseudomonas spp received special attention
as microbial control agent because of their catabolic
versatility, excellent root-colonizing abilities and
production of broad range antifungal metabolites
such as 2,4-diacetylphloroglucinoal (DAPG),
pyoluteorin, pyrrolnitrin and phenazines (Chin-A-
Woeng et al., 2001; Raaijmaker et al., 2002). The
mechanisms through which Pseudomonas spp.
control plant diseases involve (i) competition for
niches and nutrients, (ii) antibiosis, (iii) predation,
and (iv) induction of plant defense responses.
Present research work was undertaken to i)
isolate the phytopathogen from infected groundnut
part ii) search for effective microbial control agent,
particularly Pseudomonas spp. against the stem rot
pathogen, Sclerotium rolfsii iii) find out the
mechanism of microbial control agent that could be
used to kill the phytopathogen, Sclerotium rolfsii.
MATERIALS AND METHODS
Isolation of Sclerotium rolfsii: -
The phytopathogen, Sclerotium rolfsii was
isolated from infected groundnut stem (Plate 1) in
Department of Microbiology, Shri Guru
Buddhiswami Mahavidyalaya, Purna (Jn.) -
431401(India) on potato dextrose agar (PDA) plates
and incubated at 25 °C for 4 – 6 days. Stock culture
of S. rolfsii was maintained on PDA slants and
stored at 4 °C.
Plate 1: Infected Groundnut Stem
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Isolation of Pseudomonas spp. from rhizospheric
soil Rhizospheric soil from different healthy
plants such as groundnut etc. were collected in
poly-ethylene bags and brought to the research
laboratory. A 1 gm of soil sample was inoculated
into 100 ml nutrient broth and kept for incubation at
room temperature (28± 2°C) for 24 h. For isolation
of Pseudomonas spp, 1ml of this nutrient broth was
transferred to selective enrichment media,
Cetrimide broth and kept for incubation at room
temperature for 24 h. From enriched Cetrimide
broth, a loopful of culture was streaked on
Cetrimide agar (Brown and Lowbury, 1965 and the
plates were incubated at room temperature till
colonies were observed (24 – 48 h). The isolated
colonies developed were then purified on nutrient
agar slants and used for screening against the
phytopathogen, Sclerotium rolfsii for microbial
control ability. All the Pseudomonas isolates were
tentatively named during this research to avoid
confusion.
In Vitro Screening for Potential Biocontrol
agents against phytopathogen All rhizospheric Pseudomonas isolates
were screened for potential antagonistic activity
against phytopathogen, Sclerotium rolfsii on King’s
B agar (Ran, et al., 2003) using dual culture
technique (Rangeshwaran and Prasad, 2000). In
this technique, an agar disc (5 mm dia.) was cut
from an actively growing (96 h) phytopathogen, S.
rolfsii culture and placed on the surface of fresh
King’s B agar medium at the one side of the Petri
plates. A loopful of actively growing Pseudomonas
isolates (each) was placed opposite to the fungal
disc. Plates inoculated with phytopathogen and
without bacteria were used as control. Each
experiment was carried out in triplicates. Plates
were incubated at room temperature for 7 days.
Degree of antagonism was determined by
measuring the radial growth of pathogen with
bacterial culture and control and percentage
inhibition was calculated by the following equation
(Riungu et al., 2008).
[ Radial growth of Pathogen – [Radial growth of Pathogen
(Control) Plate] + Antagonist (Test Plate)]
Inhibition (%) = × 100
[Radial growth of Pathogen (Control Plate)]
Identification of Biocontrol agent
An efficient microbial control agent
obtained during screening was identified by 16S
rDNA sequencing carried out at Agharkar Research
Institute (ARI) Pune, Maharashtra.
In vitro Characterization of biocontrol features
To search for the biocontrol mechanism, the
efficient rhizospheric Pseudomonas isolate was
tested for its ability to produce Siderophore.
Analysis of Siderophore Production
Inoculum Preparation
The inoculum of Pseudomonas spp was
prepared in King’s B medium and incubated at 28
°C on rotatory shaking incubator (120 rpm) for 18-
20 h.
Culture and Cultivation
Siderophore production was studied using
modified Succinate medium (Meyer and Abdallah,
1978) consisting (g/l) Succinic acid (4), K2HPO4
(6), KH2PO4 (3), (NH4)2 SO4 (1), MgSO4 (0.2), and
pH (7.0). 0.1ml of inoculum was separately
inoculated in 250 ml Erlenmeyer flask containing
Succinate medium and then incubated on rotatory
shaker for 48 h at 28°C. A supernatant was
harvested by centrifuging the culture at 10,000 rpm
in cooling centrifuge at 4°C for 10 minute.
Quantitative Detection of Siderophore
Quantitatively siderophores in culture
filtrate were detected as per Payne, (1994) where
0.5 ml of culture filtrate was mixed with 0.5 ml of
CAS solution. A reference was prepared using,
uninoculated succinate medium (used for
siderophore production by Pseudomonas). Both the
test and reference were read at 630 nm and %
siderophore units in the culture filtrate were
calculated.
Ar - As
% Siderophore Units = × 100
Ar
Where,
Ar = Absorbance of reference at 630 nm.
As = Absorbance of test sample at 630 nm.
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Dalvi and Rakh
In Vivo Pot Assay for biocontrol of stem rot:
To check the biocontrol ability of
Pseudomonas cf. monteilii 9 in vivo against
Sclerotium rolfsii, a pot assay was set up using two
groundnut varieties SB XI and TAG-24. Mass
multiplication of S. rolfsii was carried out in Potato
Dextrose broth at room temperature for 3 weeks
(Ordentlich et al., 1988) and then the numbers of
sclerotia produced were used for the preparation
sick pots. This experiment was carried out in three
sets of pots and triplicates. All pots were first
disinfected with 5 % CuSO4 solution. First set
(Positive Control) of three pots, was filled with 150
gm sterilized soil sand (1:1) mixture with sclerotia
of S. rolfsii artificially inoculated at 1 sclerotia/ gm
soil (Fouzia Yaqub and Saleem Shahzad, 2005).
The pots containing inoculum were incubated for 15
days at room temperature, frequently stirred and
watered for colonization of fungus in the soil. Then
5 surface disinfected untreated seeds of groundnut
varieties, TAG-24, and SB XI each were sown in
pots. Second set (Negative Control) of three pots,
was inoculated with 150 gm of sterilized soil sand
mixture and 5 untreated seeds of SB XI and TAG-
24 each were sown in pots. Third set (Test) of three
pots, was filled with 150 gm of sterilized soil sand
mixture artificially inoculated with sclerotia of S.
rolfsii at rate of 1 sclerotia/ gm of soil. These pots
were sown with 5 treated groundnut seeds of each
variety with Pseudomonas culture. The pots
containing inoculum were incubated for 15 days at
room temperature, frequently stirred and watered
for colonization of fungus in the soil and then 5
biocontrol agent’s formulation (15 gm/ kg of seeds)
treated seeds of groundnut varieties, SB XI and
TAG-24 each were sown in pots. All these pots
were kept at room temperature and watered
regularly. Percent germination, shoot length, root
length, and chlorophyll content were recorded after
15, 30, 45 and 60 days. Vigour index was calculated
by using the formula as per Baki and Anderson
(1973).
Vigour index = (Mean shoot length + mean root
length) x Germination (%)
RESULTS AND DISCUSSION
Isolation of Phytopathogen:
After 7 days incubation on PDA plates, the
fungus produced abundant white septate mycelia,
1.5–3.0 μm diameter with clamp connections at
each septation, aerial hyphae and also numerous
spherical, or ellipsoidal, white sclerotia, 0.5–2.0
mm diameter, which turned brown on maturation,
(Plate 2). Based on morphological and culture
characteristic, the disease-causing organism was
identified as Sclerotium rolfsii (Mesquita et al.,
2007).
Plate 2: Isolated Sclerotium rolfsii from infected groundnut stem
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Bioscience Discovery, 8(3): 546-555, July - 2017
Isolation of Pseudomonas spp. from rhizospheric
soil During this research work, 21
Pseudomonas spp were isolated from rhizospheric
soil of different healthy plants such as Soybean,
Neem, Groundnut, Tur etc. All the rhizospheric
isolates were tentatively named (Table 1) and
maintained on Nutrient Agar slants for further
screening.
Screening for Potential Biocontrol agents against
phytopathogen It was observed that Pseudomonas isolate
that was named as RSPGN11 found to be efficient
antagonist against S. rolfsii in dual culture
technique (Plate 3) while none of the other
Pseudomonas isolates found efficient (Table 1).
Plate 3: In vitro screening for potential biocontrol agents against Sclerotium rolfsii
It was revealed that Pseudomonas culture
RSPGN11 inhibits Sclerotium rolfsii (94 %). Our
results when compared with the results earlier
reported by Kishore et al., (2005), for control of
Sclerotium rolfsii with Pseudomonas aeruginosa in
dual culture. It was found that our results with
Pseudomonas are far better than the above-
mentioned results because there was only 32-74 %
inhibition recorded where as 94 % inhibition was
recorded for S. rolfsii.
Table 1: Primary Screening of Pseudomonas isolates against Sclerotium rolfsii by dual culture
technique
Tentative Name of
Bacteria
Inhibition of S. rolfsii (%) Tentative Name of
Bacteria
Inhibition of S. rolfsii (%)
RSPGN 1 21.2 RSPGN 12 33.0
RSPGN 2 35.3 RSPGN 13 27.0
RSPGN 3 43.1 RSPGN 14 36.4
RSPGN 4 12.1 RSPGN 15 20.2
RSPGN 5 21.0 RSPGN 16 16.0
RSPGN 6 30.4 RSPGN 17 11.0
RSPGN 7 19.0 RSPGN 18 14.0
RSPGN 8 12.5 RSPGN 19 23.0
RSPGN 9 21.4 RSPGN 20 26.9
RSPGN 10 16.9 RSPGN 21 25.0
RSPGN 11 94.0
TEST CONTROL
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Dalvi and Rakh
Identification of Biocontrol agent:
The efficient RSPGN11 was identified by
16S rDNA gene sequencing. The sequence was
edited and aligned with sequence in the public
domain GenBank by BLAST Programm which
showed 98 % similarity with Pseudomonas cf.
monteilii 9 with accession number AF181576.
In vitro Characterization of biocontrol features:
To investigate the biocontrol mechanism of
the selected strains namely, Pseudomonas cf.
monteilii 9 was tested for production of
siderophore.
Siderophore Analysis:
Quantitative Detection of Siderophore
A supernatant of the culture was harvested
after 12, 24, 36 and 48 h by centrifuging the culture
at 10,000 rpm in cooling centrifuge. Quantitatively
siderophores in supernatant was detected as per
Payne, (1994) where 0.5 ml of culture filtrate was
mixed with 0.5 ml of CAS solution. A reference
was prepared using, uninoculated Succinate
medium. Absorbance of both the test and reference
were read at 630 nm and % siderophore units in the
culture filtrate were calculated. Pseudomonas cf.
monteilii 9 gave instant color change of CAS
reagent from blue to classical golden orange (Plate
4) and produced maximum siderophore whose
absorbance was read at 630 nm. % siderophore unit
of culture was calculated after 12, 24, 36 and 48 h
(Graph 1).
Plate 4: liquid CAS assay for siderophore production
Graph 1: Siderophore productions by Pseudomonas cf. monteilii with respect to time
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Bioscience Discovery, 8(3): 546-555, July - 2017
In the time course of siderophore production,
maximum siderophore secretions by Pseudomonas
cf. monteilii 9 (85.51%) was recorded after 36 h.
Siderophore being a secondary metabolite produced
under iron stress condition hence maximum
siderophore was recorded 36 h. thereafter, re-
incubation showed decline in the % siderophore.
Hence for siderophore production the batch needs to
be harvested after 36 h. for maximum recovery of
siderophore. Siderophores produced by certain
strains of fluorescent Pseudomonas spp. have been
reported to link in suppression of soil-borne plant
diseases (Bashan and de-Bashan, 2005). It has been
suggested that siderophores act antagonistically by
sequestering iron from the environment, restricting
growth of the pathogen. Convincing evidence for
the involvement of siderophores in disease
suppression is readily available (Bashan and de-
Bashan, 2005). For example, a mutant strain of P.
putida that overproduces siderophores has been
shown to be more effective than the wild bacterium
in controlling the pathogenic fungus Fusarium
oxysporium in tomato. Many wild strains that lose
their siderophore trait also lose biological control
activity. Pseudomonas siderophores have also been
implicated in inducing systemic resistance (ISR) in
plants (Leeman et al., 1996), i.e. enhancement of
the defence capacity of the plant against a broad
spectrum of pathogens. Exposure to pathogens,
non-pathogens, PGPR and microbial metabolites
stimulates the plants natural self-defence
mechanisms before a pathogenic infection can be
established, effectively `immunizing' the plant
against fungal, viral and bacterial infections
(Bashan and de-Bashan, 2005). Above results
showed that Pseudomonas cf. monteilii 9 produced
siderophore that played a vital role in biocontrol of
S. rolfsii.
In Vivo Pot Assay for Biocontrol of Stem rot:
It is evident from Plate 5 and 6 that the
vigor of groundnut in artificially infested soil
(Positive control) was poor as compared to the
negative control. Also, it was observed that,
Pseudomonas treated groundnut seeds showed good
over all vigor as compared to positive control.
Treatment with the Pseudomonas cf. monteilii 9,
increased % seed germination, shoot length, root
length No. of leaves and chlorophyll content of
groundnut, as depicted in Table 2. It was also
revealed that incidence of the disease symptoms in
the sick pot (positive control) after 60 days of
sowing was observed where as no symptoms were
recorded in the test pots (bacterized seeds) even
after 60 days.
The percent disease control due to
Pseudomonas cf. monteilii 9 treated seeds compared
to the untreated check (Positive control), ranged
from 45.45 to 66.67 %. These results were
somewhat like that of Kishore et al., 2005 where
groundnut endophytes Pseudomonas aeruginosa
GSE 18 and GSE 19 reduced the seedling mortality
by 54 % and 58 %, compared to the control.
Plate 5: Pot Assay (In Vivo) with Pseudomonas cf. monteilii 9 against Sclerotium rolfsii (TAG 24 ) after
60 days
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A) Dalvi and Rakh
B) Positive control (TAG 24 Seeds + Sclerotium rolfsii)
C) Test (Pseudomonas cf. monteilii 9 + TAG 24 Seeds + Sclerotium rolfsii)
Plate 6: Pot Assay (In Vivo) with Pseudomonas cf. monteilii 9 against Sclerotium rolfsii (SB XI) after 60
days
A) Positive control (SB XI Seeds + Sclerotium rolfsii)
B) Test (Pseudomonas cf. monteilii 9 + SB XI Seeds + Sclerotium rolfsii)
The data obtained during in vivo experiment was
analyzed by One-Way (Unstacked) ANOVA using
Minitab 15 and Statplus 2008 Statistical Software.
It was revealed by data analysis that variations in
values of % germination, shoot length, root length
No. of leaves and chlorophyll content of both the
treatment by using Pseudomonas cf. monteilii 9 as
biocontrol agents against Sclerotium rolfsii, causing
stem rot disease in groundnut, was found to be
significant at 5 % level because the calculated F
values (64.97) was found to be more than the Table
Value (2.27) which clearly indicates that the results
obtained, are not mere by chance but due to the
effective treatment.
Acknowledgment
The author acknowledges the financial
support provided by University Grants
Commission, New Delhi, as Major Research Project
grant.
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How to Cite this Article:
Dalvi SM and Rakh RR, 2017. Siderophore Producing Pseudomonas cf. monteilii 9 for Assured
Biological Control of Sclerotium rolfsii Causing Stem Rot of Groundnut. Bioscience Discovery, 8(3):546-
555.