siderophore producing for assured biological control of

http://jbsd.in 546 ISSN: 2229-3469 (Print)

Bioscience Discovery, 8(3): 546-555, July - 2017

© RUT Printer and Publisher

Print & Online, Open Access, Research Journal Available on http://jbsd.in

ISSN: 2229-3469 (Print); ISSN: 2231-024X (Online)

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

*[email protected]

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.

http://biosciencediscovery.com 547 ISSN: 2231-024X (Online)

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



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.


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



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


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



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


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.

REFERENCES

Baki Abdul AA, and Anderson J D, 1973. Vigour

determination in Soybean seed by multiple criteria.

Crop Science, 13:630–633.

Bashan Y, and de-Bashan LE, 2005. Bacteria. In:

Encyclopedia of Soils in the Environment. Hillel D.

(ed.). Elsevier, Oxford, U.K, 1, pp. 103-115.

Brown VI, and Lowbury JL, 1965. Use of an

improved cetrimide agar medium and other culture

methods for Pseudomonas aeruginosa. J. Clin.

Path., 18:752-756.

Chet I, 1975. Ultrastructural basis of sclerotial

survival in soil. Micro. Ecol. 2: 194-200.

Chin-A-Woeng TFC, Thomas-Oates JE,

Lugtenberg BJJ, Bloemberg GV, 2001. Introduction of the phzH gene of Pseudomonas

chlororaphis PCL1391 extends the range of

biocontrol ability of phenazine-1- carboxylic acid-

producing Pseudomonas spp. strains. Molecular

Plant–Microbe Interactions, 14: 1006–1015.

Fouzia Yaqub, and Saleem Shahzad, 2005. Pathogenicity of Sclerotium rolfsii On Different

Crops and Effect of Inoculum Density on

Colonization of Mungbean and Sunflower Roots.

Pakistan Journal of Botany, 37(1):175-180.

Kishore GK, Pande S, and Podile AR, 2005. Biological control of collar rot disease with broad-


Dalvi and Rakh

spectrum antifungal bacteria associated with

groundnut. Can. J. Microbiol,. 51(2):123-32.

Leeman M, den Ouden F M, van Pelt J A, Dirkx

F P M, Steijl H, Bakker P A H M, and Schippers

B, 1996. Iron availability affects induction of

systemic resistance to Fusarium wilt of radish by

Pseudomonas fluorescens. Phytopathology, 86:149-

155.

Mark GL, Morrissey JP, Higgins P, O'Gara F,

2006. Molecular-based strategies to exploit

Pseudomonas biocontrol strains for environmental

biotechnology applications. FEMS Microbiology

Ecology, 56: 167-77.

Mesquita, ER, OL Pereira, and JAS Grossi, 2007. Basal rot of arum lily (Zantedeschia

aethiopica) caused by Sclerotium rolfsii in Brazil.

Australasian Plant Disease Notes, 2: 91–92.

Meyer JM and Abdallah MA, 1978. The

fluorescent pigment of Pseudomonas fluorescens:

biosynthesis, purification and physico-chemical

properties. J. Gen. Microbiol., 107:319-328.

Nair J R, Singh G, and Sekar V, 2002. Isolation

and characterization of a novel Bacillus strain from

coffee phyllosphere showing antifungal activity.

Journal of Applied Microbiology, 93: 772-780.

Ordentlich A, Y Elad, and I Chet, 1988. The role

of chitinase of Serratia marcescens in biocontrol of

Sclerotium rolfsii. Phytopathology, 78: 84-88.

Payne SM, 1994. Detection, isolation and

characterization of siderophores. In, Methods in

Enzymology, 235:329-344.

Punja ZK, 1985. The biology, ecology and control

of Sclerotium rolfsii. Ann. Rev. of Phyto. 23: 97-

127.

Raaijmakers JM, Vlami M, and de Souza JT,

2002. Antibiotic production by bacterial biocontrol

agents. Antonie van Leeuwenhoek, 81: 537–547.

Ran LX, Xiang ML, Van Loon LC, and Bakker

P A HM, 2003. Siderophore-mediated suppression

of Grey Mould in Eucalyptus urophylla by

Fluorescent Pseudomonas spp.6th International

PGPR Workshop, 5-10 October 2003, Calcutta,

India. 558-562.

Rangeshwaran R, and Prasad RD, 2000. Biological control of Sclerotium rot of sunflower.

Indian Phyto. 53: 444-449.

Ray S, Datta R, Bhadra P, Chaudhuri B and

Mitra AK, 2012. From space to earth: Bacillus

aryabhattai found in the indian sub-continent.

Bioscience Discovery. 3 (1): 138-145.

Riungu GM, JW Muthorni, RD Narla, JM

Wagacha, and JK Gathumbi, 2008. Management

of Fusarium Head Blight of Wheat and

Deoxynivalenol Accumulation using Antagonistic

Microorganisms. Plant Pathology Journal, 7(1): 13-

19.

Shadi Selseleh-zakeri, Abbas Akhavan-sepahy,

Anita Khanafari, Sara Saadat-mand, 2015. Investigation of native Bacillus strains antifungal

activity against some native species of Fusarium

fungi. Bioscience Discovery, 6(2):59-69.

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.

siderophore producing for assured biological control of

Documents