ORIGINAL PAPER

Entomopathogenicity of endophytic Serratia marcescens strainSRM against larvae of Helicoverpa armigera(Noctuidae: Lepidoptera)

Muthugounder Mohan • Govindan Selvakumar •

Satya Nand Sushil • Jagdish Chandra Bhatt •

Hari Shankar Gupta

Received: 16 June 2010 / Accepted: 17 March 2011 / Published online: 7 April 2011

� Springer Science+Business Media B.V. 2011

Abstract An endophytic Serratia marcescens strain SRM

(MTCC 8708) isolated from the flowers of summer squash

was found to be entomopathogenic against the larvae of

Helicoverpa armigera. Natural epizootic of this bacterial

strain on the larvae collected from summer squash flowers

ranged from 19.9 to 72.3%. Under laboratory conditions, a

dose of 6 9 1010 c.f.u./ml diet induced 66.3% mortality of

first instar H. armigera larvae. Similarly all other growth and

development parameters of the insect were severely retarded

in a dose-dependent manner. The bacterium invaded the

entire alimentary canal and haemolymph with successful

replacement of all other gut-associated microflora. There

was a great reduction in midgut proteinase activity due to

inhibition of five major proteinase isozymes by S. marces-

cens infection. Further, a synergistic interaction between

chitinases isolated from this strain and Bacillus thuringien-

sis Cry1Ac toxin was observed. The present findings suggest

that this plant-associated S. marcescens strain SRM could be

suitably exploited for the management of H. armigera.

Keywords Serratia marcescens � Entomopathogenicity �Endophytic � Summer squash � Helicoverpa armigera

Introduction

The members of the genus Serratia (Family: Enterobacte-

riaceae) are Gram negative, rod shaped bacteria that are

ubiquitous in nature. They are commonly found in soil,

water, plants and animals and are known to produce plant-

growth-promoting substances, anti-fungal substances,

encourage the establishment of nitrogen-fixing symbionts

and cause insect pathogenesis (Ashelford et al. 2002; Jeong

et al. 2010). Serratia marcescens is the most studied

member of this genus and it has been widely known to

cause diseases in plants, invertebrate and vertebrate hosts

(Grimont and Grimont 1978).

The insect pathogenic, red-pigmented strains of Serratia

marcescens have been frequently recovered from healthy,

diseased or dead insects associated with insects of many

orders by several workers (Grimont and Grimont 1992). The

majority of the S. marcescens strains isolated from human

hosts are non-pigmented, while pigmented strains isolated

from insects and other environments are rarely responsible

for an outbreak of nosocomial infection in humans. Serratia

marcescens is normally considered to be opportunistic or

facultative pathogen, as it is often avirulent to insects when

present in the digestive tract but becomes lethal on entering

insect hemocoel following injury or stress. Serratia mar-

cescens produces several virulence factors that may be

harmful to insects (Escobar et al. 2001; Nunez-Valdez et al.

2008). Epizootics of S. marcescens are common among

laboratory reared insects but rarely found under field con-

ditions. Eggs, larvae, pupae, and adults of heliothines reared

under laboratory conditions are known to be infected by

S. marcescens and sometimes entire colonies is eliminated

very quickly (Sikorowski and Lawrence 1998).

Recent investigations have focused on role of endophytic

strains of S. marcescens in plants. They reportedly act as

M. Mohan (&) � S. N. Sushil � J. C. Bhatt

Division of Crop Protection, Vivekananda Institute of Hill

Agriculture, (Indian Council of Agricultural Research),

Almora, Uttarakhand 263601, India

e-mail: mohan_iari@yahoo.com

G. Selvakumar � H. S. Gupta

Vivekananda Institute of Hill Agriculture, (Indian Council

of Agricultural Research), Almora, Uttarakhand 263601, India

Present Address:M. Mohan

Directorate of Rice Research (ICAR), Hyderabad 500 030, India

123

World J Microbiol Biotechnol (2011) 27:2545–2551

DOI 10.1007/s11274-011-0724-4

plant-growth-promoters, anti-fungal agents (Gyaneshwar

et al. 2001; Queiroz and Melo 2006) and as phytopathogens

causing cucurbit yellow vein disease in cucurbits and decay

of stored bulbs in onion (Escobar et al. 2001; Rascoe et al.

2003). This gains significance, since endophytic microor-

ganisms are currently being viewed as potential natural pest

control agents (Azevedo et al. 2000). Through biotechno-

logical interventions genes encoding pesticidal factors from

other microbes have also been transferred to endophytic

microorganisms for effective pest control (Nian-Long et al.

2005).

The present study reports the entomopathogenic activ-

ity of a cucurbit endophytic S. marcescens strain SRM

(MTCC 8708) against the larvae of Helicoverpa armigera

(Lepidoptera: Noctuidae) and its synergistic interaction

with Bacillus thuringiensis. The strain has been isolated and

previously been characterized with plant growth-promoting

potential. (Selvakumar et al. 2008).

Materials and methods

Culture media and strain isolation

The S. marcescens strain SRM was grown in Luria–Bertani

liquid medium (10 g tryptone, 5 g NaC1, and 5 g yeast

extract, in 1 l of distilled water, pH 6.8) at 30�C in an

orbital shaker with 150 rev/min for 24 h. For induction of

chitinolytic activity, it was grown in a chitinase-inducing

medium supplemented with 0.4% (wt/vol) colloidal chitin.

The fermented culture was centrifuged at 8,000 rev/min for

10 min and the cells were suspended in sterile distilled

water for testing its insecticidal activity.

Pathogenicity bioassays on H. armigera

Helicoverpa armigera larvae were collected from different

host plants at weekly intervals beginning at 50% flowering

of the crops and maintained separately under laboratory

conditions on an artificial diet to observe for the natural

incidence of S. marcescens. Laboratory-reared disease-free

larvae of H. armigera maintained on a chickpea-based

semi-synthetic artificial diet were used to test the insecti-

cidal activity of the cucurbit endophytic S. marcescens

strain SRM. Optimum rearing conditions (27 ± 2�C tem-

perature, 60–80% relative humidity and 14L: 10D h pho-

toperiod) were provided throughout rearing and bioassay

period. Desired concentrations of 24-h-old cultures of

S. marcescens strain SRM were incorporated into the

artificial diet prior to solidification according to the pro-

cedure of Navon (2000). About 2% of the water in the diet

was replaced with bacterial preparations and addition of

antibiotics was avoided to preserve bacterial activity. The

diet was mixed well by a magnetic stirrer and then poured

into small plastic cups. At least 50 neonates (24 h old)

were tested for each concentration. The larval mortality

was counted after 6 and 11 days of continuous feeding on

the treated diet with addition of fresh treated diet at regular

intervals. The surviving larvae after day 6 were separated

and maintained individually on plastic containers to avoid

any cannibalism. The weight of larvae after 6 and 11 days

of feeding, larval duration, pupation rate and adult lon-

gevity were also recorded. The larvae in control were fed

with untreated diet throughout the bioassay period. The

experiment was repeated twice on alternate days.

Estimation of bacterial load and midgut proteinase

activity

Third instar larvae weighing approximately 30 mg were

fed with a diet containing 6 9 1010 c.f.u./ml for 4 days. At

the end of day 4, the larvae were starved for 2 h, and

immobilized on ice for 1 h. Such immobilized larvae were

surface sterilized in 70% ethanol for 1 min, rinsed in sterile

water and dissected by cutting both ends in ice-cold sterile

100 mM Tris–HCl buffer (pH 7.0). The foregut, midgut

and hindgut from 20 diseased and healthy larvae were

excised separately and stored in 500 ll of 100 mM Tris–

HCl buffer (pH 7.0) at -20�C. Similarly 500 ll haemo-

lymph was also collected from diseased and healthy larvae

after pricking the first pair of prolegs and stored in the same

way.

The guts were sonicated (30 Hz, 1.0 Amps) (SANYO,

Soniprep 150, UK) for 30 s, macerated with a plastic

pestle, and vortexed for 30 s to separate bacterial cells from

the gut wall. The supernatant was made up to 2 ml with the

same buffer and used for further analysis. Aliquots (100 ll)

from gut and haemolymph samples were plated onto

nutrient agar plates after appropriate dilutions and incu-

bated for 24 h at 30�C. The population load of S. mar-

cescens and other bacterial species (if any) on the agar

plates was estimated and expressed as c.f.u. per 100 ll of

homogenate.

The general proteinase activity present in the midgut

supernatant obtained by centrifugation at 12,000 g for

15 min, was studied by the azocasein digestion method

(Marchetti et al. 1998). A trypsin standard curve was

constructed using the methodology of Kunitz (1947). The

assay mixture contained 100 ll of 2% azocasein, 50 ll of

100 mM Tris–HCl, pH 10.0, and varying concentrations of

enzyme. After incubation for 1 h at 30�C, the reaction was

stopped by addition of 0.5 ml of 10% trichloroacetic acid

(TCA). In control, TCA was added before substrate. Fol-

lowing centrifugation, 500 ll of supernatant was mixed

with an equal volume of 1 M sodium hydroxide and the

absorbance was measured at 420 nm. The enzyme activity

2546 World J Microbiol Biotechnol (2011) 27:2545–2551

123

was expressed as tryptic units (TU), after comparing with a

standard curve obtained with bovine trypsin.

Zymogram analysis of midgut proteinases

Gut homogenates (10 lg protein) of S. marcescens infected

and normal larvae were subjected to SDS–PAGE on 12%

non-reducing gels and processed for zymogram analysis as

described by Garcia-Carreno et al. (1993). The gel was

washed with 2.5% Triton X-100 to remove SDS. The gel

strip containing proteinase lanes were incubated in

100 mM glycine–NaOH buffer (pH 10.0) containing 2%

casein for 2 h. Then the gel strip gel was stained with 0.5%

Coomassie brilliant blue R-250 overnight and destained

briefly with methanol–acetic acid–water (40:10:50 v/v).

Proteolytic activity was revealed as a zone of white

clearing in a dark blue background.

Isolation of chitinase and synergistic studies

with B. thuringiensis Cry1Ac toxin

Serratia marcescens culture was cultured in a chitinase-

inducing medium consisting of colloidal chitin (0.4%),

yeast extract (0.06%), (NH4)2SO4 (0.1%), MgS04 (0.03%),

KH2PO4 (0.6%), K2HPO4 (1.0%). at 30�C for 3 days with

constant shaking. The cells were centrifuged, and the

supernatant was filtered through a 0.2 lm membrane filter.

The protein component was then precipitated by 70%

ammonium sulfate saturation. The precipitate was dis-

solved in 10 mM phosphate buffer (pH 6.0), dialysed and

lyophilized. Chitinase activity was measured using a

standard procedure with colloidal chitin as the assay sub-

strate (Brurberg et al. 1996). The toxin from cry1Ac gene

expressed in E. coli strain JM103 (BGSC, Colombus, USA)

was produced and purified according to Lee et al. 1992.

The protein content of the activated toxin preparation was

estimated by the Bradford Coomassie brilliant blue G-250-

binding method using bovine serum albumin as standard.

The chitinase preparation and Cry1Ac toxins were diluted

appropriately in distilled water and then mixed with arti-

ficial diet before solidification. Seven to ten neonate

H. armigera larvae were released in each diet preparation

and replicated three to six times. Assay was maintained at

28�C and 60% RH and the percent larval mortality and

larval growth inhibition were observed after 6 days.

Statistical analysis

The data on field survey, larval mortality and growth

inhibition were subjected to analysis of variance according

to Gomez and Gomez (1984). Differences among treatment

Table 1 Damage caused by H. armigera and their natural infection by S. marcescens under field condition on different host plants

Host plant Per cent pod/flower/fruit damage Per cent larval infection by S. marcescens

Week 1 Week 2 Week 3 Week 1 Week 2 Week 3

Chickpea 19.1 (25.8)c 20.0 (26.4)cd 24.5 (29.6)c 0.0 (0.57)c 0.0 (0.57)c 0.0 (0.57)c

Summer squash 84.2 (66.6)a 75.7 (60.7)a 60.5 (51.1)a 58.2 (49.7)a 72.3 (58.2)a 19.9 (26.5)a

Cucumber 45.7 (42.5)b 51.5 (45.9)b 44.5 (41.8)b 11.1 (19.5)b 5.5 (13.6)b 5.0 (12.9)b

Tomato 5.8 (13.8)d 17.0 (24.3)d 20.5 (26.9)cd 0.0 (0.77)c 0.0 (0.97)c 0.0 (0.97)c

Capsicum 17.8 (24.9)c 25.6 (30.4)c 17.3 (24.6)d 0.0 (0.97)c 0.0 (0.97)c 0.0 (0.97)c

LSD (P = 0.05%) 3.7 5.6 3.8 3.2 2.2 1.3

Means within a column followed by the same letter are not significantly different from each other at 5% level of significance

Table 2 Effect of S. marcescens on growth and development of H. armigera larvae

Treatments Larval mortality (%) day 6 Larval weight (mg) Larval period (days) Normal pupation (%) Adult longevity (days)

Day 6 Day 11 Female Male

6 9 1010 (c.f.u./ml) 66.3a 7.1c 218.1d 18.0a 48.7d 1.9e 1.1d

6 9 108 (c.f.u./ml) 21.3b 15.9b 329.6c 14.9b 60.6c 5.4d 4.9c

6 9 106 (c.f.u./ml) 12.5c 20.2a 372.6ab 13.7bc 68.6b 6.5d 5.4c

6 9 104 (c.f.u./ml) 2.5d 20.5a 409.4a 12.7cd 71.7a 8.3c 5.9c

6 9 102 (c.f.u./ml) 0.0e 20.5a 409.8a 12.4 cd 74.1a 10.6b 6.9bc

Control 1.3e 22.4a 391.6ab 12.1cd 74.2a 12.3a 10.5a

LSD (P = 0.05%) 5.2 4.2 28.0 1.5 4.6 1.2 1.0

Means within a column followed by the same letter are not significantly different from each other at 5% level of significance

World J Microbiol Biotechnol (2011) 27:2545–2551 2547

123

means were tested with the LSD test at 5% level of

significance.

Results and discussion

Pathogenicity of S. marcescens on H. armigera

The incidence of H. armigera on different host plants

grown at VPKAS experimental farm was monitored in the

months of April-July during 2005–2008. Damage on eco-

nomic parts of summer squash and cucumber was severe.

The range of larval mortality caused by S. marcescens on

H. armigera larvae collected from summer squash and

cucumber was 5.0–72.3%. However, the larvae collected

from other host plants such as chickpea, tomato and cap-

sicum were totally free of S. marcescens infection

(Table 1). The laboratory bioassay with S. marcescens

strain SRM resulted in 66.3% larval mortality at the

highest dose of 6 9 1010 c.f.u./ml tested. Similarly all

other growth and development parameters of the insect

were severely retarded in a dose-dependent manner

(Table 2). Several Serratia marcescens strains have been

shown to be lethal to insect pests. In a previous study,

treatment of Heliothis virescens with S. marcescens was

shown to produce sublethal effects, such as reduced adult

longevity and a decreased ratio of oviposition and egg

hatching (Sikorowski and Lawrence 1998). Similarly

strains of S. entomophila and S. proteamaculans have been

shown to kill various species of Scarab larvae (Jackson

et al. 2004; Dodd et al. 2006; Nunez-Valdez et al. 2008).

Effect of S. marcescens infection on insect gut

and haemolymph

S. marcescens was the only bacterial inhabitant of the gut

and haemolymph of infected fourth instar larvae, and the

population levels were higher in the midgut compared to

the fore and hind guts. However, in healthy larvae no

S. marcescens-like colonies were found in any of the larval

tissues but other bacterial species (probably symbiotic

organisms) were found in abundance (Table 3). Analysis

of gut-associated bacteria through the disease development

process indicates that the S. marcescens strain SRM has the

ability to overcome the insect host’s defenses and prolif-

erate rapidly in the entire gut and hemocoel, thereby

replacing other beneficial microflora in the insect system.

Dillon et al. (2005) also reported a significant negative

relationship between the density of Serratia marcescens

and the number of symbiotic gut bacteria in the desert

locust, Schistocerca gregaria.

The midgut general proteinase activity of fourth instar

larvae was determined after 4 days of S. marcescens Ta

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2548 World J Microbiol Biotechnol (2011) 27:2545–2551

123

infection. The larvae treated with S. marcescens at

6 9 1010 c.f.u./ml diet stopped feeding 4 days after treat-

ment. Upon dissection of moribund larvae, the entire ali-

mentary canal was found to be amber coloured due to

S. marcescens infection, as compared to normal greenish

yellow coloration of the alimentary canal of healthy larvae.

The infected larvae turned reddish and the body contents

were liquefied (Fig. 1). The gut clearance in larvae, 4 days

post infection was succeeded by reduction in midgut pro-

teinase activity. The midgut homogenate from normally

fed and starved fourth instar H. armigera larvae showed

very high level of midgut proteinase activity, whereas in

S. marcescens-infected larvae, the midgut proteinase

activity was found to be 18.9-fold less as compared to

normally fed larvae (Fig. 2). The amber disease of the New

Zealand grass grub, Costelytra zealandica caused by a

strain of S. entomophila, shows a similar disease symptom

(Jackson et al. 2001, 2004).

The zymogram analysis of midgut proteinases from

normally fed, starved and S. marcescens-infected fourth

instar H. armigera larvae using casein as substrate

revealed five proteinase activity bands in normal and

starved larvae with approximate molecular weights of

68.0, 59.5, 55.0, 44.5 and 41.0 kDa. Interestingly, the

activity of these midgut proteinase isozymes was almost

inhibited in S. marcescens-infected larvae (Fig. 3). Bosa

et al. (2004) also observed a positive correlation between

N-acetylglucosaminidase and quimoelastase protease

activities and S. marcescens biocontrol activity, suggest-

ing that the activity of these enzymes could be related to

the mode of action of entomopathogenic bacteria of the

genus Serratia.

Fig. 1 Symptoms of

S. marcescens infection in

H. armigera larvae a Normal

(top) and S. marcescens infected

(bottom) alimentary canals

b Normal and c S. marcescensinfected larvae

Normal Starved Infected0

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4000

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Fig. 2 Midgut proteinase activity in normal and S. marcescensinfected H. armigera larvae

Fig. 3 Zymogram analysis of midgut proteinases from normal and S.marcescens infected H. armigera larvae Lanes 1 & 2: Proteinases

from S. marcescens infected larvae; Lane 3 & 4: Proteinases from

starved and normal fed H. armigera respectively; Lane 5; Protein

molecular weight marker

World J Microbiol Biotechnol (2011) 27:2545–2551 2549

123

Synergism between S. marcescens chitinase and B.

thuringiensis Cry1Ac toxin

Synergism tests were carried out with 0–10 ppm of

B. thuringiensis Cry1Ac toxin and 0–100 ppm of S. mar-

cescens chitinase preparation. In combination with S. mar-

cescens strain SRM chitinase preparation, there was an

enhanced larval growth inhibitory activitiy (Table 4). The

synergism occurred in a dose-dependent manner of chiti-

nase preparation. The Serratia marcescens strain SRM

secretes a variety of bioactive metabolites, including gel-

atinase and chitinase. The chitinases of S. marcescens are

one of the best characterized chitinolytic machineries

known to date (Sanchez et al. 2005). The chitinases of

S. marcescens, B. thuringiensis and other chitinolytic

bacteria have biocontrol activities, and reportedly enhance

the insecticidal activity of B. thuringiensis (Asano et al.

1999; Sampson and Gooday 1998; Downing et al. 2000).

Many endophytic Gram-negative and Gram-positive

bacterial species have been isolated from cultivated tropi-

cal plant species such as cabbage, maize, coffee etc., with

antagonistic activities against plant pests (Azevedo et al.

2000). S. marcescens is capable of thriving in diverse

habitats and ecological niches. The wide range of roles

played by S. marcescens include those of diverse origin

such as epiphytes, endophytes, soil inhabitants, nematode

symbionts, insect and plant pathogens, bacterial and fungal

antagonistics, plant growth promoters and nosocomial

human pathogens (Ashelford et al. 2002; Zhang et al.

2009). In addition to cold-tolerant and plant growth-pro-

moting abilities (Selvakumar et al. 2008), the entomo-

pathogenic nature of S. marcescens strain SRM is unique,

since endophytic S. marcescens strains so far reported are

either plant pathogenic, fungal antagonistic or plant growth

promoters (Gyaneshwar et al. 2001; Queiroz and Melo

2006; Rascoe et al. 2003).

The findings from the present study hints that the tox-

icity of B. thuringiensis Cry toxin produced in crop plants

could be synergized by coexpression of a suitable chitinase

gene from the S. marcescens strain reported here for

effective control of H. armigera. Further it has the potential

to be exploited as plant growth promoter as well as bio-

control agent. However endophytic S. marcescens strains

have unique relationships with insects, some as entomo-

pathogens and others in a vector–host relationship as in the

case of transmission of CYVD by the squash bug, Anasa

tristis, a cucurbit insect pest (Bruton et al. 2003). Hence, a

greater understanding of the ecology of this bacterium is

required if the impact of Serratia marcescens on insect

control is to be adequately understood.

Acknowledgments The authors are grateful to Indian Council of

Agricultural Research (ICAR) for funding the study.

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100 5 79.5 ± 3.7 93.3 ± 5.9

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