Aquaculture Research, 2015, 1–10
12746
Exclusion of Vibrio spp. by an antagonistic marine
actinomycete Streptomyces rubrolavendulae M56
Deepthi Augustine, Jimly C Jacob & Rosamma Philip
Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science
and Technology, Kochi, Kerala, India
Correspondence: R Philip, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin
University of Science and Technology, Fine Arts Avenue, Kochi 682016, Kerala, India. Emails: rose@cusat.ac.in;
rosammap@gmail.com
Abstract
A marine actinomycete Streptomyces rubrolavendulae
M56 isolated from the sediments of Bay of Bengal
and displaying biogranulation property was used
for the study. The strain showed antagonistic prop-
erty against vibrios, the opportunistic pathogens in
aquaculture. The efficacy of the biogranules of
actinomycete M56 in competitive exclusion of
Vibrio spp. was tested both in vitro and in vivo. Strep-
tomyces rubrolavendulae M56 biogranules could
significantly exclude the pathogenic Vibrio spp. in
co-culture experiments (in vitro). In vivo exclusion of
Vibrio spp. in a Penaeus monodon postlarval rearing
system was evaluated by treatment of the rearing
water with biogranules of S. rubrolavendulae M56.
The experiments proved that S. rubrolavendulae
M56 biogranules could reduce the pathogenic
Vibrio spp., while maintaining total heterotrophic
bacterial count. Therefore, the actinomycete biog-
ranules (M56) can be used as a promising alterna-
tive to antibiotics in the shrimp larval production
system which is often affected by vibriosis.
Keywords: actinomycetes, Streptomyces, bio-
granule, Vibrio, antagonistic, shrimp farming
Introduction
Shrimp farming represents a substantial source of
revenue in many developing countries, particularly
Asia. However, the shrimp culture industry is con-
stantly under threat due to water quality problems
and outbreak of infectious diseases. Vibrios are
indigenous marine microflora and constitute the
majority among the natural flora of farmed and
wild penaeid shrimps. They become opportunistic
© 2015 John Wiley & Sons Ltd
doi:10.1111/are.
pathogens when the animal is under stress and
defence mechanisms are suppressed (Lightner
1993). The major species causing vibriosis in
shrimps are V. harveyi, V. alginolyticus, V. anguilla-
rum and V. parahaemolyticus (Goarant, Merien,
Berthe, Mermoud & Perolat 1999). Indiscriminate
use of antibiotics has led to wide spread drug resis-
tance, and ecological damage necessitating more
effective and environment friendly alternatives to
control vibriosis. Competitive exclusion of potential
pathogenic bacteria effectively reduces the need for
antibiotic prophylaxis in intensive larviculture sys-
tems (Garriques & Arevalo 1995).
Antagonism is a widespread trait implicated in
competition and ecological success of many mar-
ine bacteria and is thus considered an important
attribute of aquaculture probionts (Long & Azam
2001; Gram, Melchiorsen & Bruhn 2010). Marine
actinomycetes particularly Streptomyces spp. are
recommended as promising candidates for inhibi-
tion of vibrios in marine aquaculture (You, Cao,
Liu, Zhou, Tan & Lin 2005; You, Xue, Cao, Lu,
Wang, Zhang & Zhou 2007). Actinomycetes have
been extensively screened for the exploitation of
their secondary metabolites. Actinomycetes are fil-
amentous and when cultured in shake flask cul-
tures, the filaments aggregate to form bead like
granules. Microbial communities in the form of
biofilms, or biogranules have also been employed
in environmental bioremediation technologies, e.g.
waste water treatment for removal of nitrogen,
phosphorus, heavy metals and hydrocarbons (Ve-
nugopalan, Nancharaiah, Mohan & Narasimhan
2005). In this regard, focus has been given in
exploiting the antagonistic potential and the gran-
ulation property of marine actinomycetes for the
control of vibriosis in aquaculture.
1
Vibrio exclusion by a marine Streptomycete D Augustine et al.
Materials and methods
Micro organisms used for the study
Marine actinomycete M56 isolated from the sedi-
ments of Bay of Bengal and maintained in the Micro-
biology Laboratory of School of Marine Sciences,
Cochin University of Science and Technology (CU-
SAT) were used for this study. This isolate was
selected based on its property to inhibit vibrios and
the biogranulation property when cultured in nutri-
ent broth and kept in an incubator shaker set at
150 rpm, 28°C for 7 days. Vibrio spp. used as patho-
gens for the study was isolated from diseased larvae
from a prawn hatchery in Kochi. The cultures were
maintained as part of the culture collection at the
National Centre for Aquatic Animal Health, Cochin
University of Science and Technology. The patho-
gens included Vibrio harveyi (MCCB 111), Vibrio
parahaemolyticus (MCCB 133), Vibrio alginolyticus
(MCCB 112) and Vibrio fluvialis (MCCB 130). All the
Vibrio spp. was streaked on prawn flesh agar med-
ium (Singh & Philip 1993) to improve virulence.
Molecular identification of the antagonistic
actinomycete strain M56
Spore suspension of the antagonistic actinomycete
strain M56 was inoculated into nutrient broth and
incubated in an orbital shaker at 28°C, 150 rpm
for 48–72 h to form a pellet of vegetative cells
(pre-sporulation). Genomic DNA of the strain was
extracted as per standard Proteinase-K digestion
method (Sambrook, Fritsch & Maniaties 1989).
The universal eubacterial primers 27F (50 -AGA
GTTTGATCTGGCTCAG-30 ) and 1492R (50 -
0
GGTTACCTTGTTACGACTT-3 ) (Lane, 1991) were
used to amplify nearly full length 16S rDNA
sequences. PCR amplification of 1 lL of the geno-
mic DNA was performed in a 25 lL reaction vol-
ume containing 19 standard Taq buffer (10 mM
Tris-HCl, 50 mM KCl, pH 8.3), 2.5 mM MgCl2,
200 lM dNTPs, 0.4 lM each primer and 1 U Taq
DNA polymerase (Fermentas Inc., Hanover, MD,
USA). The PCR programme consisted of an initial
denaturation of 95°C for 5 min, 35 cycles of dena-
turation (94°C for 20 s), annealing (58°C for
20 s), extension (72°C for 1 min 30 s) and a final
extension at 72°C for 10 min. The partial sequence
of the PCR products was ascertained using the
same primers with an ABI Prism Cycle Sequencing
Kit (Big Dye Terminator Cycle) at SciGenom, India.
2 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Aquaculture Research, 2015, 1–10
Phylogenetic analysis
The 16S rRNA gene sequence was compared with
GenBank entries using NCBI-BLAST (http://
www.ncbi.nlm.nih.gov/BLAST) to assess the degree
of similarity for identification of the strain. The
sequences were deposited in the GenBank using
the web based data submission tool, BankIt. Multi-
ple sequence alignment with reference sequences
obtained from GenBank was performed using CLU-
STALW and phylogenetic tree was generated using
the neighbour-joining method with MEGA 5.2
package (Tamura, Peterson, Peterson, Stecher, Nei
& Kumar 2011). Tree topology was evaluated
using bootstrap analysis of 1000 replicates.
Characterization of the actinomycete strain (M56)
Morphological, physiological and biochemical
characterization
The antagonistic strain (M56) with biogranulation
property was streaked on to starch casein agar,
glycerol asparagine agar and nutrient agar and
the colony characteristics were noted. Coverslip
culture of the isolates were done using casein
starch peptone yeast extract malt extract agar
medium and morphology of spore bearing hyphae
were recorded according to International Strepto-
myces Project (Shirling & Gottlieb 1966; Nonom-
ura 1974).
The biochemical characterization of the antago-
nistic strain (M56) was performed by the methods
of Berd (1973) and Bergey’s manual of systematic
bacteriology (Williams, Goodfellow & Alderson
1989).
The strain M56 was screened for various hydro-
lytic enzymes viz; protease, amylase, lipase, DNase,
phosphatase, ligninase and chitinase following the
methods of Holding and Collee (1971).
In vitro exclusion of vibrios by actinomycete
biogranule (co-culture experiment)
The efficacy of actinomycete biogranule (M56) in
the exclusion of vibrios in aquaculture was tested
using co-culture experiments.
Co-culture experiments with actinomycete iso-
late (M56) and aquaculture pathogens of Vibrio
spp. viz., Vibrio harveyi (MCCB 111), Vibrio parahae-
molyticus (MCCB 133), Vibrio alginolyticus (MCCB
112), Vibrio fluvialis (MCCB 130) were carried out
following the method of Gram, Melchiorsen,
Aquaculture Research, 2015, 1–10
Spanggaard, Huber and Nielsen (1999) with slight
modifications. The vibrios were pre-cultured sepa-
rately in 100 mL flasks at 28°C on a shaker at
120 rpm overnight. For the preparation of actino-
mycete granules, spore suspension was inoculated
into nutrient broth and incubated at 28°C on a
shaker at 150 rpm for 3–5 days. The medium-sized
granules were harvested, washed in sterile seawa-
ter and 15 granules of approximate weight 0.5 g
was inoculated into 100 mL fresh nutrient broth of
1/10th strength, with 1.5% NaCl to support the
viability of Vibrio spp. and actinomycetes. The opti-
cal density of overnight culture of different Vibrio
spp. was adjusted so as to inoculate a cell density
of approximately 105 CFU mL1 of vibrios sepa-
rately with actinomycete granules. Respective vib-
rio controls were set up with 105 CFU mL1 of
Vibrio sp. without actinomycete granules. All the
experiments were set up in triplicates. Flasks were
incubated at 28°C on a shaker at 120 rpm and
samples (1 mL) were withdrawn at 24 h intervals
to determine the viable vibrio count. The samples
were serially diluted 10-fold and 0.1 mL aliquots
were spread plated on Thiosulphate citrate bile
salts sucrose (TCBS) agar plates in triplicates. The
plates were incubated at 28°C, and colonies formed
on TCBS were counted and expressed as Log10
CFU mL1 of vibrios in co-culture.
Biomass estimation of biogranule
The actinomycete, Streptomyces rubrolavendulae M56
was streaked on to starch casein agar plates and
incubated at 28°C for 5–7 days until sporulation.
These spores were scraped from the agar medium
and inoculated into nutrient broth (50 ml) prepared
with sea water, in a 250 ml conical flask on an
incubator shaker set at 150 rpm, 28°C for 7 days.
To estimate the biomass of the granules, ATP esti-
mation was carried out according to Parsons, Maita
and Lalli (1984). Actinomycete granules approxi-
mately 4–5 mm diameter was added to boiling Tris-
HCl buffer (0.02 M, pH 7.8). The supernatant
obtained by centrifugation after boiling for 5 min
was used for ATP analysis. The ATP concentration
was determined by the luciferin-luciferase reaction,
using an ATP bioluminescent assay kit (Sigma
Chemicals Co., St. Louis, MO, USA). ATP at a con-
centration of 10–50 ng mL1 was used as the stan-
dard. The factor value was calculated from the ATP
standard. Factor value = Concentration/Absor-
bance (Karl & Holm-Hansen 1978).
© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Vibrio exclusion by a marine Streptomycete D Augustine et al.
ATP Concentration per wet weight of
biogranule (fg/g) ¼ Relative Light Units (RLU)
 Factor Value  correction factor
 Dilution factor
=wet weight of granule
In vivo exclusion of Vibrio spp. by Streptomyces
rubrolavendulae M56
Experimental animals
A batch of apparently healthy postlarvae of Pena-
eus monodon (PL-18; mean body weight 0.04–
0.05 g; PCR negative for WSSV) were brought
from a commercial prawn hatchery in Kochi
(India). They were transferred to aquarium tanks
of 30 L capacity and acclimatized for 1 week
under laboratory conditions. These larvae were
maintained on control diets for a period of 1 week.
Pathogenicity test of S. rubrolavendulae (M56)
Pathogenicity of S. rubrolavendulae (M56) was
tested on larvae of P. monodon. Apparently healthy
larvae were distributed, 50 each, to 30 L fibre glass
tanks containing 20 L, autoclaved seawater. Spores
of actinomycete strain M56 was inoculated into
nutrient broth medium and incubated for 3–4 days
at 28°C in an incubator shaker until biogranula-
tion. The biogranules were harvested and washed
in sterile saline and approximately, 2 g L1 was
introduced to the test tanks and a control tank was
maintained without biogranules. Water exchange
was not done to create a stressful environment with
compromised water quality. The experiments were
done in triplicates and animals were observed up to
14 days and the survival was recorded.
Experimental design
The postlarvae (50 nos) were stocked in Fibre
Reinforced Plastic (FRP) tanks of 30 L capacity
containing 20 L seawater. Tank without the M56
biogranules was used as the control. The experi-
ments were done in triplicate for each treatment
group and the control group. Both the control
group and treatment group of animals were fed a
commercial diet (Grobest feed ‘smart’ S1). Total
heterotrophic bacterial count and total vibrio
count of rearing water were monitored periodically
every 7 days, by spread plating the samples on
nutrient agar and TCBS agar respectively. Plates
3
Vibrio exclusion by a marine Streptomycete D Augustine et al.
were incubated at 28°C for 24–72 h, and viable
bacterial counts were estimated. All experiments
were conducted in triplicates and animals were
observed for mortality up to 30 days.
Statistical analysis
The experiment data was analysed by means of
one-way ANOVA and Duncan’s multiple compari-
sons of the means. Significance level for the analy-
sis was set to P < 0.05. Statistical analysis was
carried out using the software SPSS 19.0 (SPSS
Inc., Chicago, IL, USA).
Results
Molecular Identification and phylogenetic analysis
of antagonistic strain M56
Sequence analysis revealed that M56 is more clo-
sely related to type strains of S. rubrolavendulae
with 99% similarity on BLAST analysis. Phyloge-
netic tree constructed to study the taxonomic
position with that of 16S rDNA sequences of var-
ious Streptomyces has shown that M56 clustered
with the various S. rubrolavendulae, with a boot
strap support of 100%, consistent with BLAST
analysis. (Fig. 1). The 1132 bp sequence of 16S
4 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Aquaculture Research, 2015, 1–10
rDNA determined was submitted to GenBank
database under the accession number
KJ403746.
Characterization of S. rubrolavendulae M56
Microscopic observation of coverslip culture of the
strain revealed the sporophores as spirales with
20–30 spores per chain. The strain M56 exhibited
growth on starch casein agar, glycerol asparagine
agar and nutrient agar, with pink red aerial
mycelium and off white substrate mycelium. No
diffusible pigments were produced in any of the
media. The strain M56 degraded esculin, urea,
casein, reduced nitrate, decomposition of tyrosine,
xanthine and hypoxanthine was negative. Mela-
nin was not produced in tyrosine agar and pep-
tone yeast extract iron agar. Acid was produced
only from carbohydrates glucose, trehalose and
xylose and no acid production was observed in
media supplemented with lactose, arabinose, inosi-
tol and sorbitol. Streptomyces (M56) was resistant
to antibiotics; penicillin, streptomycin and cepha-
loridine and sensitive to rifampicin, gentamicin,
neomycin and tobramycin. The strain (M56) pro-
duced protease, amylase, lipase, DNase and phos-
phatase and was negative for chitinase and
ligninase activity.
Figure 1 A bootstrapped neigh-
bour-joining phylogenetic tree
obtained using MEGA version 5.0
illustrating relationships between
strain M56 and other related spe-
cies. Values at the node indicate
the percentage with which the par-
ticular node occurred in 1000
trees generated by bootstrapping
the original nucleotide sequence.
Aquaculture Research, 2015, 1–10
Exclusion of Vibrio spp. by S. rubrolavendulae
(M56) – In vitro
In co-culture experiments with S. rubrolavendulae
M56 granules and Vibrio spp. viz., V. harveyi,
V. parahaemolyticus, V. alginolyticus and V. fluvialis,
a gradual decline in viable vibrio count could be
Log cfu mL−1
vibrio count
Log cfu mL−1
vibrio count
Log cfu mL−1
vibrio count
Log cfu mL−1
vibrio count
Figure 2 (a–d) Inhibition of Vibrio
spp. by Streptomyces rubrolavendulae
M56 granules (a) Vibrio parahaemo-
lyticus, (b) Vibrio harveyi, (c) Vibrio
fluvialis, (d) Vibrio alginolyticus.
© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Vibrio exclusion by a marine Streptomycete D Augustine et al.
observed for all co-culture groups, and became
undetectable on the 3rd day (Fig. 2a–d). Cell num-
bers of vibrios treated with M56 biogranule at 24,
48 and 72 h were significantly lower than the
respective control at 48 and 72 h (P < 0.05).
Biogranule of Streptomyces M56 showed a competi-
9 10
8
7 8
Log cfu mL−1
vibrio count
6
5 6
4 4
3
2 2
1
0 0 V.parahemolycus
0 24 48 72
V.para + M56
Time in h
10 10
9 9
8 8
7 7
Log cfu mL−1
vibrio count
6 6
5 5
4 4
3 3
2 2
1 1
0 0 V.harveyi
0 24 48 72
V.har + M56
Time in h
9 9
8 8
7 7
6 6
Log cfu mL−1
vibrio count
5 5
4 4
3 3
2 2
1 1
0 0 V.fluvialis
0 24 48 72
V.fluvialis + M56
Time in h
9 9
8 8
7 7
6 6
Log cfu mL−1
vibrio count
5 5
4 4
3 3
2 2
1 1
0 0 V.alginolycus
0 24 48 72
V.algino + M56
Time in h
5
Vibrio exclusion by a marine Streptomycete D Augustine et al.
tive exclusion effect on V. alginolyticus, V. parahae-
molyticus, V. fluvialis and V. harveyi. The viable
count of V. parahaemolyticus treated with M56
granule decreased from 105 to 102 CFU mL1
within 24 h, and to 101 CFU mL1 at an interval
of 48 h and no detectable count was observed
after 72 h. On treatment of V. harveyi with M56
granule, the viable count showed a steady state
with slight increase in cell number after 24 h and
a drastic decrease in cell count was observed after
a period of 48 h from 105 CFU to 101 CFU mL1
which was undetectable by 72 h. The viable
counts of V. alginolyticus and V. fluvialis showed a
gradual decline from 105 to 104 CFU mL1 after
24 h to a count of 102 in the case of V. alginolyti-
cus after 48 h, while V. fluvialis count decreased to
101 CFU mL1 after 48 h and both the strains
were undetectable after 72 h. The viable counts of
vibrios treated with biogranules (M56) decreased
significantly as compared with the respective
untreated controls by one-way ANOVA.
Biomass estimation
Biomass of the actinomycete biogranule S. rubrola-
vendulae M56 was estimated in terms of ATP and
the ATP concentration was found to be
7.2 9 107 fg/g wet weight of the granule.
Pathogenicity test of S. rubrolavendulae (M56)
No significant mortality of prawns could be
observed by challenge via rearing water.
All the treatments showed above 90% survival
as the control (Fig. 3). The results confirmed the
100 a a
90
80
70
Survival (%)
60
50
40
30
20
10
0 from Bay of Bengal. Actinomycetes produce bio-
PL(control) M56 + PL
Treatment groups
Figure 3 Pathogenicity test of Streptomyces rubrola-
vendulae M56 on Penaeus monodon postlarvae.
6 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Aquaculture Research, 2015, 1–10
non-pathogenicity of the selected actinomycete
M56 to P. monodon postlarvae under experimental
set up. Duncan’s multiple range analysis showed
no significant variation between different groups
and the control when challenged with biogranules
of S. rubrolavendulae (M56) in rearing water.
In vivo exclusion of vibrios in P. monodon larval
culture system
Under natural conditions, the total heterotrophic
bacterial count in actinomycete biogranule treated
group showed an initial decline during the 7th
day, while a slight increase in count was noticed
during the 14th day and a steady state was
observed during the 21st day followed by a slight
decline in count on the 28th day (Fig. 4a). There
was no significant difference (P > 0.05) in total
heterotrophic bacterial count between the control
and biogranule treated group by one way ANOVA
on 0, 7, 14, 21 and 28 days. However, a steady
decline in presumptive vibrio count was observed
in the biogranule treated group up to 28th day. In
the case of the control group, an increase was
observed on 7th day and remained more or less
the same throughout the experimental period
(Fig. 4b). There was no significant reduction in
vibrio count on day 0 (P > 0.05), but significant
reduction in presumptive vibrio count was
observed on 7, 14, 21 and 28 days (P < 0.05)
between the control and biogranule-treated group.
The survival rate after 30 days was better for bi-
ogranule treated group (91.47%), while the con-
trol group had a percentage survival of 77. 51%
(Fig. 5).
Discussion
Screening for novel antimicrobial agents is a con-
tinuous process to match unending demand for
bioactive substances to curtail the phenomenon
of drug resistance. Actinomycetes have been
intensively screened in several underexplored
environments and extreme habitats in various
parts of the world during the last few years. Yet
there are only few reports regarding the bioactive
metabolites produced by actinomycete isolates
active compounds as a response to growth limit-
ing stress conditions such as nutrient limitation
and high cell densities (Demain & Fang 1995;
Bibb 2005).
Aquaculture Research, 2015, 1–10
(a)
Log cfu mL−1
THB
(b)
Vibrio count
log cfu mL−1
Figure 4 (a) Total heterotrophic
bacterial count in Penaeus monodon
postlarval culture systems on
treatment with Streptomyces rubro-
lavendulae M56 biogranule, (b) Pre-
sumptive Vibrio count in Penaeus
monodon postlarval culture systems
on treatment with S. rubrolavendu-
lae M56 biogranule.
100
90
80 b sition and fluid mechanical forces (Braun & Vechtlif-
70
Survival (%)
60
50
40
30
20
10
0
PL(control) M56 + PL
Treatment groups
Figure 5 Percentage survival of Penaeus monodon post-
larvae after treatment with Streptomyces rubrolavendulae
M56 in rearing water.
Actinomycetes are unique among prokaryotes
that they exhibit features of bacteria as well as
fungi. Hence in shake cultures, due to secretion of
extracellular polymeric substances and cell surface
hydrophobicity, the filaments entangle and aggre-
gate to form bead like structures known as biogran-
ules. Earlier studies by Lawton, Whitaker, Odell and
Stowell (1989) have reported the diverse morpho-
logical forms of actinomycetes in submerged
© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Vibrio exclusion by a marine Streptomycete D Augustine et al.
7 7
6 6
5 5
Log cfu mL−1
4 4
THB
3 3
2 2
1 1 PL
M56 + Pl
0 0
0 7 14 21 28
4.5 4.5
4 4
3.5 3.5
3 3
Vibrio count
log cfu mL−1
2.5 2.5
2 2
1.5 1.5
1 1
0.5 0.5
0 0 PL
0 7 14 21 28
M56 + PL
Time elapsed (Days)
a cultures. These morphological forms are modulated
by environmental factors including medium compo-
shitz 1991; Whitaker 1992; O’Cleirigh, Casey,
Walsh & O’Shea 2005; Dobson, O’Cleirigh & O’Shea
2008). Moreover, several works have reported the
correlation between the amount of antibiotic pro-
duced and morphological forms existing within a
culture (Jonsbu, McIntyre & Nielsen 2002; Mante-
ca, Alvarez, Salazar, Yague & Sanchez 2008; Singh,
Wangikar & Jadhav 2012). A high concentration
tends to produce a dispersed form of growth while a
low concentration normally results in pellet forma-
tion (Lawton et al. 1989).
The identification of the bacterial isolates to the
species level based on 16S rDNA sequence analysis
is vital since this provides informative insight
about the organism and novelty of bioactive com-
pounds produced (Adegboye & Babalola 2012).
The strain was identified as S. rubrolavendulae
based on molecular approach. Only very few
reports are available on the occurrence and
distribution of antagonistic Streptomyces in the
marine environment. In the present investigation,
it was observed that S. rubrolavendulae M56 exhib-
ited antagonistic activity against V. harveyi,
7
Vibrio exclusion by a marine Streptomycete D Augustine et al.
V. alginolyticus, V. parahaemolyticus and V. fluvialis
and produced medium sized biogranules.
Application of Streptomyces (M56) in the form of
biogranules could effectively eliminate all of the
four pathogenic Vibrio spp. The reduction in viable
counts of pathogenic vibrios by in vitro exclusion
experiments clearly indicated competitive exclusion
by the selected marine actinomycete biogranule
(M56). There was no apparent inhibition of
selected actinomycete strain by the pathogenic
Vibrio spp. These observations supported the previ-
ous works (Nogami & Maeda 1992; Dalisay, Tor-
res, Penaverde & Rosales 1997) in which, niche
competition between beneficial bacteria and patho-
gens were noticed. Action of marine bacteria
appears to be significant in protecting the host
shrimp against pathogenic bacteria as earlier
reports by Banerjee, Devaraja, Shariff and Yusoff
(2007). Strain M56 produced five different
enzymes, i.e. protease, amylase, lipase, DNase and
phosphatase that could be an antagonistic factor
inhibiting the growth of the Vibrio spp. Similar
observation was reported for Bacillus spp. which
produced enzymes including protease, amylase,
lipase and gelatinase inhibiting growth of the
pathogenic bacteria (Banerjee et al. 2007).
Gregg (1991) reported that ATP estimation of
cell numbers by bioluminescence may agree clo-
sely with colony counts on agar plates for Gram-
positive bacteria. According to Stanley (1989), a
bacterium contains approximately 1015 g (1 fg)
ATP per cell. Hence, it can be estimated that a
M56 biogranule contains approximately
7.2 9 107 cells/g wet weight of biogranule.
According to Liu and Tay (2002) a self-immobi-
lized granule contains millions of organisms per
gram of biomass. The biogranules of S. rubrola-
vendulae (M56) at an initial biomass of
72 9 106 fg/g could significantly eliminate the
Vibrio spp. within 48 h of co-culture. Representa-
tive isolates of V. alginolyticus VaM11 and V. para-
haemolyticus VpM1, when tested for competitive
exclusion using the marine Bacillus spp., showed
decreased viable counts from 108 to 102
CFU mL1 (Banerjee et al. 2007). Likewise, co-cul-
ture experiments with Bacillus subtilis BT23 and
V. harveyi revealed that the growth of V. harveyi
was inhibited by B. subtilis at an initial cell density
of 105–109 CFU mL1 (Vaseeharan & Ramasamy
2003). The results of the present study also sup-
port the previous studies that antagonists are
required at higher concentration than the patho-
8 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–10
Aquaculture Research, 2015, 1–10
gens for elimination (Jayaprakash, Pai, Anas, Pre-
etha, Philip & Singh 2005; Banerjee et al. 2007;
Goulden, Hall, Bourne, Pereg & Hoj 2012).
The better survival of postlarvae in tanks inocu-
lated with strain M56 biogranule could be due to
the extracellular products of S. rubrolavendulae
(M56) preventing the entry of luminescent vibrios
present in the natural system, into the postlarvae,
which could prevent their multiplication in vivo in
postlarvae. This could also be due to the competi-
tion for colonization by the luminescent vibrios
and actinomycete strain (M56) in the natural sys-
tem, which effectively reduces the concentration
of the luminescent vibrios invading the postlarvae.
Although water exchange is not done, degrada-
tion of organic compounds by actinomycetes
enhances mineralization and maintains water
quality in the aquaculture system. This in turn
favours the better survival of P. monodon larvae in
the biogranule treated tank compared to control.
Results of this study indicate that although the
marine actinomycete S. rubrolavendulae M56
reduce the vibrio count in the culture system, the
heterotrophic bacterial count remains more or less
the same.
Conclusion
The present investigation suggests that S. rubrola-
vendulae M56 antagonistic to Vibrio spp. isolated
from marine sediments of Bay of Bengal is a prom-
ising alternative to the use of antibiotics and can
contribute significantly as a biocontrol agent in
prawn larval production systems.
Acknowledgments
The authors are thankful to the Department of
Biotechnology (DBT), Govt. of India for the
research grants (BT/PR 13761/AAQ/03/514/
2010) with which the work was carried out. The
first author thanks the Department of Science and
Technology, Govt. of India for the Research Fel-
lowship. We are also grateful to Cochin University
of Science and Technology for providing necessary
facilities to carry out the work.
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