3. pnsb as bio-control agents - information and...
Post on 30-Mar-2018
220 Views
Preview:
TRANSCRIPT
3. PNSB AS BIO-CONTROL AGENTS
3.1 INTRODUCTION
Farming of marine shrimps has spread rapidly in South-east and
South Asia, with exception of a few countries, including Myanmar.
Myanmar's neighbors Bangladesh to the north and Thailand to the
south are both major producers of cultured marine shrimps. Culture of
marine shrimp is now spreading rapidly in India. In all these countries
export of cultured marine shrimp is a major earner of foreign exchange
(http://www.fao.org/docrep/field/376565.html). Disease outbreaks and
mass mortalities of cultured shrimp are common now and the industry
is thus crippled economically (Ramasamy, 1995).
Shrimp disease is a major constraint to shrimp aquaculture
production around the world. Since 1994, average annual production
loss due to diseases in this sector in India amounts to about Rupees 350
– 400 crores (MPEDA/NACA. 2003). Pollution/deterioration of the
pond/nutritional imbalance/predisposing lesions cause stress leading to
the secondary invasion of bacteria and result in heavy mortalities.
Bacterial diseases of shrimp are on the body surface (erosion of the
appendages, cuticle), and shell disease (black discoloration/black
spot/brown spot) and tail rot are external infection while the internal
infection occur mainly in the hepatopancreas, gills, muscles, gut etc.
The external infections not only cause mortalities of infected hosts but
also reduce the market value of the shrimp (Ramasamy, 1995). Long
term exposure to even sublethal concentration of pollutants can make
shrimps more susceptible to bacterial diseases. Organisms like Vibrio
spp, Pseudomonas spp, Aeromonas spp, and Flavobacterium spp are
the most common pathogens associated with the disease of naturally
occurring shrimp in sea.
60
Diagnosis of vibrio infection is based on clinical signs and the
histological demonstration of rod-shaped Vibrio bacteria in lesions,
nodules or haemolymph, excised organs and haemolymph may be
streaked on Robert bailey and scotts agar. (Ramasamy, 1995) followed
by TCBS agar.
3.1.1 Vibriosis in shrimps
Vibriosis is a serious problem in shrimp aquaculture, as they are
responsible for higher incidence of shrimp mortality . Species of Vibrio
viz., V. harveyi , V. fischeri, V.parahaemolyticus, V. alginolyticus,
V.anguillarum V. vulnificus and V.splendidus are associated with the
diseased shrimp (Lightner and Lewis, 1975; Lightner, 1993; Huervana
et al., 2006) . Shrimps suffering with vibriosis display localised
lesions of the cuticle typical of bacterial shell disease, localised
infections from puncture wounds, loss of limbs, cloudy musculature,
localised infection of the gut or hepatopancreas and/or general
septicemia, (Takahashi et al., 1985; Lightner, 1993). Mass mortalities
in hatcheries and grow-out ponds of shrimp attributed to outbreaks of
vibriosis, have been recorded from many regions of the world where
shrimp cultivation is done (Couch, 1978; Lightner, 1983; 1985; 1988;
1996; Jiravanichpaisal et al., 1994; Mohney et al., 1994).
Vibriosis is also rampant in the Indian region where brackish
water shrimp farming is the main aquaculture activity. The disease
problem is particularly severe in hatcheries, and in the past few years
many units were shut down due to invasion by luminous vibrios
(Karunasagar et al., 1994; Hameed et al., 1996; Shome et al., 1999).
Vibriosis is also having its impact in grow-out ponds and is frequently
responsible for the morbidity and mortalities of shrimp (Hameed,
1994; Abraham and Manley 1995; Jayasree et al., 2000). Surveys
undertaken on diseases caused by Vibrio spp. in Penaeus monodon
61
from culture ponds of coastal Andhra Pradesh, India, recorded the
occurrence of five types of diseases: tail necrosis, shell disease, red
disease, loose shell syndrome (LSS), and white gut disease (WGD).
Among these, LSS, WGD, and red disease caused mass mortalities in
shrimp culture ponds. Six species of Vibrio viz., V. harveyi, V.
parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus, and
V. splendidus are associated with the diseased shrimp. The number of
Vibrio spp., associated with each disease ranged from two to five.
Additionally, shrimp with red disease had concurrent infections with
white spot syndrome virus. Vibrio harveyi in the case of LSS and
WGD, V. parahaemolyticus for red disease, and V. alginolyticus for
shell disease are the major etiological agents (Jayasree et al., 2000).
The situation is aggravated by the emergence of antibiotic-resistant
strains (Karunasagar et al., 1994) and the ability of Vibrio spp. to form
biofilms (Karunasagar et al., 1996).
Damage to shrimp stocks is frequently associated with bacterial
diseases that are mostly caused by luminous bacteria (Ruangpan et al.,
1997). Luminous vibriosis causes mortalities and termination of grow-
out activities especially in the first 45 days of culture (Lavilla-Pitogo et
al., 1998). Among the luminous Vibrio spp., prevalent in shrimp
ponds, Vibrio harveyi is more pathogenic to shrimps and causes mass
mortalities, and they are oppourtunistic pathogens, when the shrimps
are in a stressed state (Lavilla-Pitogo et al., 1990; 1998; Abraham et al,
2007).
3.1.2 Chemotherapeutic agents against the control of vibriosis in
shrimp ponds
Antibiotics are applied to shrimp pond to reduce the severity of
vibriosis. The normally used antibacterial agents in aquaculture are
Benzylpenicillin, Nitrofurans (furazolidone), Chloramphenicol, and
62
Rifampicin. (Hernandez, 2005) The massive (mis)use of antibiotics to
control infections in aquaculture has resulted in the development of
resistant strains (Defoirdt et al., 2007).
Repeated usage of the antibacterial and other therapeutics in
coastal aquaculture has the potentiality to result in chemical residues
appearing in wild fauna of the local environment. The horizontal
transfer of resistance determinants to human pathogens and the
presence of antibiotic residues in aquaculture products for human
consumption constitute important threats to public health. In recent
years the use of nitrofurans in shrimp culture has been banned in USA,
UK, and other European countries (Hernandez, 2005; Defoirdt et al
2007). Consequently, antibiotics are no longer effective in treating
luminescent vibriosis (Karunasagar et al., 1994). In recent years, there
has been growing interest in biocontrol of microbial pathogens in
aquaculture to make the industry more sustainable.
3.1.3 Biological control of Vibrio spp.,
Sea weed, sea sponges and marine algal extracts have been used
to control luminescent Vibrio spp., shrimp ponds (Selvin and Lipton
2004; Kanagasabhapathy et al., 2005; Huervana et al., 2006; Isnasetyo
et al., 2009). Vibriostatic activity has been reported in several bacterial
members such as, Bacillus spp., (Watchariya and Nontawith, 2007),
Flavobacterium spp., (Qi et al., 2009), Lactobacillus spp., (Ajitha et
al., 2004 and Vieira et al., 2007), Pseudomonas spp., (Bushra et al.,
2009), Phaeobacter spp., (Prado et al., 2009). Some of these bacteria
were reported to be effective in controlling vibriosis, where they were
also used as probiotics. Moriarty (1998), reported that probiotic strains
of Bacillus spp., were used in shrimp culture ponds, which were
effective in controlling luminous Vibrio spp. Apart from Bacillus spp.,
other probiotic bacterial strains, such as Lactobacillus spp., and
63
Pseudomonas spp., are also commonly employed in shrimp culture
ponds to protect shrimp from vibriosis.
Abraham et al. (2001) suggested the use of co-existing bacteria
in the shrimp pond as potential biocontrol agents. Likewise, the
utilization of photosynthetic bacteria as probiotics is a common
practice in many fish or shellfish hatcheries and aquaculture farms in
China and are claimed to have multifunctional effects such as
improvement of water quality, enhancement of growth rate and
prevention of disease (Qi et al., 2009).
3.1.4 Antagonistic potentials of purple non sulfur bacteria
Some facultative anaerobic purple non sulfur bacteria such as
Rhodobacter sphaeroides, Rhodobacter capsulatus and
Rhodopseudomonas palustris have been found to produce bacteriocins
and exhibit inhibitory activity among the closely related species of
purple non sulfur bacteria (Guest, 1974; Wall et al., 1975; Willison et
al., 1987). Kaspari and Klemme (1977) reported about the antibiotic
effects of Rhodobacter sphaeroides against Bacillus subtilis, and found
that the antagonistic activity is not caused by bacteriocins, but by the
metabolites extractable with organic solvents, and further suggested
that the inhibitory substance from the PNSB may be a high molecular
(or) lipophillic substance.
The production of antibiotics by marine bacteria is well
documented (Rosenfeld, and Zobell, 1947; Krassilnikova, 1961;
Burkholder et al., 1966; Andersen et al., 1974; Gauthier, 1976;
Isnansetyo and Kamei, 2003), although, until now, no studies on the
antagonistic potentials of marine photosynthetic purple non sulfur
bacteria, against pathogenic Vibrio spp., have been performed, despite
their wide spread occurrence in shrimp ponds.
64
Keeping this in mind, the present work aims in screening the
shrimp pond havest discharges from brackish and direct sea water
shrimp ponds and screening diseased shrimps in each shrimp farm for
pathogenic Vibrio spp., and studying the antagonistic potentials of
native Purple non sulfur bacterial strains against the pathogenic Vibrio
spp., responsible for vibriosis in Penaeus monodon.
In the light of the above, the present work was carried out with the
following objectives.
1. To isolate and characterize pathogenic Vibrio spp., from harvest
discharges and diseased shrimps.
2. To study the antagonistic potential of native PNSB strains
isolated from shrimp ponds, against pathogenic strains of Vibrio
spp.
65
3.2 MATERIALS AND METHODS
3.2.1 Microbiological screening of shrimps and harvest discharges
with reference to heterotrophic bacteria
3.2.1.1 Selection of diseased shrimps
The tissues of Tiger prawn (Penaeus monodon), from shrimp
ponds exhibiting characters like, erratic / lethargic swimming, eroded
black or brown coloured cuticle, cloudy muscular tissues with necrosis
(Fig. 14a-14f) were chosen for the isolation of heterotrophic bacteria.
3.2.1.2 Selection of healthy shrimps
The tissues from healthy shrimps (Fig.15) that were growing in
other ponds in the same shrimp farm without any sign of infection
were also collected in order to compare them with regard to the
heterotrophic bacterial population with that of the infected shrimps.
3.2.1.3 Isolation and enumeration of heterotrophic bacteria from
infected and healthy tissues of Penaeus monodon. (Ramasamy
1995)
The surfaces of the infected tissues as well as the healthy tissues
were sterilized with a cotton swab soaked with rectified spirit. The
infected as well as healthy tissues were washed with (autoclaved)
0.85% physiological saline solution. One gram of the tissue was
homogenized using a glass tissue homogenizer. The tissue
homogenates were serially diluted and 100µl of homogenized serially
diluted tissue extracts was spread plated on Robert Bailey & Scott‟s
(1966) media (Appendix) and incubated overnight at 37°C. The
resulting colonies were enumerated.
66
3.2.1.4 Isolation and enumeration of heterotrophic bacteria from
shrimp pond harvest discharge
One ml of harvest discharge was serially diluted in 9 ml of
(0.7% NaCl w/v) saline and was spread plated on modified Anderson‟s
marine agar media (Appendix) and incubated overnight at 37°C. The
resulting colonies were enumerated.
3.2.2 Screening of Vibrio spp., using TCBS agar
The colonies arising from Robert bailey and Scott‟s media
(1966) (shrimp tissue homogenate/extract) and modified Andersons
marine agar plates (harvest discharge), with different colony characters
were individually picked and streaked onto the plates containing
Thiosulphate citrate bile salts sucrose (TCBS) agar (Hi-Media,
India)(Appendix) and incubated overnight at 37°C. The presence of
green/greenish-yellow coloured colones indicated the presence of
Vibrio spp.
3.2.3 Purification of test strains
The green/greenish-yellow coloured colonies from TCBS agar
plates were picked and the purification of test strains was done by
repeatedly streaking onto modified Anderson‟s marine agar.
3.2.4 Characterization and identification of Vibrio spp.,
The purified test strains were identified based on simple
taxonomic key (Fig.) developed by Abraham et al. (1999). The key
was designed using the following tests on the basis of their
discrimination power and ease of application. These include growth
on modified Anderson‟s marine agar with and without 3% sodium
chloride (Nacl), gelatinase, arginine dehydrolase (ADH), lysine
decarboxylase (LDC), ornithine decarboxylase (ODC), yellow orange
pigmentation and luminescence on modified Anderson‟s marine agar,
67
utilization of lactose, maltose and Voges-Proskauer reaction.
Additional 5 tests viz. D- glucosamine, growth at 8% Nacl, indole,
arabinose and ONPG (o-nitrophenyl-β-galactopyranoside) were done
to differentiate the stray luminous strains of Vibrio spp. All tests
proposed in the key were performed as per standard methods outlined
in Bergeys manual of systematic bacteriology (Bauman and Schubert,
1984).
3.2.4.1 Morphological, physiological and biochemical
characterization
3.2.4.1.1 Colony characteristics
The colonies of the purified test strains grown on modified
Andersons marine agar was observed for luminescence and colony
characteristics like colony shape, colony colour, texture, pigmentation
(yellow orange) and presence of swarming colonies.
3.2.4.1.2 Physiological and biochemical characters
3.2.4.1.2.1 Growth at various NaCl concentrations
The isolated pure cultures were grown at various NaCl
concentration (0% to 10%) at 37°C in modified Anderson‟s Marine
broth (Appendix) in 10 ml test tubes and growth was observed after 24
hours by analyzing the optical density (OD 600) using a colorimeter at
600 nm. The uninoculated medium with varying NaCl concentrations
served as the blank.
3.2.4.1.2.2 Decarboxylases and arginine dihydrolase test (ODC,
LDC, ADH)
Moeller Decarboxylase broth base (Hi-Media, India)
(Appendix) was prepared and separately added 1.0 gram each of, L-
Ornithine mono-hydrochloride, L-Lysine hydrochloride and L-
Arginine hydrochloride and the medium was heated at 60°C to
68
solubilize all the contents. Dispensed 5 ml of the broth into 10 ml
tubes and 0.5ml of 20% NaCl was added to each test tube, with pH
adjusted to 6.8 and sterilized by autoclaving the tubes at 15 lbs
pressure (121°C) for 15 min. Two loops full of purified test strains
were inoculated into the medium and overlaid the inoculated broth
with sterilized paraffin oil up to 2mm thickness and incubated at 35 ±
2°C for up to 4 days. A tube of basal medium without amino acid was
inoculated in parallel with the test media. A tube containing basal
medium with respective amino acids served as the control. Incubated
tubes were observed daily after 24 hours of incubation and observed
for color change from purple to yellow and then from yellow to purple,
for up to 4 days.
3.2.4.1.2.3 Gelatin Liquefaction
Gelatin salt agar (Hi-Media, India) (Appendix) was prepared
and poured into sterile petri plates. A loop full of culture was streaked
on to the media and incubated at 37°C for 24 – 48 hours. Production of
gelatinase was seen as either a cloudy or clear zone around the area of
bacterial growth. The plates were flooded with ammonium sulphate
which assisted with clear view of zones of clearing. The plate was held
up to the light and read against a darkish background.
3.2.4.1.2.4 Voges-Proskauer Test
MRVP broth (Hi-media, India) was prepared and 5ml of the
broth was evenly distributed into 10 ml test tubes and each tube
containing MRPV broth (Appendix) was supplemented with an
additional 0.5 ml of 20% NaCl, and autoclaved. A loop full of culture
was inoculated and the tubes were incubated aerobically at 35 ± 2°C
for 72 hours. Six drops of Barrit‟s reagent –A (Appendix), and 2 drops
of Barrit‟s reagent –B (Appendix), were added and mixed gently and
69
allowed for colour development up to 1 hour. The appearance of red
colour indicated a positive result.
3.2.4.1.2.5 Indole test
Tryptone broth (Appendix) (Hi-Media, India) was prepared
and 5 ml of the broth was evenly distributed into 10 ml test tubes and
each of the test tubes containing the tryptone broth was supplemented
with an additional 0.5 ml of 20% NaCl and autoclaved. Two loop full
of pure culture was inoculated into the test tubes containing 5ml of
sterile tryptone broth, and incubated aerobically for 48 h at 37°C. After
48 hours, 6–7 drops of Kovac‟s reagent (Appendix) was added to the
test tubes and shaken. Development of a cherry red colour in the upper
reagent layer on top of the broth medium indicates positive result while
no colour development indicates a negative result.
3.2.4.1.2.6 Utilization of organic substrate as carbon sources
The test for sole carbon source utilization was done by
preparing the basal medium according to Bauman et al. (1984)
(Appendix). About, 5ml of basal medium was dispensed into 10 ml test
tubes and autoclaved at 15 lbs pressure (121°C) for 15 minutes. L-
arabinose, lactose, maltose and D-glucosamine were used as test
carbon sources. The test carbon source solutions were prepared at the
conc. of 2% using sterile deionized water and was filter sterilized using
a 0.22 mm membrane filter. Each test carbon source was added in 0.5
ml volumes into the individual test tubes containing sterile basal
medium. The purified test strains measuring 2µL with a McFarland
cell density of 0.5 was aseptically inoculated into tubes containing the
sterile basal medium along with individual test carbon source and
incubated aerobically at 35±2°C for 48 hours. After the incubation
period, the optical density was measured at 600 nm (OD600),
uninoculated tubes served as blank.
70
3.2.4.1.2.7 ONPG (o-nitrophenyl-β-galactopyranoside) Test
Phosphate-ONPG solution was aseptically added to the peptone
water, and dispensed 2.5 ml of ONPG broth (Appendix) into sterile test
tubes. Inoculated a tube of ONPG broth with a loop of culture and
incubated at 35 ± 2 °C for 24–48 hours. The presence of yellow colour
was indicative of a positive result and indicated the presence of the
enzyme, β-galactosidase.
3.2.5 BIOCONTROL OF VIBRIO SPP., USING PNSB STRAINS
3.2.5.1 Screening of PNSB strains for antagonistic activity
3.2.5.1.1 Preparation of crude aqueous extracts from PNSB
strains (Burgees et al., 1991)
The purified PNSB strains, were cultured en-masse, using
modified Biebl and Pfennig‟s (1981) broth filled in 6 litre high grade
glass jars and sealed with noncorrosive stainless steel lid –with
scilicone rubber lining. The medium was poured up to the brim,
leaving no head space, bubbled with argon for 5 minutes and kept
under constant illumination at 2400lux at 30 ± 2°C, for 7 days until the
colour changed to red - brick red/ brown. Mass cultured photosynthetic
bacterial strains were harvested by centrifugation at 4000 rpm for 6
minutes and re-suspended in deionized water and placed in a boiling
water bath for 90 minutes. The cleared supernatant was recovered at
different time intervals, after cooling and used for the bioassay against
Vibrio spp., by the agar well diffusion method.
3.2.5.1.1.1 Agar well diffusion method
The petriplates containing Muller Hinton agar (Appendix) were
seeded with 24hr broth cultures of purified Vibrio spp by swabbing
them on the surface of medium. Agar wells measuring 6 mm in
diameter were cut with the help of sterilized cork borer. Using a
micropipette, 500µL of aqueous PNSB extracts were added into the
71
wells. The plates were incubated in an upright position at 37° for 24
hours. The diameter of inhibition zones was measured in mm and the
results were recorded. The aqueous extracts of PNSB strains showing
inhibitory action against Vibrio spp., were further subjected to disc
diffusion assay and broth tube dilution method to determine their
antagonistic efficiency and MIC (minimum inhibitory concentration)
to bring about vibriocidal activity, by preparing crude intracellular
solvent extracts.
3.2.5.1.2 Extraction of crude intracellular extracts from positive
PNSB strains using chemical solvents (Burgees et al., 1991)
Mass cultured positive PNSB strains (50ml) were evenly
distributed in sterile glass centrifuge tubes and centrifuged at 4000 rpm
for 6 minutes, the supernatant was discarded and the cell pellet was
washed twice by centrifugation, with double deionized distilled water.
The cell pellets was subjected to solvent extraction. Solvents like (E1)
Chloroform: methanol: water (1:2:0.8), (E2) Acetone: methanol (7:2)
and (E3) Toluene: methanol (3:1) were used in the extraction of
intracellular extracts. The intracellular extracts were obtained by
vortexing the cell pellets for 10 minutes suspended in solvents along
with presterilized 0.5mm glass beads and filtered using ultrafine nylon
filter. The solvent extracts were collected and then concentrated in
vacuum at 40°C using a rotary evaporator (Photonix, India), the dry
residues were collected and stored in dry eppendorf tubes. The dry
residues (0.5grams) were further dissolved in 10 ml of 2% dimethyl
sulfoxide (DMSO) (SRL chem. India) for studying the antibacterial
activity by disk diffusion and broth tube dilution method.
3.2.5.1.3 Bioassay of crude PNSB extracts
The sensitivity of purified strains of Vibrio spp., to solvent
extracts like (E1) chloroform: methanol: water (1:2:0.8), (E2) acetone:
72
methanol (7:2) and (E3) toluene: methanol (3:1) of PNSB was tested
by measuring the zone of inhibition of a given concentration of the
extract by the disk-diffusion method (Bauer et al., 1966) and by
determining the minimal inhibitory concentration (MIC) by Broth tube
dilution method.
3.2.5.1.3.1 Bioassay of crude extracts by disc diffusion (Bauer et al.,
1966)
The broth cultures containing various Vibrio spp., isolated from
infected shrimps and harvest discharges were streaked on to Andersons
Marine agar plates and incubated aerobically at 35±2°C for 24 hours
and the individual colonies were picked and diluted to approximately
108 CFU/mL, according to the 0.5 McFarland standard (Roe-Carpenter,
2007) (Appendix). 0.1ml of the inoculum were spreaded on Mueller-
Hinton agar incorporated with 2 % Nacl and sterile paper disks (6 mm
in diameter) were laid on the inoculated substrate after being soaked
with 15 μL of PNSB extract at a concentration of 50 mg/ml. The plates
were incubated for 24 h at 37°C. Antimicrobial activity was
determined by measuring the diameter of the zone of inhibition around
the disk. As a positive control of growth inhibition, Chloramphenicol
30 μg/ml (Hi-media) antibiotic disc (6mm) was used.
3.2.5.1.3.2 Bioassay of crude extracts by broth tube dilution
(Qaiyumi, 2007 modified)
The broth tube dilution assay was performed to determine the
minimal inhibitory concentration (MIC) of the solvent extracts over
Vibrio spp.Twelve sterile screw capped test tubes were labeled from 1
to 12. One ml of solvent (DMSO) was taken in tube 1 (negative
control). One ml of sterile Mueller Hinton broth was taken in each of
the tubes from 3-12. About 1 ml of working test extract (dissolved in
2% DMSO with a conc. of 50mg. /ml) was taken in tube 2 and another
73
Broth tube dilution test
1 m
L o
f M
&H
bro
th +
1m
L o
f t
est
extr
act
so
luti
on
1m
l o
f te
st e
xtr
act
fro
m P
NS
B i
sola
te
at t
he
con
c. (
50
mg
/mL
) +
1m
L i
no
culu
m
1m
l o
f D
MS
O +
1m
L i
no
culu
m
1ml 1ml 1ml 1ml 1ml 1ml 1ml Discard 1 ml
1 2 3 4 5 6 7 8 9 10 11 12
1mL Mueller Hinton broth
Final Conc. (mg/mL) 50 25 12.5 6.25 3.12 1.56 0.78 0.39
Note: Inoculum of the test pathogen was added in all the tubes except the tube 12.
Tube 11 serves as control and receives no test extract solution.
Tube 12 contains only 1 ml of M&H broth without inoculum, i.e broth control
The concentration of the working test extract solution due to the doubling dilutions from the tube 3 to tube 10, ranges from 50mg/ml to 0.39 mg/ml
74
1ml of the test extract solution was added to tube 3. One ml of the
solution (working test extract + Mueller Hinton broth) was transferred
from tube 3 to 4 and this process of transfer was continued through
tube 10. About 1ml from tube 10 was discarded. Tube 11 serves as
control and receives no working test extract solution.
Isolated colonies from an overnight culture of the test pathogen
growing in Andersons marine agar plates were diluted in the Mueller
Hinton broth to a turbidity comparable to that of a 0.5 Mc Farland
turbidity standard (approximately 1.0 × 108 CFU/mL). This suspension
was further diluted 1:100 (106) with Mueller Hinton broth. Within 15
minutes of the preparation of this suspension of test pathogen 1ml of
the bacterial broth suspension was added to each tube except the 12th
(last) tube which serves as broth control tube.
The tubes were incubated at 37°C for 24 hours. The presence of
turbidity in the tubes indicates growth. The boundary dilutions having
lowest concentration of test extract dilution containing tubes without
any visible growth was taken as the minimal inhibitory concentration
(MIC) against test Vibrio spp. Cfu/ml was determined by spreading
10µl of test pathogen suspension from tube 11, onto Andersons marine
agar plate and incubating overnight at 37°C.
75
3.3 RESULTS
3.3.1 Microbiological screening of shrimps and harvest discharges
with reference to heterotrophic bacteria
The diseased shrimps and harvest discharges, when subjected to
microbiological screening using Robert Bailey & Scott‟s (1966)
medium and modified Anderson‟s marine agar plates yielded bacterial
colonies with different colony colour (pale white, greyish white,
orange, pale orange-yellow), size and colonial morphology . The
tissue extracts from the healthy, diseased shrimp samples and harvest
discharges from various stations that were subjected to serial dilution
and spread plating onto Robert Bailey & Scott‟s (1966) and modified
Anderson‟s marine agar plates respectively (Fig.16a-17), yielded
microbial colonies ranging from 8.90 ×105 to 2.07 × 10
6(tissue
extracts) and 1.0 × 106 to 2.13 × 10
6 (harvest discharges) (Tables 21
and 22).
Heterotrophic bacterial count was more in diseased shrimp
samples than from healthy shrimp samples in all four stations.
However in the Karankadu station, shrimps showing erratic swimming
(indicating diseased nature) had significant heterotrophic bacterial
counts. Harvest discharge samples also contained heterotrophic
bacterial counts comparable to diseased shrimp samples. Types of
shrimp diseases prevalent in various stations are as follows. Red
disease was found in 3 stations viz., Vadakkupoygainallur, Pappakovil
and Karankadu. Shell disease and necrotic lesions in the abdomen was
found in 2 stations viz., Vadakkupoygainallur and Pappakovil. Loose
shell disease and erratic swimming was found in 2 stations viz.,
Sethubavachatram and Karankadu. White spot disease was found only
in one station, Karankadu.
76
3.3.2 Screening for Vibrio spp., using TCBS agar
All the colonies representing samples from shrimp tissues and
harvest discharge were either green/yellow or yellowish green and thus
indicated the presence of Vibrio spp., (Tables 23 – 26; Fig.18).
3.3.3 Purification of test strains
Green/yellow or yellowish green colonies from TCBS agar
plates were purified by streaking onto plates containing modified
Andersons marine agar. A total number of 66 strains were reported
after purification. The details of the purified strains with reference to
their growth in modified Anderson‟s marine agar are given in tables 23
to 26.
3.3.4 Characterization and identification of strains
The characterization details of the 66 strains are given in tables
27 to 30. The identification of the strains based on the simple
taxonomic key developed by Abraham et al. (1999) revealed that
majority of the strains (60) belonged to Vibrio spp., and the remaining
(6) strains belonged to Photobacterium spp. Details regarding the
species identification of the 66 strains are given in table 31, and their
prevalence across the stations are given in table 32. List of shrimp
pathogens in each station is given in the table 33. For the biological
control of shrimp pathogens using indigenous PNSB strains three
species of shrimp pathogens namely Vibrio harveyi, Vibrio fischeri and
Vibrio alginolyticus were used as test pathogens.
77
3.3.5 Antagonistic activity of purple non sulfur bacterial strains
against Vibrio spp.,
3.3.5.1 Screening of PNSB strains for Antagonistic activity by agar
well diffusion method
Screening of antagonistic activity of PNSB strains using their
hot water extracts (aqueous) by agar well diffusion (Fig.19) revealed
the following. BRP1, BRP2, BRP3, BRP4, BRP6, BRP10 and BRP11
did not show antagonistic activity against Vibrio harveyi and Vibrio
fischeri. Besides the above mentioned strains BRP5 and BRP7 also
did not show antagonistic activity against Vibrio alginolyticus. Among
the strains showing antagonistic activity, the aqueous extracts of BRP9
require a minimum boiling time of 30 minutes, BRP8 requires 40
minutes, BRP5 requires 50 minutes and BRP12 requires an hour of
heating to show antagonistic activity. However prolonged heating up to
90 minutes did not have any negative effect on the antagonistic
activity. Among the strains showing antagonistic activity against
Vibrio harveyi, BRP9 (26.03±0.33) ranks first followed by BRP12
(20.60±0.65), BRP8 (15.87±0.62), BRP5 (12.10±0.62) and BRP7
(11.83±0.5). Against Vibrio fischeri, again BRP9 showed maximum
antagonistic activity (22.50±0.17) followed by BRP12 (16.20±0.41),
BRP7 (11.40±0.24), BRP8 (9.40±0.22) and BRP5 (9.07±0.26). In the
case of Vibrio alginolyticus again BRP9 registered maximum
antagonistic activity (23.40±0.24) followed by BRP8 (9.23±0.05) and
BRP12 (9.13±0.4). The PNSB strains exhibiting antagonistic activity
against 3 strains of Vibrio spp., is summarized in the tables 34 to 36.
As only 5 strains of PNSB (BRP5, BRP7, BRP8 and BRP12) showed
antagonistic activity against the test Vibrio strains, they alone were
considered for further bioassay against test Vibrio strains using disk
diffusion method and broth tube dilution method.
78
3.3.5.2 Bioassay of crude PNSB extracts by disk diffusion
From the disk diffusion assay (Fig.20) for assessing antagonistic
activity of different solvent extracts of PNSB strains, the following
observations were made. Among the PNSB stains BRP9 showed
maximum antagonistic activity (27.50±0.34), followed by BRP12
(21.23±0.17), BRP8 (21.17±0.12), BRP5 (12.20±0.08), and BRP7
(12.17±0.05) against Vibrio harveyi. Against Vibrio fischeri, BRP9
(23.83±0.26) was found to be more effective, followed by BRP12
(18.07±0.74), BRP5 (12.20±0.22), BRP7 (11.20±0.22), and BRP8
(10.17±0.17). BRP9 showed maximum inhibition (23.17±0.17) against
Vibrio alginolyticus followed by BRP8 (11.07±0.09) and BRP12
(9.00±0.22). Of the different solvents used for extracting PNSB
extracts E1 (Chloroform:methanol:water (1:2:0.8)) was found to be
more effective, since it showed maximum antagonistic activity against
all the three Vibrio spp. Against Vibrio harveyi and Vibrio fischeri, the
E1 extracts of BRP9 registered significant antagonistic activity and it is
almost on par with the antibiotic standard viz. Chloramphenicol
(30µg). However E2 (acetone:methanol (7:2)) and E3
(Toluene:methanol (3:1)) extracts also showed inhibitory potentials.
The inhibitory potentials of the various PNSB strains varied depending
on the solvents used (Table 37).
3.3.5.3 Bioassay of crude extracts by broth tube dilution
Broth tube dilution test performed to find out the minimum
inhibitory concentration (MIC) of the different solvent extracts of
PNSB strains revealed the following (Table 38). Among the strains
BRP9 was the most effective, confirming the results of earlier two
assays. Solvent E1 could bring out the inhibitory potential of the PNSB
strains better compared to E2 and E3. The various tests undertaken for
the study of antagonistic activity of PNSB strains brought to light that
79
Rhodobacter sphaeroides (BRP9) could be considered as a potential
antagonistic agent for the control of Vibrio spp., that are pathogenic to
shrimps.
80
3.4 DISCUSSION
In shrimp culture ecosystem, most of the bacteria play a
negative role as they compete with shrimps for food and oxygen,
causing stress and disease (Moriarty, 1997). Due to the influence of
turbidity, heavy organic matter plays a major role in the distribution of
bacterial population in pond water (Sharmila et al., 1996).
In the present study the enumeration of total heterotrophic
bacteria from the harvest discharge and shrimps (healthy and diseased)
was done in order to quantify the total heterotrophic bacterial counts
using modified Andersons marine agar for harvest discharge and
Robert Bailey & scott‟s (1966) media for shrimp tissue extracts. The
shrimp tissue extracts and harvest discharge yielded bacterial cells
ranging from 8.9 × 105 to 1.05 × 10
6 cell/mg(healthy shrimps), 1.04 ×
106 to 9.30 × 10
6 cells /mg (Diseased), likewise the total heterotrophic
bacteria from harvest discharges from the four stations yielded
bacterial cells ranging from 1.00 × 106
to 2.13 × 106 cells /ml of
sample. From the results obtained one can ascertain that the
heterotrophic bacterial count is lesser in the healthy shrimps than that
of the diseased shrimp and the level of the heterotrophic bacteria in the
harvest discharge is also higher. This may be due to the fact that the
population of heterotrophic bacteria tends to increase, as the days of
culture progressed (Abraham et al., 2004; Ganesh et al., 2010). The
higher heterotrophic bacterial counts in the harvest discharges may due
to the abundance and availability of nutrients derived from excess feed,
shrimp excreta and other dead and decaying organic matter, which is
conducive for bacterial growth, as observed by Abraham et al., (2004).
Among the total heterotrophic bacteria (THB), the members of
the Genus Vibrio spp., play a vital role in shrimp culture ecosystem
(Ruangpan and Kitao, 1991) as they damage water quality causing
81
diseases and mortality to the shrimp as primary and secondary
pathogens. Vibrio spp., is part of the autochthonous flora of the coastal
pond ecosystem (Aiyamperumal, 1992; Ruangpan and Tabkaew,
1994).
Bacteria of the genus Vibrio are ubiquitous in marine and
estuarine aquatic ecosystems in which shrimp occur naturally and are
farmed. Several Vibrio spp. form part of the natural biota of fish and
shellfish (Vanderzant et al., 1971; Colwell, 1984; Ruangpan and Kitao,
1991; Otta et al., 1999). Some of the Vibrio species such as V. harveyi
and Vibrio parahaemolyticus are also associated with bacterial
infections in shrimp (Lightner, 1993; Jiravanichpaisal and Miyazaki,
1995; Lavilla-Pitogo, 1995) and are generally considered to be
opportunistic pathogens causing disease when shrimps are stressed
(Gopal et al., 2005). In shrimp farms from India, Otta et al., (1999) and
Vaseeharan and Ramasamy (2003) noted that Vibrio spp., accounted
for 38–81% of the bacterial biota.
In the present study, the shrimps showing symptoms of various
types of shrimp diseases could be observed. For example, Shell disease
was observed in shrimps collected from the stations
Vadakkupoygainallur and Pappakovil, Red disease could be observed
in shrimps in the station Vadakkupoygainallur , Pappakovil and
Karankadu, necrosis in the abdomen could be observed in the stations
Vadakkupoygainallur and Pappakovil, loose shell in
Sethuvbavachatram and Karankadu, White spot disease could be found
in only one station viz., Sethubavachatram and erratic swimming in
diseased shrimps could be observed in two stations viz.,
Sethubavachatram and Karankadu. Shrimp diseases, like shell disease,
red disease, necrosis, loose shell and white spot disease are commonly
found in many regions of the world where shrimp cultivation is done.
82
(Lightner, 1988; Sindermann, 1990; Nash et al., 1992; Yang et al.,
1992; Hameed, 1994; Abraham and Manley, 1995; Hameed et al.,
1996). In India too the above mentioned diseases are common and
prevalent in shrimps as observed by Jayasree et al. (2006), in the
shrimp ponds of Andhrapradesh. Different species of Vibrio are
associated with shrimp diseases.
3.4.1 Purification, Isolation and characterization of pathogenic
Vibrio spp.,
The colonies arising in Robert Bailey and Scotts media and
modified Andersons marine agar were streaked onto TCBS agar and all
the colonies representing samples from shrimp tissue and harvest
discharge from all stations were either green/yellow or yellowish green
and thus indicated the presence of Vibrio spp. The occurrence of
various Vibrio spp., in water, sediment and shrimp samples from
multiple shrimp farm environments was also reported from the east and
west coast of India by Gopal et al. (2005). Further purification of
green/yellow /yellowish green colour colonies on modified Andersons
marine agar, yielded a total of 66 strains.
These purified (66) strains of Vibrio spp., were characterized
and identified using the simple taxonomic key developed by Abraham
et al. (1999), as they had better discriminatory power and ease of
application. The species identified include, Vibrio fischeri, V.
mediterranei, V.harveyi, V.orientalis, V.splendidus, V. logei, V.
alginolyticus, V.damsela and V. marinus. Among them the
nomenclature of last two species has been revised and the new names
are Photobacterium damselae for V. damsela (Osorio et al., 1999) and
Moritella marina for V. marinus (Urakawa et al., 1998) respectively.
The percentage of prevalence of various strains of Vibrio spp., in all
the 4 stations are as follows, Vibrio fischeri (7.69%), Vibrio
83
mediterrranei (28.84%),Vibrio harveyi (34.61%), Vibrio orientalis
(3.84%), Vibrio splendidus (7.69%),Vibrio logei (13.46%) and Vibrio
alginolyticus (3.84%). The prevalence of some of the Vibrio spp., viz.,
Vibrio harveyi (94.86%), V. splendidus (4.57%) and V. fischeri
(0.57%) reported from shrimp farms by Abraham et al. (2007), show a
pattern that has been replicated in the present study also.
Among the strains of Vibrio isolated in the present study, the
percentage of luminous Vibrio spp., was 37.87% and the non luminous
Vibrio spp., was 62.12%. The higher prevalence of non luminous
vibrios in shrimp ponds was also reported by Ruangpan (1998). With
regard to the prevalence of pathogenic Vibrio spp., in each station, V.
fischeri and V. splendidus could be isolated in the healthy shrimps
from two stations viz., Vadakkupoygainallur and Pappakovil (brackish)
respectively, but pathogenic Vibrio spp., could not be isolated from the
healthy shrimps collected from the stations Sethubavachatram and
Karankadu (direct sea water). Vibrio harveyi could be isolated from the
shrimps exhibiting shell disease from the stations
Vadakkupoygainallur and Pappakovil. Likewise Vibrio fischeri and
Vibrio splendidus, could be isolated from the shrimps showing shell
diseases from the station Vadakkupogainallur.
Vibrio fischeri, though considered to be pathogenic to shrimps,
were reported to be isolated from shrimp pond only. However isolation
of the same species from the tissues of diseased shrimp has been
reported by Chandrasekaran and Kumar (2011), which to the best of
our knowledge is the first report on the isolation of V. fischeri from
diseased shrimps in the natural environments though Manilal et al.
(2010) reported the pathogenic ability of the above species under
invitro conditions. Among the three stations showing red disease viz.,
Vadakkupoygainallur, Pappakovil, and Karankadu, only Vibrio harveyi
84
could be isolated, and that too in one station (Karankadu) only. The
shrimps exhibiting necrosis were found only in two stations viz.,
Vadakkupoygainallur and Pappakovil. Vibrio harveyi and Vibrio
splendidus could be isolated from the necrotic sample collected from
Vadakkupoygainallur, but shrimp sample exhibiting necrosis collected
from Pappakovil yielded Vibrio alginolyticus.
Loose shell syndrome could be observed only in two stations
viz., Sethubavachatram and Karankadu and both the samples yielded
Vibrio harveyi. White spot disease could be observed only in one
station (Sethubavachatram) and Vibrio harveyi could be isolated. In the
shrimps showing erratic swimming obtained from two stations viz.,
Sethubavachatram and Karankadu, Vibrio harveyi could be isolated
only from Sethubavachatram. The number of pathogenic Vibrio spp.,
isolated from each type of shrimp disease ranged from 1 to 3, of which
Vibrio harveyi was prevalent in all the diseased shrimps, but non
prevalent in healthy shrimps. V. harveyi, could be isolated from the
harvest discharges in stations viz., Vadakkupoygainallur, Pappakovil,
and Sethubavachatram. Likewise Vibrio fischeri could be isolated in
the harvest discharge only from one station (Vadakkupoygainallur).
Vibrio alginolyticus and Vibrio splendidus could not be isolated from
harvest discharges from any of the stations.
The disease wise prevalence of pathogenic Vibrio spp., are as
follows, Vibrio harveyi was isolated from shrimps exhibiting shell
disease, red disease, necrosis of the abdominal region, loose shell,
white spot syndrome, and also in the shrimps exhibiting erratic
swimming. Vibrio alginolyticus was isolated in shrimps exhibiting
necrosis of the abdominal region, Vibrio fischeri was isolated from
shrimps exhibiting shell disease and Vibrio splendidus from the
shrimps exhibiting shell disease and necrosis of the abdominal region.
85
In an earlier report on the isolation of pathogenic Vibrio spp., from
diseased shrimps in India by Jayasree et al. (2006), Vibrio harveyi and
Vibrio alginolylticus was isolated from shrimps showing loose shell
syndrome, and red disease, Vibrio splendidus was isolated from loose
shell syndrome alone. Likewise Vibrio fischeri, was isolated from
shrimp pond water, where there was incidence of shell disease by
Manilal et al. (2010) and established that Vibrio fischeri as moderate
opportunistic pathogen. In other words, none of the species of Vibrio
could be termed as specific to any particular type of vibriosis.
Among the pathogenic Vibrio spp., isolated from diseased
shrimps Vibrio harveyi was dominant and have been documented in
the previous studies of Abraham et al. (2003;2004;2007), possibly
because of its ability to grow well in shrimp pond environment with
wide ranging conditions of physicochemical parameters, which might
have favored the prevalence of Vibrio harveyi in cultured shrimps also.
The occurrence of luminous bacteria shows the inherent fertility of the
shrimp pond environment providing media, for bacterial growth and
favoring luminescent vibrios. The effect of pond soil on water quality
had been recognized by Chen et al. (1992) and its fertility was
apparently influenced by the accumulated excess feed, waste products,
and shrimp exuviae. Although it was shown that thorough drying of
soil eliminates Vibrio, populations that get established within ponds
always enter with the incoming water (Moriarty, 1997; Lavila-pitogo et
al., 1998).
Traditionally antibiotics have been used in attempts to control
bacterial disease in aquaculture (Defoirdt et al., 2007). But antibiotics
are no longer effective and the prevalence of antibiotic resistant strains
in the shrimp aquaculture has become an issue of concern
(Karunasagar et al., 1994; Alderman and Hastings, 1998). The massive
86
usage of commercially available antibiotics results in natural
emergence of antibiotic-resistance bacteria, which can transfer their
resistance genes to other bacteria that have never been exposed to
antibiotics (Austin et al., 1995; Moriarty, 1997).
This has led to the, suggestion on the usage of non pathogenic
bacteria as probiotic biocontrol agents instead of antibiotics (Fuller,
1978; Gatesoupe, 1999; Mishra et al., 2001). The use of probiotics is
prevalent in the aquaculture industry (particularly in shrimp culture) as
a means of controlling disease and in turn improving water quality by
balancing bacterial populations and nutrient (e.g., nitrogen and
phosphorus) concentrations. Probiotics that have been examined for
use in shrimp aquaculture include bacteria, yeasts, bacteriophage, and
microalgae (Park et al., 2000; Iriano and Austin, 2002; Ajitha et al.,
2004; Balcazar et al., 2006; Farzanfar, 2006). Vibriostatic activity has
been observed by Abraham (2004) in bacterial members belonging to
the Genera Alteromonas spp., and they were effective against Vibrio
harveyi. Prado et al. (2009) reported on the inhibitory activity of
Phaeobacter strain pp154 against shrimp pathogens like Vibrio
alginolyticus, Vibrio fischeri and Vibrio harveyi.
The antibacterial effect of probiotic bacteria results from factors
such as the production of antibiotics, bacteriocins, sideropheros,
lysozyme, protease, hydrogen peroxide, the alteration of pH values,
and the production of organic acids and ammonia (Verschuere et al.,
2000). Lactic acid bacteria and Bacillus produce several compounds
that may inhibit the growth of competing bacteria. Among these
compounds, the bacteriocins are the most important (Gildberg et al.,
1997; Ali, 2000). These are proteins, or protein complexes, produced
by certain strains of bacteria that can have an antagonistic action
against species that are closely related to the producer bacterium.
87
Bacteriocins are divided into four classes: class-I antibiotics; class-II
small hydrophobic, heat-stable peptides; class-III large heat-stable
peptides; and class IV complex bacteriocins: probiotics with lipid
and/or carbohydrate (Fooks and Gibson, 2002).
Purple non sulfur bacteria do produce bacteriocins and
antibiotics (Guest, 1974; Wall et al., 1975; Willison et al., 1987;
Kaspari and Klemme, 1977). A study on the role of antagonistic
bacteria, especially the co-existing bacteria as biocontrol agents
appears worthwhile in lieu of the negative impacts of antibiotics
(Abraham et al., 2001). PNSB are found to co-exist in shrimp ponds
along with Vibrio spp., in the present study and this has prompted us to
investigate the antagonistic potentials of PNSB members against
pathogenic Vibrio spp. The present study focuses on the biocontrol of
three, pathogenic Vibrio spp., viz., V. harveyi, V. fischeri, and V.
alginolyticus, isolated from shrimps and harvest discharges, using
native purple non sulfur bacteria.
3.4.2 Antagonistic activity of purple non sulfur bacterial strains
against Vibrio spp.,
The antagonistic study was performed by subjecting pathogenic
Vibrio spp., to the aqueous extracts of 12 PNSB strains and the
influence of boiling time was also checked for the antagonistic effect
on the pathogenic Vibrio spp. The aqueous extracts of 12 PNSB strains
(BRP1 to BRP12), were tested for their antagonistic activity by agar
well diffusion method. The strains BRP1, BRP2, BRP3, BRP4, BRP6,
BRP10 and BRP11 could not elicit antagonistic activity against the 3
strains of pathogenic Vibrio spp., viz., V. harveyi, V. fischeri, and V.
alginolyticus. Likewise BRP5 and BRP7 did not show any antagonistic
activity against the pathogen Vibrio alginolyticus. Among the 12
PNSB strains screened for antagonistic activity against the 3 strains of
88
pathogenic Vibrio spp., only 5 PNSB strains showed antagonistic
activity. The positive PNSB strains are as follows: Rhodobacter
spheroides (BRP9), Rhodobacter capsulatus (BRP12), Rhodobium
marinum (BRP8), Rhodovulum sulphidophilum (BRP5) and
Rhodobium orientis (BRP7). However the last two species were
negative in their antagonistic response to V.alginolyticus. Against
Vibrio harveyi, the PNSB strains BRP9 showed highest inhibitory
activity followed by BRP12, BRP8, and BRP5and BRP7. Against
Vibrio fischeri BRP9 showed maximum antagonistic activity followed
by BRP12, BRP7, BRP8, and BRP5. Against Vibrio alginolyticus the
only 3 PNSB strains viz., BRP9, BRP8 and BRP12 showed maximum
antagonistic activity. The inhibitory activity of the aqueous extracts of
the PNSB isolates was influenced by heating and found to be time
dependent in eliciting antagonistic activity against the pathogenic
Vibrio spp.
Thus the supernatants of BRP9 started to inhibit the V. harveyi
strains only after 30 min of boiling, BRP8 after 40 min., BRP5 and
BRP7 after 50 min. and BRP12 after 60 min. For the antagonistic
activity against Vibrio fischeri the aqueous extracts of BRP9 and BRP7
required a minimum heating time of 50 min to inhibit followed by
BRP12, BRP8 and BRP5, which required 60 minutes of heating to
produce antagonistic activity. In the case of antagonistic activity
against Vibrio alginolyticus the aqueous extract of BRP8 showed
inhibiton after 40 minutes of heating, BRP12 after 60 min and BRP9
after 70 min. of heating. In all the positive strains of PNSB, the zones
of inhibition increased with the time of heating up to 90 min.
This indicates that the antibacterial compounds are intracellular
as observed by Burgees et al. (1991) and the bioactive compounds are
thermostable, even during prolonged periods of boiling (upto 90 min.),
89
a characteristic feature observed in non protein compounds of
Pseudomonas aeruginosa CMG1066 by Bushra et al. (2009), thus
suggesting that the bioactive compound might be a non-protein
compound.
Among the PNSB strains that tested positive for antagonistic
interaction against pathogenic Vibrio spp., by disk diffusion method,
Rhodobacter sphaeroides (BRP9) was found to be most efficient
strain, in terms of its antagonistic activity against all the three species
of Vibrio tested using E1 solvent extracts (chloroform:methanol:water)
than others.
Vibriostatic activity has been observed in Bacillus spp.
(Watchariya and Nontawith, 2007), Flavobacterium spp. (Qi et al.,
2009), Lactobacillus spp. (Ajitha et al., 2004; Vieira et al., 2007),
Pseudomonas spp. (Bushra et al., 2009) and Phaeobacter spp. (Prado
et al., 2009). The reports available on the utilization of PNSB against
marine Vibrio sp., or any other shrimp pathogen are rather limited. The
effectiveness of utilizing these anoxygenic purple non-sulphur bacteria
depends on the outcome of extensive research in this perspective in the
coming future. Now with the outcome of this study it is possible to
consider using PNSB as candidates to control Vibriosis in Penaid
shrimps and the scope is wide open to harness the bio-pharmaceutical
potential of Rba. sphaeroides as well. However invivo studies are
required to confirm the above findings in the pond environment.
top related