International Journal of Advanced Biotechnology and Research
ISSN 0976-2612, Online ISSN 2278–599X,
Vol 3, Issue 3, 2012, pp 703-710
http://www.bipublication.com
EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON
ISOLATED STRAINS OF PECTOBACTERIUM SPP.
FROM BANANA PLANTS
Vijay J.Pachupate1 and Pallavi T.Kininge2
1
Department of Biotechnology Engineering,
Tatyasaheb Kore Institute of Engineering and Technology, Warananagar. India
2
Department of Biotechnology Engineering,
Kolhapur Institute of Technology, College of Engineering, Kolhapur.India
*
Corresponding author: Email: pachupate@gmail.com Tel: +91 9890659079
[Received-27/07/2012, Accepted-07/08/2012]
ABSTRACT:
Pectobacterium is one of the most common pathogen affecting banana plant. One of the major species of
Pectobacterium infecting banana plant is Pectobacterium caratovora which causes soft rot at the crown region of
root and the plant eventually dies within 3 months of infection. This infection mainly affects young plants which are
about 1-2 months old. Pectobacterium spp. were isolated on Pectobacterium isolation media from the infected
banana plant tissue. The morphological and cultural characteristics of isolates were studied. After obtaining rich
population of Pectobacterium spp., its pathogenecity on healthy plant was checked. Effect of chemical compounds
namely Streptomycin and Copper Sulphate with variable concentrations were checked on Pectobacterium spp. Two
isolates of Pectobacterium spp. showed resistance to Streptomycin and Copper sulphate.
Keywords: Pectobacterium spp., Banana soft rot, Copper Sulphate, Streptomycin.
INTRODUCTION
Bacterial diseases of plants are usually very
difficult to control and concern over potential
toxicity of pesticides and over the continuing loss
of appropriate, effective pesticides available for
bacterial plant disease control have continued to
increase since the 1970s. Most of the chemicals
are used to control bacterial diseases of the
foliage and of the aboveground parts of plants.
Others are used to disinfect and to protect seeds,
tubers, and bulbs or stored fruits and vegetables
from infection. Some chemicals are used for soil
treatment or disinfection. Chemicals applied on
plants or plant organs can only protect them from
subsequent infection and cannot stop or cure a
disease after it had started, some of these
chemicals have local action, others have a
EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
therapeutic-eradicative-action and several are
translocated systemically by the plants [15,16].
Rhizome rot disease is caused by Pectobacterium
spp. a bacterial genus known for its ability to
cause soft rots on a wide range of host plants and
tissues. These bacteria infect banana plants
through leaves and pseudostems, causing the
following symptoms: wilting or death of leaves
before fruit has ripened, vascular discoloration,
and internal rot of the pseudostem (usually
accompanied by a characteristically foul odor).
Pectobacterium species like E.caratovora,
E.amylovora & E.chrysanthemi mainly causes the
rhizome rot and soft rot disease. Management of
this disease is done through cultural control
tactics such as planting of disease free material,
and prompt identification and eradication of
diseased plants (preferably by burning). [14]
Fire blight, caused by the Gram-negative
bacterium Pectobacterium amylovora, is the most
destructive disease of many rosaceous plants
including apple (Malus domestica), pear (Pyrus
communis) and quince (Cydonia oblonga) [1].
Susceptible plants can be severely damaged or
killed in the nursery and commercial orchards
[2]. No single measure is available for controlling
fire blight [3] but careful plant management and
quarantine measures, together with reliable
disease detection and monitoring, may help
minimizing its spread [4]. Copper containing
sprays are routinely used against a number of
diseases, including fire blight [5] because since
1900, copper-based compounds have been
established as effective bactericides against fire
blight on apples and pears [1]. Copper ranks after
silver and mercury for toxicity, accumulating in
the outer membrane or in the cytoplasm of Gramnegative bacteria. Its toxic effect is expressed as a
growth inhibition of bacterial cultures [6]. If
allowed, the application of an aminoglycoside
antibiotic such as streptomycin would represent
an additional tool for controlling fire blight [7].
Streptomycin limits the normal growth of the
cells by interfering with the proper reading of
Vijay J.Pachupate and Pallavi T.Kininge
messenger RNAs. Streptomycin-resistant strains
of E. amylovora have been detected in several
countries [8] and the genetic basis for resistance
has been extensively studied [9-11]. To cope with
streptomycin, several strategies are deployed by
the pathogen, including alteration of the target
site and production of streptomycin-degrading
enzymes [10]. E. amylovora is currently present
in 46 countries [12,13]. Among chemicals
copper compounds are recommended for the
control of fire blight. Out of several formulations
applied the most common are: copper hydroxide
copper sulphate and lime (Bordeaux mixture) and
copper oxychloride. In addition phosetyl
aluminum had also some effects [17,18].
Although the above mentioned compounds are
quite good preventive bactericides they may
cause rusting problems on leaves and fruits
presumably due to weather conditions, mainly
temperature [2]. Screening for resistance to
streptomycin was according to Burr et al.[19].
Consequently, the objectives of this study was to
assess the effects of copper sulfate
and
streptomycin-resisitivity on isolated stains of
Pectobacterium spp.
MATERIALS AND METHODS
2.1.Screening and collection of infected banana
plants:
Survey of area for plants infected with
Pectobacterium spp. was done.Sample collection
was done from Seema biotech, Talsande. Infected
plant parts and soil samples were brought to the
laboratory in polythene bag and proceeded
immediately for isolation of Pectobacterium spp.
2.2. Sample preparation:
Infected plants were uprooted from bags and was
washed in tap water followed by sterile water,
and dried by paper towelling. Surface sterilization
(3 min in dilution of 0.1% Mercuric chloride)
was done. Again the surface was washed with
sterile D/W to remove traces of mercuric
chloride. Affected tissue (mainly root, leaves)
were removed from young lesion by scalpel,
tissue was crushed in saline.
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EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
2.3. Enrichment and Isolation:
The processed samples were transferred into
Pectobacterium enrichment broth in aseptic
conditions and kept in rotary shaker at room
temperature for 3 days. 0.1 ml of sample from the
enrichment
broth
was
transferred
to
Pectobacterium isolation media by spread plate
method for isolation and kept for incubation at
room temperature for 24 hrs.
2.4.
Characterization
of
isolated
microorganisms:
Isolated microorganisms were confirmed by
Specific Morphological and Biochemical
characteristics.
2.5 Morphological tests:
Gram Staining:
Isolated bacteria were stained with the gram stain
to know the gram nature. Motility of the prepared
suspension was done by hanging drop method. A
loopful suspension was streaked on plate of
nutrient agar and Pectobacterium isolation media.
Plates were incubated at room temperature for
36-48 hours. The colony characteristics of
isolated colonies were studied.
2.6. Biochemical tests:
i. Catalase test:
Some part of the colony of isolate test
microorganism was lifted with sterile loop and
dip in the tube containing 2ml of Hydrogen
peroxide solution, and observed for bubble
evaluation, no bubbling indicates negative test.
ii. Indole production test:
Buffered peptone water medium was inoculated
and incubate at 37ºC. After 24 hours of
incubation few drops of Kovac’s reagent was
added. Formation of a red or pink colored ring at
the top is taken as positive.
2.7.Resistivity Tests
Streptomycin resistivity test:
Colonies grown for 48 hrs. on nutrient agar were
resuspended in phosphate buffer saline and
adjusted spectrophotometrically to a cell density
of 1x 108 CFU/ ml. A suspensions of 100 µl was
spread on nutrient agar plates.
Vijay J.Pachupate and Pallavi T.Kininge
Sterile filter paper discs impregnated with 0, 10,
50, 100 and 500µg/ml was placed on nutrient
agar plates. Nutrient agar plates were kept in a
refrigerator (about 40C) for 10 min to facilitate
diffusion.The plates were then incubated at room
temperature for 24hrs. This experiment was
repeated twice.[19]
Copper sulphate resistivity test:
Test tubes containing10ml nutrient broth of
variable concentrations as 0.08, 0.64, 1.2 and 2.4
mM of copper sulphate were prepared.These
tubes were inoculated with 0.1ml suspension
containing 1x106 CFU, enriched cultures of
Pectobacterium isolates.10 ml of sterile nutrient
broth was used as control.These tubes were
incubated at room temperature for 24 hrs.This
experiment was repeated twice.[19]
2.8. Physiological testing:
(For formation of soft rots in banana plants):
Banana plants were treated with enriched samples
of isolated pure culture. The experiment was
divided in to two parts:
a. Banana plants exposed to Pectobacterium
without injury
b. Banana plants exposed to Pectobacterium
with injury.
RESULTS AND DISCUSSION :
Colour change was observed in Pectobacterium
selective broth after inoculation with samples and
incubation for 2 days at room temperature.
OD was taken at 530nm of Pectobacterium
selective media:
Before
0.18
After
0.43
Ssmall
Fig .1 a
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EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
Catalase test:
The colony of isolate test microorganism was
lifted with sterile loop and dipped in the tube
containing
2ml
of
hydrogen
peroxide
solution.There was evolution of
bubbles
indicating positive test .
Indole production test:
After 24hours of incubation, on addition of few
drops of Kovac’s reagent, red layer was formed
on the surface of the medium ,indicating the
reaction is positive.
3.3 Physiological test results:
Rsoil
Table: 02
Fig .1 b :
Sr
No
Fig .1 a and 1 b. Screening of S small and Rsoil on
Pectobacterium Selective plates
3.1 Colony Characteristics of isolates:
Pinpoint colonies were obtained which formed
light purple on selective media.Isolates were
opaque with moist consistency.These were gram
negative, rod shaped and occurred singly or in
pairs; also motile . A total of 08 isolates were
obtained Sm1 ,Sm2 ,Ssmall , Sb1 ,Sb2 ,Sb3 ,Ssoil
(Smooth) and Rsoil (Rough) .Standard culture
of Pectobacterium caratovora was ordered from
MTCC, culture no: 1428 and was named as Epure.
3.2 Biochemical characterestics:
Table: 03
Samples
Epure
Catalase test
+
Indole production test
+
Sm1
+
+
Sm2
+
Sample
name
No. of plants
treated with
cultured
sample
Period of
infection in
Days
No of
plants
infected
1
E pure
25
45-60
12
2
Sm1
25
45-60
16
3
Sm2
25
45-60
12
4
Small
25
45-60
18
5
Sb1
25
45-60
03
6
Sb2
25
45-60
00
7
Sb3
25
45-60
03
8
Rsoil
25
45-60
15
9
Ssoil
25
45-60
06
TOTAL
225
85
Table: 2. Observation of banana plants after treating
with enriched samples of isolated pure culture
(Infected without injury).37 % of non injured plants
showed infection within 45-60 days .
Table: 03
Sr
No
Sample
name
No. of plants
treated with
cultured sample
Period of
infection in
Days
No of
plants
infected
+
1
Epure
25
7-14
19
2
Sm1
25
7-14
25
3
Sm2
25
7-14
23
Small
+
+
Sb1
+
+
4
Small
25
7-14
20
Sb2
+
+
5
Sb1
25
7-14
20
Sb3
+
+
6
Sb2
25
7-14
25
7
Sb3
25
7-14
23
8
Rsoil
25
7-14
24
9
Ssoil
25
7-14
22
Rsoil
+
+
Ssoil
+
+
Table 1: Results of biochemical tests
+: Positive test
-: Negative test
Vijay J.Pachupate and Pallavi T.Kininge
TOTAL
225
201
Table: 3.Observation of banana plants after treating with
enriched samples of isolated and pure culture (Infected by
706
EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
pseudostem injector). 89% of injured plants showed
infection within 7-14 days.
From tables 01 and 02, it can be concluded that
the plants showed higher rate of infection when
injury is present indicating that Pectobacterium
spp can show increased activity when the plant
rhizomes have injuries.
3.4 Isolation of resistant strains of
Pectobacterium spp:
Total of 08 isolates and pure culture were
checked for resistance against Streptomycin and
Copper Sulphate, out of which two strains – Ssmall
and Rsoil showed resistance.
Streptomycin resistivity test
Fig .4 : Pure culture Epure showed no resistance to 10 ug , 50
ug and 100 ug of Streptomycin.
Sr
No
Conc. in µg/ml
Sample name
0
10
50
100
500
1.
Pectobacterium
caratovora Epure
-
16
22
26
36
2
S- Small
-
-
-
10
30
3
R- Soil
Zone of inhibition in mm
-
-
-
09
22
Table 04 : Zone of inhibition in mm obtained by treating
with Streptomycin
Fig .2 : Isolate Ssmall showing resistance to 10 ug , 50 ug and
100 ug of Streptomycin.
The pure culture showed clear zones of inhibition
for streptomycin at concentrations of 10, 50 and
100 µg indicating sensitivity.Ssmall and Rsoil
showed complete resistance at 10 and 50 µg
concentrations, partial resistance to 100 µg and
sensitivity to 500 µg concentration.
Effect of Streptomycin
40
30
Zone
of Inhibition
in mm
Epure
20
Ssmall
10
Rsoil
0
0
Fig .3: Isolate Rsoill s Isolate Ssmall showing resistance to 10
ug , 50 ug and 100 ug of Streptomycin.
Vijay J.Pachupate and Pallavi T.Kininge
10
50
100
500
Streptomycin Concentration in µg
On X axis: streptomycin conc(µg)
On Y axis: Zone of inhibition (mm)
Graph 01: Comparison of resistivity of Epure , Ssmall and
Rsoil against streptomycin.
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EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
Copper Sulphate resistance test:
Fig:8
Fig:5
Fig:6
Fig 5,6,7,8: Test tubes showing variations in turbidity
at different concentrations of CuSO4
After incubation of Sm1 ,Sm2 ,Ssmall , Sb1 ,Sb2 ,Sb3
,Ssoil, Rsoil and Epure isolates with 0.08 , 0.64,1.2
and 2.4
mM Copper sulphate at
room
temperature for 24hrs, the tubes of isolates Sm1
showed turbidity
,Sm2 ,Ssmall ,Ssoil and Rsoil
indicating resistance. While tubes of isolates , Sb1
,Sb2 ,Sb3 and Epure isolates showed no turbidity
indicating sensitivity.
Sr
Fig:7
Conc. in mM
No
Sample name
1.
Epure
0
0
0
0
2
Sm1
0.2
0.1
0
0
3
Sm2
0.1
0.1
0
0
4
Small
0.6
0.38
0.22
0
5
Sb1
0
0
0
0
6
Sb2
0
0
0
0
7
Sb3
0
0
0
0
8
Rsoil
0.4
0.23
0.1
0
9
Ssoil
0.3
0
0
0
0.08
0.64
1.2
2.4
Table 05 : Optical Density (OD) showing variations in
turbidity at different concentrations of CuSO4
Vijay J.Pachupate and Pallavi T.Kininge
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EFFECT OF COPPER SULPHATE AND STREPTOMYCIN ON ISOLATED STRAINS OF PECTOBACTERIUM SPP
REFERENCES
Effect of Copper Sulfate
1.
0.6
Sm1
0.5
Sm2
0.4
Ssmall
0.3
Sb1
O. D
Sb2
0.2
Sb3
0.1
Ssoil
Rsoil
0
0.08
0.64
1.2
2.4
Epure
Concentration of Copper Sulphate
On X axis: CuSo4 Concentration ( mM)
On Y axis: O.D at 600 nm
Graph 02 : Comparison of resistivity of isolates against
CuSO4
CONCLUSION
Micro organisms responsible for soft rot disease
in banana plants were successfully isolated from
infected plant specimen.Mode and approximate
time of infection was studied and found that rate
of infection by pathogenic Pectobacterium spp. is
more when plant rhizome is injured.Antibacterial
activity of streptomycin and copper sulphate was
checked and found that two strains Ssmall and Rsoil
were resistant to these chemical compounds.
ACKNOWLEDGEMENT
The author is thankful for constant support and
encouragement provided by Dr. S.V. Anekar,
Principle, T.K.I.E.T, Warananagar. Dr.M.
M.Pillai, Head, Department of Biotechnology
Engg, and Dr. Monica Sanandam, Asst. Prof,
KIT’s College of Engg,
is gratefully
acknowledged for their moral support .The author
would also like to thank Mr. Vishwas Chavan,
Chief executive and Mr. Dhumal from Seema
biotech for their consistent support for this
project.
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