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IJPRD, 2014; Vol 6(08);October-2014 (070 - 075)
International Standard Serial Number 0974 – 9446
-------------------------------------------------------------------------------------------------------------------------------------------------IN-VITRO INHIBITORY ACTIVITY OF COW URINE WITH HELICTERES ISORA LINN. FRUIT EXTRACTS AGAINST
COPPER RESISTANT XANTHOMONAS SP
Suryavanshi Mangesh V.1*,
Hingamire Tejashri B.2 AND Shaikh Nasir R.3
1
Microbial Culture Collection, National Centre for Cell Science, NCCS Complex,
University of Pune Campus, Ganeshkhind, Pune, M.S.(INDIA)-411 007.
2
Biochemical Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, M.S.(INDIA)-411 008.
3
P.G. Department of Microbiology, Yashwantrao Chavan College of Science, Karad, M.S.(INDIA)-415 124.
ABSTRACT
In this study, an herbal preparation containing aqueous and
methanolic extracts of Helicteres isora Linn. fruit with cow urine,
was evaluated for its inhibitory potential against phytopathogenic strains of Xanthomonas isolated from Bacterial
Blight Disease of Pomegranate (Punica granatum Linn.). These
isolates showed resistance towards Cupric Sulphate (130 μg ml-1)
and were sensitive to Streptomycin (MIC – 9.0 μg ml-1). Growth
inhibition activity of cow urine, aqueous and methanolic extracts
of Helicteres isora Linn. fruit and there combinatorial
formulations were tested against standard Streptomycin towards
Xanthomonas isolate by agar well diffusion assay. The present
work reports that combination of cow urine with fruit extracts
have potential antibacterial activity and can be used as a potent
antiseptic preparation for prevention and treatment of Bacterial
Blight Disease of Pomegranate in an eco-friendly manner.
Correspondence Author
Suryavanshi Mangesh V
Microbial Culture Collection, National
Centre for Cell Science, NCCS
Complex, University of Pune Campus,
Ganeshkhind, Pune,
M.S.(INDIA)-411 007.
Keywords- Copper resistant, Xanthomonas sp, Bacterial Blight
Disease, Cow urine, Helicteres isora Linn. fruit etc.
INTRODUCTION:
Xanthomonas axonopodis pv. punicae bacterium is
known to produce Bacterial Blight Disease, a major
constraint of export quality pomegranate (Punica
granatum Linn.) production in India and worldwide
as well; which is also known as ‘oily spot’ or ‘Tailya’
in Maharashtra1. Since three decades products
containing copper have been used to manage this
disease throughout the year. But these days such
products are failing to protect the pomegranate
crops from infection. The ineffectiveness report of
such disease management products to many plant
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International Journal of Pharmaceutical Research & Development
pathogenic bacteria, including Xanthomonas
pathovars have developed resistance to copper as
well as Streptomycin2, 3, 4. Because of easy
availability, eco-friendly nature of bioactive
substances derived from cow urine and the
medicinal plants; which are used to restrict the
infection have been a great interest to researchers.
Cow urine has provided massive toxic potential to
kill drug resistant bacterial strains5 and also shows
adequate bio-enhancer activity6. As the part of
medicinal plant, Helicteres isora Linn. fruits are
known to have astringent, also provides inhibitory
activity against enteric pathogenic bacteria7, fungal
vegetation with greater efficiency8, 9.
Considering developing resistance to copper, the
present study was carried out to determine the
sensitivity to copper and streptomycin sulfate of
Xanthomonas strains isolated from Solapur district
(Maharashtra) during the years 2008-2009.
Consequently, the inhibitory potential of cow urine
and fruit extracts of Helicteres isora Linn. against
these isolates independently and in combinatorial
manner.
MATERIALS AND METHODS
Isolation and Identification of Xanthomonas sp.
The diseased fruits of pomegranate collected from
affected orchards from Boholi, Pandharpur Tahsils
of Solapur district. Isolation and Identification for
the isolates were done by using standard test
methods as described in Laboratory Guide for
Identification of Plant Pathogenic Bacteria10 and
Xanthomonadin pigment detection method11.
Screening for sensitivity
streptomycin sulfate
to
copper
ISSN: 0974 – 9446
Fruit extract, Cow urine and Combinational
Formulations for antibacterial activity:
The fruits of Helicteres isora Linn. were collected
from Botanical garden which authenticated by
Department of Botany, Yashwantrao Chavan
College of Science, Karad. The preparation of
aqueous and methanolic extracts from shade dried
fruits of H. isora Linn. were done by earlier
report15. Fresh cow urine sample was collected
from “Kandhar” variety cow from local Cow yard
established in the vicinity of college area (at Karad,
Maharashtra) using sterile container and filter
sterilized with 0.22 μm Millipore membrane and
this stored at 5ºC for further uses.
Combinatorial formulation preparation i.e. the
various combinations of cow urine with aqueous &
methanolic extract of Helicteres isora Linn. fruits
prepared as 1:1, 1:2,1:3, 3:1, 3:2 ratios for isolate &
tested for antibacterial activity along with standard
streptomycin.
Antibacterial
activity
was
determined by agar well diffusion method on
nutrient agar medium16.
RESULTS AND DISCUSSION
The three bacterial isolates viz. P1, P2 and P3 were
isolated from the three different diseased fruit
samples on Sucrose Peptone Agar (SPA). From
Gram
properties,
morphological,
cultural,
biochemical and physiological characteristics with
reference to Laboratory Guide for Identification of
Plant Pathogenic Bacteria10, the isolates were
identified as Xanthomonas axonopodis pv. puniciae
which was similar to previously characterized by
Higorani and Singh 19591 (data were not shown
here).
and
Minimum Inhibitory Concentration (MIC)
for
isolates was determined using Serial tube dilution
technique12 and the Xanthomonas strains were
screened for sensitivity to copper and streptomycin
sulfate (Sigma Aldrich Inc) as described method13, 14
.
Sensitivity to copper and streptomycin sulfate
Tube Dilution Method accordingly gave the
absence of growth of all isolates in tube containing
09 μg ml-1 and above Streptomycin concentration,
which represents the MIC of Streptomycin sulphate
against isolates. And SPA amended with different
concentrations of cupric sulfate (30-150 μg ml-1
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
range) were used for screening and it was also
found that the Xanthomomas isolates grows on
130 μg ml-1cupric sulphate but not on 135 μg ml-1.
Copper resistance screening data were similar to
results reported by Ritchie and Dittapongpitch14,
and Sahin and Miller17 on Xanthomonas campestris
pv. vesicatoria copper sensitivity and found to
resistant. Interestingly, all the isolated strains
altered their colony morphology characters on SPA
amended with 130 μg ml-1 cupric sulfate as yellow
coloured, round, rough with irregular margin
(Figure 1).This phenotypic variation may be due to
the elevated copper concentration, where as
copper-induced colony morphology change was
traced in Saccharomyces cerevisiae18 and genotypic
level in Xanthomonas pathovars19.
Figure 1: Colony morphology of Xanthomonas
isolates- a) Isolates on SPA (without cupric
sulphate) and b) Isolates on SPA (with 130 μg ml1
cupric sulphate).
Figure 2: Standard Streptomycin inhibition curve
(regression) against P3 isolate.
Figure 3: Inhibitory activity by cow urine, fruit
extract and their combinational formulations
against P3 isolate.
Inhibitory activity by cow urine, fruit extract and
their combinational formulations
As all isolates belonging to same strain, only P3
isolate was selected for the inhibitory assays. The
inhibitory activity of cow urine, aqueous and
methanolic extracts of H. isora Linn. fruit, against
the P3 isolate as shown in Table 1. Aqueous and
methanolic extract of H. isora Linn. fruit showed
about 8 mm and 10 mm zone of inhibition
diameter respectively which was equivalent to
tested std. streptomycin concentration of 10.1 ±0.8
μg ml-1and 25.2 ±0.5 μg ml-1 for the P3 isolate
(standard inhibitory curve by streptomycin is
shown in Figure 2).
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
Table 1: Antibacterial activity of cow urine, aqueous & methanolic extracts of Helicteres isora Linn. Fruit and in
combinatorial formulations against P3 isolate.
Gr.A Formulations
Diameter Zone of Inhibition
(Mean± SD)$
a) Cow urine
Equivalent to Std
streptomycin activity in mL
(Mean± SEM)$
7.01 ±0.03 mm
9.5 ±0.4 μg
b) Aqueous extract of H. isora Fruit
8.05 ±0.03 mm
10.1 ±0.8 μg
c) Methanolic extract of H. isora Fruit
10.1 ±0.04 mm
25.2±0.5 μg
d) 10% Methanol
Gr.B Combinatorial
formulations¥
6.01 ±0.02 mm
U:Ae
ND
U:Me
U:Ae
U:Me
a)
1:1
9.01 ±0.03 mm
12.1 ±0.1 mm
20.1 ±0.3 μg
40.1 ±0.5 μg
b)
1:2
8 .07 ±0.02 mm
13.04 ±0.03 mm
11.3 ±0.9 μg
51.8 ±0.6 μg
c)
1:3
8.08 ±0.03 mm
11.01 ±0.03 mm
12.4 ±0.3 μg
27.1 ±0.7 μg
d)
2:1
9.02 ±0.03 mm
11.05 ±0.02 mm
15.1 ±0.5 μg
28.3 ±0.8 μg
e)
3:1
10.09 ±0.03 mm
10.12 ±0.03 mm
25.1 ±0.7 μg
25.9 ±0.4 μg
¥
$
Concentration in ratio
Mean of three replications
ND = Non Detectable
U:Ae = Cow urine : Aqueous extract of H.isora Linn.fruit
U:Me = Cow urine : Methanol extract of H.isora Linn.fruit.
In combinatorial way, formulations viz. cow urine
to aqueous extract of H. isora Linn. fruit and cow
urine to methanolic extract of H. isora Linn. fruit in
varied ratio were tested against P3 isolate. As in
1:1 ratio formulation, methanolic extract gave
higher equivalent activity 40.1 ±0.5 μg ml-1 to that
of aqueous extract and 1:2 ratio formulation gave
higher activity to that of all formulations, which
showed equivalent activity to 51.8 ±0.65 μg ml-1 std
streptomycin concentration. The inhibitory activity
was observed lower in aqueous extract of H. isora
Linn. fruit at 1:2 ratio, this could be possibly due to
the cow urine rendering the effects in different
proportion, comparative with lower ratio
formulations like 1:1 ratio and with higher ratios.
Representation of inhibitory activity in campanile
format generated through ‘ggplot2’ in R- statistical
program20 (shown in figure 3). In regards to above
observation, suggestively we can hypothesize that
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the cow urine can enhance the bio-activity of
extract in methanol than aqueous phase.
Interestingly, 3:1 ratio formulation of aqueous as
well as methanol extract of H .isora Linn. fruit has
given notably the equal activity.
The inhibitory activity of cow urine, H. isora fruit
extracts
and
their
herbal
combinatorial
formulations was found significant against copper
resistant strains of Xanthomonas isolates. In our
knowledge, there are no previous reports dealing
with antibacterial activity of the tested plant
extracts selected against phyto-pathogens in cited
literature. In parallel, Yadav et. al. 9 were used
herbal preparations containing Dalbergia sissoo
and Datura stramoium with cow urine (DSDS),
evaluated for its antibacterial potential against
human pathogenic bacterial strains and the result
shows that the cow urine extract of DSDS may be
used as a potent antiseptic preparation. Secondly,
73
International Journal of Pharmaceutical Research & Development
some herbal extracts with cow urine were
demonstrated antibacterial activity against
Xanthomonas axonopodis21.
SUMMARY
Our data demonstrates:
1. Emergence of copper resistant been observed in
Xanthomonas strain and further studies needs to
be done in other production regions.
2. H. isora fruit extracts have great potential as
antimicrobial
compounds
against
human
22
pathogenic microorganisms . Similarly, they can
be used in the treatment of Bacterial Blight Disease
caused by copper resistant phyto-pathogenic
Xanthomomas species.
3. The effect of cow urine with H. isora fruit
extracts against resistant bacteria may leads to
new remedies for controlling phyto-pathological
diseases consequences. A conjoint use of naturally
occurring antibacterial substances and their
consortium might be proving useful in control of
disease. Development of commercially valued
combinatorial formulation will be the future
prospect of these studies, which may be achieved
through extensive field trials and experiments.
ACKNOWLEDGEMENT
This work has been taken as a part, from M.Sc.
dissertation work carried by Mangesh Suryavanshi.
All authors are thankful to Head, Department of
Microbiology and Management of Yashwantrao
Chavan College of Science, Karad (India) for the
facilities and help rendered in this work.
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