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Journal of Agricultural Technology
317
Anti-bacterial activity of Caesalpinia coriaria (Jacq.) Willd.
against plant pathogenic Xanthomonas pathovars: an eco-
friendly approach
D.C. Mohana and K.A. Raveesha*.
Agricultural Microbiology Laboratory, Department of Studies in Botany, University of
Mysore, Manasagangotri, Mysore. India.
Mohana, D.C. and Raveesha, K.A. (2006). Anti-bacterial activity of Caesalpinia coriaria
(Jacq.) Willd. against plant pathogenic Xanthomonas pathovars: an eco-friendly approach.
Journal of Agricultural Technology 2(2): 317-327.
Powdered leaf and pod material of Caesalpinia coriaria (Jacq.) Willd. was extracted with
water and successively with different solvents viz., petroleum ether, benzene, chloroform,
methanol and ethanol. Anti-bacterial activity assays of all the extracts against the important
phytopathogenic Xanthomonas pathovars, known to cause diseases in tomato, french bean and
cotton, were carried out by cup diffusion method. Aqueous pod extract showed significant
activity. Among the five solvents extracts tested, methanol extract of both leaf and pod was
most active against all the test bacteria, followed by ethanol extract. Comparison of the
inhibitory activity of the extracts with the antibiotics bacterimycin 2000 and streptocycline
revealed that methanol and ethanol extract of both leaf and pod and aqueous extract of pod
were significantly higher than that of the antibiotics tested. Phytochemical analysis of leaf and
pod materials revealed that antibacterial activity is due to the presence of phenolic and acidic
fraction. Further separation of active fraction resulted in the loss of anti-bacterial activity,
indicating a synergistic effect of the isolated active fraction. The results suggest that C.
coriaria is a potential candidate plant for the management of phytopathogenic Xanthomonas
which are known to cause diseases on cotton, french beans and tomato.
Keywords: anti-bacterial activity, Caesalpinia coriaria, Xanthomonas pathovars
Introduction
Pesticides are an essential input for preventing pre and post harvest crop
losses (Mathur and Tannan, 1998; Saksena, 2001; Wheeler, 2002). Synthetic
pesticides are commonly used to control phytopathogenic microorganisms
(Agrios, 1997). Incessant and extensive use of these synthetic pesticides are
posing serious problem to the life supporting systems due to their residual
toxicity (Ferrer and Cabral, 1991; Gassner et al., 1997; Andrea et al., 2000;
*Corresponding author: Raveesha, K.A.; e-mail: raveesha@sancharnet.in
318
Harris et al., 2001; Campos et al., 2005). It is estimated that hardly 0.1% of the
agro-chemicals used in crop protection reaches the target pest, leaving the
remaining 99.9% to the environment to cause hazards to non target organisms
including humans (Pimentel and Levitan, 1986). The large numbers of
synthetic pesticides have been banned in the western world because of their
undesirable attributes such as high and acute toxicity, long degradation periods,
accumulation in the food chain and an extension of their power to destroy both
useful and harmful pests (Barnard et al., 1997; Wodageneh and Wulp, 1997;
Ortelli et al., 2005).
In-spite of use of all available means of plant protection, about 1/3 of the
yearly harvest of the world is destroyed by pests and loss due to this is
expected to be nearly $300 billion per year (Chandler, 2005). Many
phytopathogenic bacteria have acquired resistance to synthetic pesticides
(Sundin et al., 1994; Clarke et al., 1997; Williams and Heymann, 1998; White
et al., 2002). Pathovars of Xanthomonas are known to cause diseases on
several vegetable and cash crop and are reported to have developed resistance
to kanamycin, ampicillin, penicillin and streptomycin (Weller and Saettler,
1980; Nafade and Verma, 1985; Verma et al., 1989; Bender et al., 1990;
Rodriguez et al., 1997). This seriously hinders the management of diseases of
crops and agricultural products (Dekker, 1987).
Considering the deleterious effects of synthetic pesticides on life
supporting systems, there is an urgent need to search for alternative approaches
for the management of plant pathogenic microorganisms (Hostettmann and
Wolfender, 1997). Green plants represent a reservoir of effective
chemotherapeutants and can provide valuable sources of natural pesticides
(Balandrin et al., 1985; Mahajan and Das, 2003). Biopesticides has been
suggested as an effective substitute for chemicals (Verma and Dubey, 1999;
Kapoor, 2001). Reports are available on the use of several plant by-products,
which posses antimicrobial properties, on several pathogenic bacteria and fungi
(Dorman and Deans, 2000; Parameswari and Latha, 2001; Rath et al., 2001;
Britto and Senthilkumar, 2001; Bylka et al., 2004; Shimpi and Bendre, 2005;
Kilani, 2006), but reports are not available on the evaluation of inhibitory
action of plants extract on phytopathogenic bacteria particularly in different
pathovars of Xanthomonas which are known to cause many diseases in a wide
variety of crops, causing considerable losses in yield and quality. This led the
authors to screen in vitro, a large number of plants for antibacterial activity
against important seed borne phytopathogenic Xanthomonas pathovars, with
the ultimate aim of developing plant based formulations for plant disease
management (Satish et al., 1999; Mohana et al., 2006; Kiran and Raveesha,
2006; Raghavendra et al., 2006).
Journal of Agricultural Technology
319
Caesalpinia coriaria (Jacq.) Willd. distributed in tropical and subtropical
region belongs to the family Caesalpinaceae is used in traditional medicine.
Pods are used in the treatment of bleeding piles. This plant is good for
emollient properties useful in treating freckles and alleviates acute colic pain
(Anon, 2000). Considering these, a detail investigation was conducted to test
the efficacy of the different solvent extracts against important phytopathogenic
bacteria and to identify the bioactive compound responsible for the
antibacterial activity.
Materials and methods
Collection of plant materials
Fresh leaves and pods of Caesalpinia coriaria free from diseases were
collected from Mysore (India), washed thoroughly 2-3 times with running tap
water and once with sterile water, shade-dried, powdered and used for
extraction. A voucher specimen of the plant is deposited in the herbarium of
Department of Studies in Botany, University of Mysore, Mysore.
Preparation of the aqueous extracts
Fifty gm of shade dried, powder of leaves and pods of C. coriaria were
macerated separately with 100 ml of sterile distilled water in a Waring blender
(Waring International, new Hartford, CT, USA) for 10min. The macerate was
first filtered through double layer muslin cloth and then centrifuged at 4000 g
for 30 min. The supernatant was filtered through Whatman No. 1 filter paper
and heat sterilized at 120ºC for 30 min. The extract was preserved aseptically
in a brown bottle at 5ºC until further use. The extract was subjected to
antibacterial activity assay.
Preparation of solvent extractions
Fifty gm of shade dried, powder of both leaf and pod of C. coriaria were
filled separately in the thimble and extracted successively with 200 ml each of
Petroleum ether, Benzene, Chloroform, Methanol and Ethanol using a Soxhlet
extractor for 48 hours. All the extracts were concentrated using rotary flash
evaporator. After complete solvent evaporation, each of these solvent extract
was weighed and preserved at 5ºC in airtight bottles until further use. One gm
of each solvent residue was dissolved in 10 ml of methanol which served as the
test extracts for antibacterial activity assay.
320
Phytochemical analysis of methanol extract
Methanol extract that showed highest antibacterial activity was subjected
to phytochemical analysis (Anon, 1985; Harborne, 1998) and active fraction
separation such as Fraction I (Phenolic compounds), Fraction II (Neutral
compounds), Fraction III (Bases) and Fraction IV (Weaker acids) following the
procedures of Roberts et al. (1981). The active fraction was further resolved by
TLC and column chromatography using silica gel G and H (Merck)
respectively with mobile phases Chloroform : Acetone (1:1.5). All the
corresponding fractions and spots were again subjected to antibacterial activity
assay at 50 µl concentration.
Plant pathogenic bacterial cultures
Authentic pure cultures of phytopathogenic Xanthomonas axonopodis pv
malvacearum (X. a. pv. m) isolated from cotton (Gossypium herbaceum L.),
Xanthomonas axonopodis pv phaseoli (X. a. pv. p) isolated from french bean
(Phaseolus vulgaris L.) and Xanthomonas campestris pv vasicatoria (X. c. pv.
v) isolated from tomato (Lycopersicon esclentum mill.) were obtained from
DANIDA lab, University of Mysore, India.
Anti-bacterial activity assay
Antibacterial activity of aqueous extract, solvent extracts and isolated
constituents was determined by cup diffusion method on nutrient agar medium
(Anon, 1996). Cups were made in nutrient agar plate using sterile cork borer (5
mm) and inoculum containing 106 CFU/ml of bacteria were spread on the solid
plates with a sterile swab moistened with the bacterial suspension. Then 50 µl
each of all aqueous, solvent extracts and isolated constituents were placed in
the cups made in inoculated plates. The treatments also included 50 µl of
sterilized distilled water and methanol separately which served as control.
Antibiotics bacterimycin 2000 (Nitro propane hexadiol) (3 µg/ml) (Source: T.
Stanes and Company Ltd., 23, Race-cource Road, Coimbatore-641018, India)
and streptocycline (Streptomycin sulphate I.P. 90% Tetracycline
Hydrochloride I.P. 10%) (1 µg/ml) (Source: Hindustan Antibiotics Ltd.,
PIMPRI, Pune-411018, India) at their respective recommended dosage were
also treated for activity for comparative efficacy. The plates were incubated for
24 hours at 37ºC and zone of inhibition if any around the wells were measured
in mm (millimeter). For each treatment six replicates were maintained. The
data was subjected to statistical analysis using SPSS for windows software.
The aqueous and methanol extracts of both leaf and pod showed highest
Journal of Agricultural Technology
321
antibacterial activity, were further subjected to antibacterial activity assay at
10, 20, 30, 40, and 50 µl concentrations along with synthetic antibiotics
bacterimycin and streptocycline for comparison.
Results
Anti-bacterial activity
Aqueous extracts
Anti-bacterial activity of aqueous leaf and pod extracts of Caesalpinia
coriaria is presented in Table 1 and 2. Tukey HSD analysis of data revealed
that, with increasing concentration of the aqueous extract, there was increase in
antibacterial activity. Highly significant anti-bacterial activity of the aqueous
extract at 50 µl was observed against all pathovars of Xanthomonas. Among
the phytopathogenic Xanthomonas pathovars, Xanthomonas axonopodis pv.
malvacearum was highly susceptible followed by Xanthomonas axonopodis
pv. phaseoli and Xanthomonas campestris pv. vesicatoria. Pod extract
recorded higher antibacterial activity than leaf extract.
Solvent extracts
The yield of extracts from leaf and pod were petroleum ether (1.8 & 1.1
gm), benzene (0.5 & 0.4 gm), chloroform (1.2 & 0.8 gm), methanol (26.8 &
31.0 gm) and ethanol (3.2 & 2.5 gm) respectively. Anti-bacterial activity of
five different solvent extracts of both leaf and pod of Caesalpinia coriaria and
synthetic antibiotics at 50µl concentration is presented in Table 1. Among the
five solvents tested, methanol extract of both leaf and pod showed highly
significant activity against all the test pathogens followed by ethanol and
petroleum ether extract. Benzene and chloroform extracts of both leaf and pod
did not show any activity against all Xanthomonas pathovars. The anti-
bacterial activity of methanol extract of both leaf and pod of Caesalpinia
coriaria at different concentrations is presented in (Table 2). Tukey HSD
analysis of the data revealed that Xanthomonas axonopodis pv. malvacearum
was highly susceptible among the Xanthomonas pathovars, where as
Xanthomonas campestris pv. vesicatoria showed least inhibition.
322
Table 1. Zone of Inhibitory activity (in millimeter) of different extracts of
Caesalpinia coriaria and antibiotics against some plant pathogenic pathovars
of Xanthomonas at 50 µl concentration.
Extracts X. a. pv.m X. a. pv.p X. c. pv.v
1 Control aqueous C 0.00 0.00 0.00
2 Control methanol C 0.00 0.00 0.00
L 15.80±0.26 15.38±0.26 14.25±0.25
3 Aqueous extract P 21.13±0.29 19.75±036 17.50±0.29
L 12.88±0.29 10.75±0.36 12.38±0.26
4 Petroleum ether
extract P 10.00±0.26 09.00±0.26 10.13±0.29
L 0.00 0.00 0.00
5 Benzene extract P 0.00 0.00 0.00
L 0.00 0.00 0.00
6 Chloroform extract P 0.00 0.00 0.00
L 22.63±0.37 19.63±0.23 19.50±0.32
7 Methanol extract P 19.50±0.18 19.38±0.32 17.50±0.32
L 18.50±0.32 16.75±0.25 16.13±0.29
8 Ethanol extract P 14.00±0.26 15.38±0.18 14.50±0.61
L 18.66±0.33 16.66±0.33 15.66±0.33
9 Methanol extract-
Phenolic fraction P 16.33±0.33 14.33±0.33 12.33±0.33
L 0.00 0.00 0.00
10 Methanol extract-
basic fraction P 0.00 0.00 0.00
L 0.00 0.00 0.00
11 Methanol extract-
Neutral fraction P 0.00 0.00 0.00
L 14.33±0.33 14.00±0.57 12.66±0.33
12 Methanol extract-
Acidic fraction P 12.06±0.33 12.66±0.33 10.33±0.33
13 Streptocycline A 19.9±0.25 16.0±0.026 14.63±0.26
14 Bacterimycin 2000 A 10.00±0.43 11.38±0.026 11.25±0.25
Data given are mean of six replicates ± standard error, p < 0.0001
L- Leaf, P- Pod, C- Control, A-Antibiotic.
X. a. pv.m - Xanthomonas axonopodis pv malvacearum
X. a. pv.p - Xanthomonas axonopodis pv phaseoli
X. c. pv.v - Xanthomonas campestris pv vasicatoria
Journal of Agricultural Technology
323
Table 2. Zone of Inhibitory activity (in millimeter) of aqueous and methanol
extracts of Caesalpinia coriaria and antibiotics against some plant pathogenic
pathovars of Xanthomonas at different concentrations.
Concentrations
Organisms Extracts 10µl 20µl 30µl 40µl 50µl
Aq(L) 7.75±0.25a 9.00±0.26b 10.88±0.29c 13.88±0.29d 15.00±0.26e
Aq(P) 9.50±0.18a 14.00±0.26b 18.00±0.26c 19.75±0.25d 21.13±0.29e
Met(L) 14.87±0.22a 16.50±0.32b 17.88±0.35c 20.75±0.31d 22.50±0.32e
Met(P) 10.25±0.25a 13.63±0.37b 14.63±0.26c 16.13±0.29d 19.00±0.26e
Strept(A) 09.75±0.25a 12.88±0.29b 15.87±0.22c 17.62±0.26d 19.95±0.25e
X .a. pv. m
Bact(A) 00.00±0.00a 07.00±0.26b 08.88±0.35c 10.00±0.26d 10.00±0.43e
Aq(L) 00.00±0.00a 8.25±0.25b 10.13±0.22c 12.00d±0.26d 15.38±0.26e
Aq(P) 9.00±0.26a 14.88±0.29b 16.13±0.29c 18.13±0.29d 19.75±036e
Met(L) 12.25±0.25a 13.38±0.37b 16.13±0.29c 17.50±0.23d 19.25±0.25e
Met(P) 12.88±0.29a 13.38±0.12b 16.25±0.36c 12.38±0.32d 19.83±0.25e
Strept(A) 09.88±0.02a 10.88±1.31b 13.63±0.026c 14.75±0.02d 16.00±0.26e
X. a. pv .p
Bact(A) 00.00±0.00a 06.88±0.02b 08.13±0.022c 10.25±0.02d 11.38±0.26e
Aq(L) 00.00±0.00a 9.16±0.29b 11.13±0.29c 14.00±0.26d 14.25±0.25e
Aq(P) 9.75±0.25a 11.88±0.29b 14.38±0.37c 16.50±0.42d 17.50±0.29e
Met(L) 12.75±0.31a 14.75±0.25b 16.38±0.37c 12.75±0.31d 19.38±0.26e
Met(P) 10.13±0.29a 12.75±0.25b 15.00±0.26c 16.13±0.29d 17.63±0.32e
Strept(A) 08.38±0.18a 09.50±0.18b 10.75±0.25c 12.63±0.26d 14.63±0.26e
X. c. pv. v
Bact(A) 00.00±0.00a 07.13±0.29b 08.50±0.32c 09.88±0.29d 11.25±0.25e
Mean of six replicate ± standard error, The means followed by the same letter(s) are not
significantly different at P< 0.05 when subjected to Tukey HSD (row by row comparisons).
X. a. pv. m - Xanthomonas axonopodis pv malvacearum., X. a. pv. p - Xanthomonas
axonopodis pv phaseoli., X. c. pv. v - Xanthomonas campestris pv vasicatoria.
Aq- Aqueous, Met-Methanol, Strep- Streptocycline, Bact- Bacterimycin 2000, L- Leaf, P- Pod,
A-Antibiotic.
Anti-bacterial activity of the methanol and ethanol extracts (leaf and pod)
and aqueous extract (pod) was highly significant when compared with that of
synthetic antibiotics Streptocycline and Bacterimycin 2000.
Phytochemical analysis of methanol extract
The phytochemical analysis of methanol extracts of both leaf and pod
revealed the presence of carbohydrates and glycosides, protein and amino acid,
phenolic compounds, saponin, tannin, flavonoids, oils, gum and mucilage.
Further phytochemical analysis (Roberts et al., 1981) revealed that the
antibacterial activity of methanol extract is due to the presence of phenolic and
acidic compounds but the activity is less than that of crude methanol extract at
50 µl concentration (Table 1). In TLC separation phenolic fraction showed four
324
bands (Rf values 0.087, 0,333, 0.701, and 0.964) and acidic fraction showed
five bands (Rf values 0.036, 0.079, 0.434, 0.565, and 0.876). Antibacterial
activity was not observed in isolated compounds indicating the loss of
antibacterial activity on further separation of the active fraction.
Discussion
The anti-bacterial activities of aqueous and solvent extracts were
compared with standard Streptocycline and Bacterimycin 2000 and the results
are reported in Table 1 and 2. The results show that the methanol extract of the
plant parts showed more inhibitory effect than the other extracts. This tends to
show that the active ingredients of the plant parts are better extracted with
methanol than other solvents. The absence of antibacterial activity in the
benzene and chloroform extracts indicates the insolubility of the active
ingredients in these solvent. In general the activities against test bacterial
culture used have shown good activity when compared with standard
antibiotics. The phytochemical analysis of methanol extract revealed that the
antibacterial activity is due to the presence of phenolic and acidic compounds
and also observed that the activity is more in combination than separation.
Further separation of the active fraction on TLC showed that the anti-bacterial
activity was not observed in the isolated compounds. It is evident from the
present investigations that the antibacterial activity in the methanol extracts of
the leaf and pod but further separation of the methanol extract results in loss of
antibacterial activity suggesting synergistic activity of the extract. There is a
possibility of synergism between the compounds in a crude decoction than in
isolated constituents (Daniel, 1999).
Field existences of antibiotic resistant phytopathogenic bacteria are
increasing in recent years (Mandavia et al., 1999). WHO banned many
agriculturally important pesticides due to wide range of toxicity against non-
target organisms including humans which are known to cause pollution
problem (Barnard et al., 1997). Some of the developing countries are still using
these pesticides despite their harmful effects. Exploitation of naturally
available chemicals from plants, which retards the reproduction of undesirable
microorganism, would be a more realistic and ecologically sound method for
plant protection and will have a prominent role in the development of future
commercial pesticides (Verma and Dubey, 1999; Gottlieb et al., 2002). Many
reports of antibacterial activity of plants extract against human pathogens and
their pharmaceutical application are available (Cowan, 1999; Cragg et al.,
1999; Newman et al., 2000; Gibbons, 2005), but not much has been done on
the antibacterial activity of plants extract against plant pathogens (Satish et al.,
1999). This is mainly due to lack of information on the screening/evaluation of
Journal of Agricultural Technology
325
diverse plants for their antibacterial potential. Thus the present study reveals
that C. coriaria is a potential candidate plant that could be successfully
exploited for management of the diseases caused by different pathovars of
Xanthomonas which are known to cause many diseases in wide variety of
crops, causing considerable losses in yield and quality in an eco-friendly way.
In the present investigations the antibacterial activity of C. coriaria against
phytopathogenic bacteria has been demonstrated for the first time.
Acknowledgements
Authors are thankful to CSIR and AICTE New Delhi, for providing financial support.
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(Received 9 September 2006; accepted 25 October 2006)
