Assessment of the antibacterial activity of Cuscuta pedicellata Ledeb

In search for alternative ways for bacterial fruit lesions control, we evaluated the aqueous decoction and aqueous infusion extract of Cuscuta pedicellata L. for their antibacterial activity against different pathovar of Xanthomonas campestris, the causal agent of this disease. The collected leaves were washed, dried and powdered. Aqueous decoction and aqueous infusion extract were prepared and observed for their antibacterial efficacy by using the well diffusion method in vitro. The significant results obtained showed that both extracts tested inhibited the bacterial growth of pathogen with inhibition zone diameter ranging from 1.0 to 5.0 cm. The aqueous decoction revealed strongest antibacterial activity on X. campestris pv. mangiferaeindicae and X. campestris pv. punicae whereas aqueous infusion extract showed strongest antibacterial activity on X. campestris pv. citri and X. campestris pv. pinicae. The susceptibility of different pathovars of X. campestris to this plant extracts varied. The study indicates the potential of this plant extracts in the management of diseases caused by X. campestris in several important crop plants.


INTRODUCTION
At present, quick and effective management of plant diseases and microbial contamination in several agricultural commodities is generally achieved by the use of synthetic pesticides (Agrios, 2005).However, the incessant and indiscriminate application of these chemical pesticides has caused health hazards in animals and humans due to their residual toxicity (Anonymous, 1998).This seriously hinders the control of crops diseases and agricultural product.Considering the deleterious effects of synthetic pesticides on life supporting systems, there is an urgent need for alternative agents for the management of pathogenic micro-organisms (Bolkan and Reinert, 1994;Rice et al., 1998).Biological control is an alternative to chemicals in the control of plant pathogens, or in order to reduce environmental pollution.It has been described as a non-hazardous strategy to reduce crop damage caused by plant pathogens (Frommell and Pazos, 1993;Cook et al., 1995).Xanthomonas campestris can infect a wide variety of species including pepper, rice, citrus, cotton, tomato and soybeans and causes visible symptoms and shedding leaves.The disease results in reductions in market value and fruit yield (Boch and Bonas, 2010;Gagnevin et al., 1997).Recently, evaluation of plant extracts against many Xanthomonas species is becoming an important area.Thus, a focus should also be on indigenous practices of the farmer to look for their effectiveness.Especially, the indigenous knowledge on plants is important.Cuscuta pedicellata belong to the family Convolvulaceae also known as Dodder.It is a leafless, green, yellowish and thread like twinning herb.
The stem shows antimicrobial activity against many fungi and bacteria (Faiyyaz et al., 2011).
In the light of these above enumerated facts, the present study was carried out on biological control of X. campestris isolated from different fruit lesions by using aqueous extracts of C. pedicellata (in vitro).

Collection of plant materials
The fresh stems of C. pedicellata were collected from various places of University of the Punjab, Lahore, Pakistan.The stems were washed under running tap water and shade dried for three weeks.The dried leaves were then homogenized by using a grinder to make fine powder and stored in air tight bottles.

Preparation of aqueous infusion
Aqueous infusion of C. pedicellata was prepared by steeping 20 g of powder in 60 ml sterile distilled water in sterile flasks.The flasks were kept for 48 h at room temperature with occasional shaking.The contents of flasks were filtered (Chaudhry and Tariq, 2008).

Preparation of aqueous decoction
Aqueous decoction of C. pedicellata was prepared by boiling 20 g of powder in 60 ml sterile distilled water over low flame for 15 min.The flasks were then plugged and removed from heat and allowed to cool.After cooling, the contents of flask were filtered (Chaudhry and Tariq, 2008).

Isolation and identification of pathogenic X. campestris species
X. campestris was isolated from different diseased fruit samples (Table 1) by serial dilution method.The infected fruits were surface sterilized with 70% ethanol and washed thoroughly with sterile water.A small infected piece was suspended and thoroughly mixed in 10 ml of sterile water in sterilized test tube.A sterile pipette was used to transfer 1 ml of this suspension to a tube containing 9 ml of sterile water.This process was continued until ten times of dilutions were made and inoculated on Luria-Bertani (L.B) agar media plate by streak plating method and incubated at 37°C for 24 h (Waller et al., 1998).Identification of bacterial species was done by recording macroscopic and microscopic characteristics.The purified colonies were subjected to Gram staining and characterized using biochemical tests and consulting the pertinent literature (Holt et al., 2000;Koneman et al., 1997;Benson, 1996).

Screening for the antibacterial activity of the aqueous extracts
The well diffusion method was used to detect the antibacterial activity of aqueous infusion and aqueous decoction extracts (Okeke et al., 2001).The standard bacterial suspension (McFarland 0.5) was made for each isolate of X. campestris.Eighty microlitres (80 µl) of bacterial inoculum of each isolate were spread on the agar surface using sterile cotton swab.The plates were allowed to dry and a sterile cork borer of diameter 0.8 cm was used to bore wells in the agar plates.Subsequently, 60 µL volume of the each aqueous extract was introduced in wells into L.B plate and incubated at 37°C for 24 h.Distilled water was used as negative controls.At the end of the period, inhibition zones around the paper discs were measured and compared with the controls.All tests were conducted in three repetitions arranged as one well in three plates for each extract (Talibi et al., 2011).Antibacterial activity Index was calculated as: Activity index (AI) = Da/ Db -1 Where: Da is the diameter (cm) of the growth zone in the experimental dish and Db is the diameter of the growth zone in the control dish.

Statistical evaluation
The antibacterial activity was determined by measuring the diameter of zone of inhibition, that is, the mean of triplicates ± SE of three replicates.

RESULTS
The antibacterial efficacy of aqueous decoction and aqueous infusion extracts of C. pedicellata were determined in vitro using the well diffusion method.Results obtained show that the two aqueous extracts inhibited different pathovar of X. campestris with inhibition zone diameter ranging from 1.0 to 5.0 cm (Table 2 and Figure 1).

Inhibitory effect of aqueous decoction extract of C. pedicellata on pathogen
The aqueous decoction extracts show evidence of high  antibacterial activity against X. campestris pv.punicae (5.0 cm) and X. campestris pv.mangiferaeindicae with 4.0 cm inhibition zone.X. campestris pv.citri has least resistance against the extract (2.1 cm) as compared to other pathovar species.In the case of X. campestris pv.musacearum and X. campestris pv.Pruni, extract showed comparable behavior by moderately active with 2.8 and 2.5 cm, respectively.Although, X. campestris pv.vesicatoria exhibited intermediate inhibitory effect (3.0cm) against aqueous decoction extract of C. pedicellata.

Inhibitory effect of aqueous infusion extract of C. pedicellata on pathogen
The aqueous infusion extract of C. pedicellata was found to be highly active against all pathogenic organisms tested.The extract showed high activity (5.0 cm) against X. campestris pv.citri in comparison with moderate activity of X. campestris pv.mangiferaeindicae and X. campestris pv.musacearum with inhibition zone of 3.2 and 3.1 cm, respectively.The extract was also more active against X. campestris pv.punicae (4.5 cm), whereas the least activity has shown X. campestris pv.pruni (1.8 cm).The growth of X. campestris pv.vesicatoria has lesser amount and moderately inhibited by the aqueous infusion extract of C. pedicellata with the zone of inhibition of 2.2 cm.

DISCUSSION
In this study, effects of antibacterial activity of C. pedicellata were tested against different pathovar of X. campestris in in vitro condition.Both extracts appear to be promising as a biocontrol agent against the pathogen.X. campestris is a rod-shaped, Gram-negative bacte-rium which causes bacterial blight, common blight, fuscous blight, cankers and leaf spots in French bean, cotton, paddy, tomato, etc.Although losses due to the pathogen are difficult to estimate, it is known to cause significant yield loss (Satish et al., 2002).Many antibiotics are used to control these diseases.The increased awareness of the environmental problems associated with these antibiotics has led to the search for non-conventional chemicals of biological origin for the management of these diseases.Bactericides of plant origin can be one approach to disease management because of their ecofriendly nature (Bolkan and Reinert, 1994).The products of plant origin are of greater advantage to the user, the public, and the radical environmentalists.
Testing the antimicrobial activity of plants remains an area of intense interest.Many reports are available on the antiviral, antifungal, antibacterial, antihelmintic, antimolluscal and anti-inflammatory properties of plant (Samy and Ignacimuthu, 2000;Palombo and Semple, 2001;Kumaraswamy et al., 2002;Stepanovic et al., 2003;Bylka and Misra, 2004;Behra et al., 2005;Govindarajan et al., 2006;Malik and Singh, 2010).Despite serious environmental implications associated with the excessive use of chemicals, it still remains the first line of defense against plant pathogen.When these fungicides are consumed by human beings and animals through food and water, they cause various ailments in the body.Whereas search of natural herbicides from the plant sources would definitely be a better alternative to these hazardous chemicals.
Laboratory screening of Cuscuta extract have given encouraging results, indicating their potential use in the management of diseases caused by Xanthomonas species.In another study, the antimicrobial activity of Cuscuta species against Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus), Gram negative bac-teria (Escherichia coli and Pseudomonas aeruginosa), fungi (Penicillin citrium and Aspergillus niger) was done and results are summarized by Faiyyaz et al. (2011).
Extract of stem of Cuscuta species showed the presence of alkaloids, carbohydrates, some glycosides, flavonoids, tannins, phenolic compounds, steroids and shows antimicrobial activity.The current results support the findings of Faiyyaz et al. (2011) who demonstrated the presence of antimicrobial activity in C. pedicellata extracts and have also reported the presence of the above mentioned antimicrobial compounds.
Thus, it can be concluded that the present experiment demonstrates that the stem extracts of C. pedicellata exhibit antibacterial effect in the present experiment which offers a scientific basis for using C. pedicellata as a good sources of antibacterial compounds.Further work is underway in the laboratory to screen for their efficacy in managing different bacterial diseases in a variety of crops.

Figure 1 .
Figure 1.Percentage inhibition of aqueous decoction and aqueous infusion extracts of C. pedicellata on different pathovars of X. campestris.

Table 1 .
Pathovar of X. campestris isolated from diseased fruits

Table 2 .
Effect of C. pedicellata extract on the growth of different pathovars of X. campestris.