ANTIBACTERIAL SCREENING OF A FEW MEDICINAL FERNS AGAINST ANTIBIOTIC RESISTANT PHYTO PATHOGEN

ANTIBACTERIAL SCREENING OF A FEW MEDICINAL FERNS AGAINST ANTIBIOTIC RESISTANT PHYTO PATHOGEN

D. Herin Sheeba Gracelin*, A. John De Britto and P. Benjamin Jeya Rathna Kumar

Plant Molecular Biology Research Unit, Post Graduate and Research Department of Plant Biology and Biotechnology, St. Xavier's College (Autonomous), Palayamkottai - 627 002, Tamil Nadu, India

ABSTRACT:Centella asiatica is a medicinal plant. It is severely affected by leaf spot disease caused by a harmful, antibiotic resistant phyto pathogen Xanthomonas campestris pv. centellae (X. c. pv. c). Farmers used synthetic pesticides to control the disease. When pesticides are applied to control this pathogen, pesticides may cause environmental hazards and health problems in man. But traditionally plants are used to prevent or control the plant diseases in agriculture. It is an eco- friendly, biocontrol, traditional method. Hence the aim of the present study is to control the pathogen in biocontrol method by screening the phytochemicals and antibacterial activity of petroleum ether, chloroform, benzene, methanol and aqueous extracts of fronds of five ferns (Pteridophytes) Adiantum caudatum, Angiopteris evecta, Pteris confusa, Pteris argyraea and Lygodium microphyllum against X. c. pv. c. The methanol extracts of all the ferns gave successful result against the tested bacteria. Phytochemical analysis of all the extracts revealed that antibacterial activity is due to the presence of alkaloids, flavonoids and phenolic compounds. According to the results of MIC (Minimum Inhibitory Concentration) and RPI (Relative Percentage Inhibition) values, Angiopteris evecta could be used as potential plant for the management of pathogenic bacteria, X. c. pv. c which is known to cause diseases on many vegetables and cash crops particularly Centella asiatica.

INTRODUCTION:A bacterial leaf spot disease caused by Xanthomonas campestris pv. centellae (X. c. pv. c) has been the major problem in cultivation of Centella asiatica. To control the disease causing pathogens, number of synthetic pesticides and antibiotics are used by the formers. But pesticides cause environmental pollution and many unwanted effects in man.

Pesticides have made great contribution for quick and effective management of plant diseases and microbial contaminations in several agricultural commodities. Considering the deleterious effects of synthetic pesticides on life supporting system, there is an urgent need for alternative agents for the management of pathogenic microorganisms ¹.

Pteridophytes are not infected by microbial pathogens, which may be one of the important factors for the evolutionary success of pteridophytes and the fact that they survived for more than 350 million years ².

Considering the rich diversity of Indian medicinal plants including Pteridophytes, it is expected that, the screening of plant extract for antibacterial activity may be beneficial for humans and plants diseases. The synergistic interaction among crude extracts or the active compounds may be useful in the preparation of improved herbal or drug formulations. The extensive survey of antibiotic activity among the ferns conducted and reported about a hundred species having such property ³. Traditionally people used pteridophytes as medicine and anti bacterial agents. Therefore the aim of the present study is to investigate the phytochemical analysis and in vitro antibacterial activity of five solvent extracts of five ferns against gram-negative plant pathogenic bacteria.

MATERIALS AND METHODS:

Preparation of Plant Extracts: Healthy, disease free leaves of five ferns Adiantum caudatum, Angiopteris evecta, Pteris confusa, Pteris argyraea and Lygodium microphyllum (Table 1) were collected from Kothayar and their identification was confirmed with the help of herbarium specimens in XCH (Xavier’s College Herbarium), St. Xavier’s college, Palayamkottai. These samples were used for the preparation of aqueous and different solvent extracts. Thoroughly washed leaves were shade dried and then powdered with the help of a blender. 25 g of the powder was extracted successively with petroleum ether, benzene, chloroform, methanol and distilled water using a Soxhlet extractor for 48 h. All the extracts were concentrated and preserved in airtight bottle until further use.

TABLE 1: MEDICINAL FERNS SELECTED FOR ANTIBACTERIAL ACTIVITY

Phytochemical analysis: Phytochemical analysis of all the extracts of the selected fernswas conducted following the standard procedures ⁴.

Antimicrobial Assay:

Bacterial Strain: The culture of Xanthomonas campestris pv. centellae isolated from diseased Centella plant was maintained in nutrient agar slant at 4^oC.

Antibacterial Activity: The antibacterial activity of five solvent extracts of five medicinal ferns was tested in disc diffusion method ⁵.  Muller Hinton agar medium was seeded with 100µl of inoculum (1× 10⁸ CFU/ml). The impregnated discs containing the test sample (20µg/ml, 40µg/ml and 80µg/ml) were placed on the agar medium seeded with tested microorganisms. Standard antibiotic discs (Kanamycin 30µg/disc, Neomycin 10µg/disc) and blank discs (impregnated with solvent) were used as positive and negative control. The plates were then incubated at 37 ⁰C for 24 h to allow maximum growth of the microorganisms⁵. The antibacterial activity of the test samples was determined by measuring the diameter of zone of inhibition expressed in millimeter. Experiments were done in triplicate and repeated twice and mean of the three experiments was recorded.

Determination of Minimum Inhibitory Concentration (MIC): The Minimum Inhibitory Concentration (MIC) of the benzene and methanol and benzene extracts of P. confusa, A. evecta, P. argyraea, A. caudatum and L. microphyllum were determined by using serial dilution technique ⁶. 1 mg/ml of the sample solutions of all the extracts were prepared using Dimethyl Sulfoxide (DMSO).

In this technique a large number of test tubes were used and each of the test tubes was filled with 1 ml of sterile nutrient broth media and graded doses of sample solution were added. Then, these test tubes were inoculated with the selected organisms (inoculum contains 1×10⁶ cells/ml) followed by incubation at 37^oC for 24 hours to allow the growth of the bacteria. The test tubes which showed minimum concentration as well as clear content were selected. This lowest or minimum concentration was considered as Minimum Inhibitory Concentration (MIC). Another three test tubes containing medium, medium and sample, medium and inoculum were used as control. Bacterial growth observed was only in test tubes (solution content was cloudy) containing medium and inoculum and the other two were clear showing no growth ⁶. Experiments were done in triplicate and repeated twice.

Sensitivity Tests: Commercially available discs of standard antibiotics (Kanamycin 30µg/disc, Neomycin 10µg/disc, amoxicillin-25μg, chloramphenicol-30μg, and penicillin 5μg) were used to determine antibiotic sensitivity profile of multi antibiotic resistance of X. c. pv. c by the disc diffusion method. Sensitivity and resistant were evaluated by measuring the inhibition zone diameters.

Relative Percentage Inhibition (RPI): The relative percentage inhibition of the test extracts with respects to positive controls (kanamycin and neomycin) was calculated by using the following formula ^{7, 8};

Relative percentage inhibition of the test extract =

100 X (x­- y)

(z-y)

Where; x: total area of inhibition of the test extract, y: total area of inhibition of the solvent, z: total area of inhibition of the standard drug.

The total area of inhibition was calculated by using area = πr2; where, r = radius of zone of inhibition.

Statistical Analysis: All data were expressed as mean ± SD. Statistical analyses were evaluated by one-way ANOVA followed by Tukey HSD test. Values with P< 0.005 were considered statistically significant. Mean and standard deviation were also calculated using the Microsoft Excel sheet, Office Edition 2007.

RESULTS:

Phytochemical Analysis: The phytochemical analysis revealed the presence of phenolic compounds/tannins, flavonoids, in methanol extracts of the selected two ferns (Table 2). Phytochemical analysis of methanol extracts revealed that significant antibacterial activity is due to the presence of phenolic compounds.

TABLE 2: PHYTOCHEMICAL ANALYSIS OF METHANOL EXTRACTS OF SELECTED FERNS

(+): Present    (-): Absent

Antibacterial Activity: To control the isolated harmful phyto pathogen in biocontrol method, petroleum ether, benzene, chloroform, methanol and aqueous extracts of five different medicinal ferns were evaluated for their antibacterial activity against the isolated harmful pathogen in disc diffusion method in three different concentrations (20µg/ml, 40µg/ml and 80µg/ml). All the extracts showed marked inhibitory effects against the isolated bacteria (Table 3).

TABLE 3: ANTIBACTERIAL ACTIVITY OF FIVE SOLVENT EXTRACTS OF SELECTED FERNS AGAINST XANTHOMONAS CAMPESTRIS PV.CENTELLAE IN THREE CONCENTRATIONS

Data given are mean of three replicates ± standard error, p < 0.005. NS-Name of the samples, Ac- Adiantum caudatum L., Pa- Pteris argyraea T. Moore,  Pc- Pteris confusa T.G. Walker   Ae- Angiopteris evecta (Forst) Hoff.,  Lm- Lygodium microphyllum (Cav) R.Br.

Among these, the benzene and methanol extracts of P. argyraea, P. confusa, A. caudatum, L. microphyllum and   A. evecta showed significant inhibitory effect compared with other solvent extracts in 40µg/ml and 80µg/ml concentration. The test pathogen was more susceptible to the methanol extract of each plant material than the other four extracts. The highest susceptibility was recorded with the methanol and benzene extract of A. evecta, followed by the extracts of     P. argyraea A. caudatum and P. confusa and the least, being recorded with the aqueous extracts of L. microphyllum. The petroleum ether and chloroform extracts of all the samples showed marked inhibitory effects. The aqueous extracts of all the selected samples also exhibit some inhibitory effects on the isolated bacteria.

The ANOVA analysis of the data revealed that among the five samples methanol extracts. A. evecta (p<0.005) showed highly significant activity against the tested pathogen (Table 3). Tukey HSD analysis of the data revealed that X. c. pv. c was highly susceptible to methanol extracts compared with other extracts. Antibacterial activity of methanol and benzene extract of A. evecta was highly significant (p<0.005) compared to Kanamycin and Neomycin (Table 4).

TABLE 4: ZONE OF INHIBITION IN POSITIVE AND NEGATIVE CONTROLS

Minimum Inhibitory Concentration (MIC): The MIC value of methanol and benzene extracts A. evecta was 8µg/ml and 16µg/ml against X. c. pv. c respectively. Similarly the MIC value of methanol and benzene extracts of     P. confusa, and P. argyraea was 64µg/ml, 128µg/ml against X. c. pv. c respectively. The highest MIC value was found in the benzene extracts of A. caudatum, L. microphyllum i.e. 128µg/ml, 256µg/ml, and 256µg/ml respectively. Hence it is concluded that the methanol extracts of A. evecta showed inhibition of bacterial growth even at low concentrations (Table 5).

Among these five samples, the MIC value of A. evecta is the lowest against X. c. pv. c.Hence A. evecta shows significant (p<0.005) bactericidal activity compared to other samples.  According to the results of antibacterial assay, the methanol extracts of A. evecta might be used as antibacterial agent against X. c. pv. c which severely affect plants.

TABLE 5: MINIMUM INHIBITORY CONCENTRATION OF THE FERNS EXTRACT AGAINST X. C. PV. C

Relative Percentage Inhibition (RPI): The results of antimicrobial activity of methanol and benzene extracts of ferns (40µg/ml) were compared with the positive control (kanamycin and neomycin) for evaluating their relative percentage inhibition (Table 6). For kanamycin the methanol extract of A. evecta exhibits maximum relative percentage inhibition against the test inoculum (207.64%) followed by           P. argyraea (167.51%) and A. caudatum (152.86%) respectively. For neomycin the methanol extract of A. evecta exhibits maximum relative percentage inhibition against the test inoculum (200.86%) followed by P. argyraea (162.04%) and A. caudatum (147.87%) respectively.

TABLE 6: RELATIVE PERCENTAGE INHIBITION OF METHANOL AND BENZENE EXTRACTS OF FERNS COMPARED TO STANDARD ANTIBIOTIC - KANAMYCIN AND NEOMYCIN

Sensitivity of the Bacteria Standard Antibiotics: Table 7 reveals the antibacterial effectiveness of the plant materials as was compared with the choicest commercially prepared antibiotics for Xanthomonas infection.

 TABLE 7: SENSITIVITY OF X.C.PV.C TO STANDARD ANTIBIOTICS

DISCUSSION: Very less work has been done on the antimicrobial activity of pteridophytes, yet ethanobotanical importance of these plants have been investigated and studied by various authors. They have been found for their biological activity ⁹. The phytochemical composition of Adiantum radiata has been studied and found that the isolated phytochmicals were effective against the growth of microorganisms ¹⁰. Antibiotic activity of pteridophytes has been studied       by ³, while the antiviral activity of crude extracts of some pteridophytes have also been analyzed by ¹¹. The antibacterial activity of Adiantum capillus-veneris was also been studied and found that nearly all the extracts were effective against the selected microorganisms ^{12, 13}.

In general, gram-negative bacteria were more resistant to antibiotics than gram- positive  bacteria ^{14, 15}. The resistance is due to the differences in their cell wall composition. In gram-negative bacteria the outer membrane acts as a barrier to many environmental substances including antibiotics ¹⁶. Presence of thick murine layer in the cell wall prevents the entry of the inhibitors ¹⁷. But in the present study gram-negative bacteria were more susceptible to the crude extracts than gram-positive bacteria. It may be due to the presence of broad spectrum of antibiotic compounds present in the selected ferns.

An important characteristic of plant extracts and their components is their hydrophobicity, which enable them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing the cell structures and rendering them more permeable. Extensive leakage from bacterial cells or the exit of critical molecules and ions will lead to death ¹⁸. Hence, the active compounds which are present in ferns inhibit the tested antibiotic resistant gram negative bacteria by using the above mechanism. The results of the MIC showed that the plant materials are very potent against the test pathogen even at very low concentration.

The result of RPI also indicates the selected ferns act as significant antimicrobial agents compared with standard antibiotics like Kanamycin and Neomycin.

CONCLUSIONS: This study has confirmed the antibacterial potentials of ferns, thus supporting their application as a biocontrol herbal remedy for Xanthomonas infection in plants. With these, there is need for the preparation of different formulations towards ensuring acceptable dosing to field trials. It is hoped that this study would lead to the establishment of some compounds that could be used to formulate new and more potent antimicrobial agents of natural origin for the treatment of Xanthomonas infection in plants particularly causing leaf spot disease in Centella asiatica.

ACKNOWLEDGEMENT: The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi for financial support (Ref. No: 38(1260)/10/EMR-II 17/05/2010).

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