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European Journal of Medicinal Plants

1(3): 40-49, 2011

SCIENCEDOMAIN international www.sciencedomain.org

Biochemical Defense against Die-Back Disease of a Traditional Medicinal Plant Mimusops elengi Linn.

Selima Khatun1, Ugur Cakilcioglu2*, Manoranjan Chakrabarti1, Suprakash Ojha1 and Narayan Chandra Chatterjee1

UGC Centre of Advanced Study, Department of Botany, University of Burdwan, Burdwan 713104, West Bengal, India. 2 Elazig Directorate of National Education, Dogukent IOO., Elazig 23100, Turkey.

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Research Article

Received 24th February 2011 Accepted 29th March 2011 Online Ready 28th April 2011

ABSTRACT

Mimusops elengi (Linn.) commonly known as Bakul is one such tree native to the Western Ghat region of the peninsular India. However, today this tree is also found growing in other parts of the tropical and sub tropical regions of the world. The stem, barks, leaves and fruits are used in various Ayurvedic and folk medications to treat various ailments. Die-back disease caused by Alternaria lunata severely affects the plants grown in and around Burdwan, West Bengal, India. An investigation was carried out to study the phenolics along with their oxidizing enzymes that involve in defense against the activity of the pathogen. Peroxidase activity reached its peak on 14th day after infection whereas polyphenol oxidase mediated phenol oxidation was increased upto 21st day in the infected tissues. The enhancement of phenols and their oxidizing enzymes in infected plants affords resistance of the host against die-back.

Keywords: Die-back; Mimusops elengi; medicinal plant; peroxidise; polyphenol oxidase; total phenol;

____________________________________________________________________________________________ *Corresponding author: Email: [email protected];

European Journal of Medicinal Plants, 1(3): 40-49, 2011

1. INTRODUCTION

Throughout history, humans have derived many uses and benefits from the plants found in their own region. Initially, wild plants were collected from their natural habitat, followed by the cultivation of those that were used most commonly (Akan et al., 2008). As the case in the other countries of the world, in recent years, the plants ­ used traditionally for curative purposes ­ have attracted attention of the researchers (Bagci et al., 2010; Cakilcioglu et al., 2010; Khatun et al., 2008; Koyuncu et al., 2010; Parveen et al., 2007; Turkoglu et al., 2010; Yapici et al., 2009). Mimusops elengi (Linn.), commonly called `Bakul' is a medicinally (Baliga et al., 2011) important plant of family Sapotaceae. In the traditional Indian system of medicine Bakul is one of the herbs mentioned in Charaka Samhita, Sushruta Samhita, Astanga Hrudaya, Bhavaprakasa, Dhanvantari and Saligrama (Nadkarni, 1976; Mitra, 1981). The bark, flowers, fruit and seeds (Bharat and Parabia, 2010) have great medicinal value. The plant is used both externally as well as internally. It is used externally for various odontopathies. Being an astringent and styptic, it is valuable aid in dental ailments like bleeding gums, pyorrhoea, dental caries (Mitra, 1981) and loose teeth. In such conditions, the tender stems are used as tooth brushes or the powder of bark is used for cleansing the teeth. The unripe fruit is used as a masticatory and helps to fix loose teeth. The flowers are used for preparing a lotion of wounds and ulcers (Nadkarni, 1976; Mitra, 1981). The fruits are believed to be effective in preventing chronic dysentery and constipations. The aqueous concoctions of the fruits are believed to promote delivery during childbirth.The unripe fruit is used as a masticatory and is supposed to be helpful in fixing the loose teeth. It is supposed to prevent premature ejaculations, to possess antidiuretic effects and is also useful as an anti-toxin. The ripe fruit is supposed to be a general tonic and to decrease the vitiated pitta dosha (Nadkarni, 1976; Mitra, 1981). The powder of dried flowers is a brain tonic and useful as a snuff to relieve cephalalgia. The extract of flowers is salutary in heart diseases as well. Internally the bark skin and flowers being astringent and styptic in properties are benevolent in leucorrhoea and menorrhagia (Nadkarni, 1976; Mitra, 1981). It works well as an antidiuretic (Koti and Ashok, 2010; Katedeshmukh et al., 2010) in polyuria and antihyperglycemic (Jeriline et al., 2009; Ganu et al., 2010) in diabetes. The plant also shows antibacterial (Nair and Chanda, 2007) and antifungal activity (Satish et al., 2007). Increased activity of polyphenol oxidase and peroxidase in response to infection by the pathogen has been reported by many workers (Vidyasekaran, 1988; Karthikeyan and Bhaskaran, 1992; Ojha et al., 2005) and is considered to play an active role in contributing to disease resistance in certain plant host-pathogen interaction following infections. Higher levels of total phenols following infection with the pathogens have been reported by previous workers (Majumdar and Pathak, 1989; Jyosthna et al., 2004; Madhavi et al., 2005; Theerthagiri et al., 2007) where phenols may play an important role as post-infectional factors in the disease resistance. Changes in phenol and phenoloxidizing enzyme activities in die-back disease of Mimusops elengi were determined in the present communication with a view to correlate the resistance of the host through elicitation of its defence system. The authors failed to trace any early records of the occurrence of this disease; therefore, it seems to be the first record of its kind from India. As a result, the disease causes tremendous economic loss in country like India as a timber wood.

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

2. MATERIALS AND METHODS 2.1 PATHOGEN

During a recent survey in and around Burdwan, West Bengal, a hitherto unrecorded die back disease of Mimusops elengi was noticed and the causal organism, isolated from different portions of affected areas was identified as Curvularia lunata (Indian type culture collection identification no. 7961.10 by IARI, New Delhi).

2.2 PLANT SAMPLES

The young healthy Mimusops plants in the Departmental garden were inoculated with the causal pathogen. Following inoculation, symptoms appear on the leaves. After 7 days interval of inoculation, total phenols, peroxidase and polyphenol oxidase activities were measured. After 21 day of infection the content reached its peak after which it declined. That is why after 28 days of the infection, it is insignificant to go beyond.

2.3 EXTRACTION AND ESTIMATION OF TOTAL PHENOL

1 gm of healthy and infected leaf tissues were cut into pieces of 1-2 cm. They were kept in 510 ml. 80% ethyl alcohol immediately and allowed to boil for 5-10 mint in a hot water bath. The extract was cooled in a pan of cold water. The tissues were crushed thoroughly in a mortar and pestle for 5-10 minutes, then passed through a double-layered cloth. The ground tissue was extracted in boiling 80% alcohol, it was cooled and passed through Whatman's No. 1 filter paper. Total phenol was estimated by the method of Mahadevan and Sridhar (1982), 1 ml. of alcoholic extract was piptted in a graduated tubes. Then 1 ml. of folinciacalteau reagent was added followed by 2 ml. of 10% Na2CO3 solution. The tube was shaken and heated in boiling water bath for 1-2 minutes. The tube was cooled under running tap water. The blue solution was diluted to 25 ml with distilled water and absorbance of it was measured at 650 nm in UV Vis Spectrophotometer (Systronics 117). The amount of phenolics was expressed as mg catechol g-1 of dry tissue.

2.4 EXTRACTION AND ASSAY OF PEROXIDASE

To extract the enzyme, 100 mg of each of infected and healthy leaf tissues were ground separately with a pinch of neutral sand in 20 ml. cold distilled water in a mortar at 0 °C. The extract was obtained by filtering off the debris with a clean cloth and centrifuging at 3000 rpm for 15 minutes in a refrigerated centrifuge. The supernatants were recovered and kept in a tube in an ice bath until assayed. Peroxidase activity was estimated following the method of Mahadevan and Sridhar (1982). 5 ml of freshly prepared pyrogallol reagent (prepared by mixing 10 ml of 0.5 M pyrogallol solution and 12.5 ml of 0.66 M phosphate buffer and the volume made to 100 ml. with distilled water) and 1.5 ml of the enzyme extract were mixed in a spectrophotometer tube and the mixture was immediately adjusted to zero absorbance of a UV Vis Spectrophotometer (Systronics 117). 0.5 ml of 1 H2O2 solution was added to it and the content was mixed by inverting the tube. The reaction was initiated by the addition of H2O2. Enzyme activity was recorded as the change in absorbance per minute (A / mint/) at 430 nm immediately after the addition of substrate and was expressed as changes in absorbance min-1 g-1 of dry tissue.

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

2.5 EXTRACTION AND ASSAY OF POLYPHENOL OXIDASE (PPO)

100 mg of healthy and infected leaf tissues were homogenized separately with a pinch of sand in 6 ml. Phosphate buffer of 0.1 M at pH 7.0 at 0°C. The extract was filtered with a clean cloth, centrifuged at 3000 rpm for 15 minutes and stored in an ice-bath until used. Phenol oxidase activity was measured by the method of Sadasivam and Manickam (1996). 2 ml of enzyme extract and 3 ml. of distilled water were mixed together in a spectrophotometer tube and adjusted to zero absorbance of a UV Vis Spectrophotometer (Systronics 117). 1 ml of catechol solution (0.4 mg / ml) was added to the above mixture and the reactants were quickly mixed. The enzyme activity was measured as the change in absorbance per minute (A / mint.) at 490 nm immediately after the addition of catechol solution which initiated the reaction. The enzyme activity was expressed as changes in absorbance min­1 g-1 of dry tissue.

2.6 STATISTICAL ANALYSIS

Data were expressed as mean + standard deviation. Significant differences among the means were determined through one way ANOVA (P<0.05) using Stat Plus version 5.8, 2009 software.

3. RESULTS AND DISCUSSION 3.1 SYMPTOMS

The symptoms of the disease (Picture 1) are thinning of leaves and crown, drying up of the ends of branches, table topped conditions and stag headness in extreme cases.

Picture 2. Healthy Mimusops elengi (Linn.) Picture 1. Diseased Mimusops elengi

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

Picture 3. Pure culture of Curvularia lunata Small drying twigs keep on falling continuously and the tree look like a blunt stub containing thick branches. Several months may elapse from the time of the first symptoms appear to the complete die back and death of the plant. The die back of this potential herb causes enormous economic loss in our country like India. The pathogenicity tests were conducted which fully satisfied the Koch's postulates and the pathogen was identified as Curvularia lunata (Picture 2). Since the authors failed to trace any early records of the occurrence of this disease, the present finding seems to be the first record of its kind in India. 3.2 TOTAL PHENOL The results revealed that the phenol content was found to be significantly (P<0.05) increased simultaneously in diseased leaf tissues in comparison to the healthy (Picture 2) tissues with increase in the period of infection (Figure 1). Total phenol content was significantly (P<0.05) higher in Curvularia lunata infected (105.84 ± 0.089) leaves of M. elengi on 21st days after infection that the healthy (15.60 ± 0.009) ones. Total phenols increased in infected plant than the healthy ones and it is well known that phenolic compounds are fungitoxic. Moreover, they increase the physical and mechanical strength of the host cell wall and thus inhibit fungal invasion (Theerthagiri et al., 2007).

3.3 PEPOXIDASE AND POLYPHENOL OXIDASE

The activity of both the phenol oxidizing peroxidase and polyphenol oxidase was significantly (P<0.05) higher in infected leaf tissues than in uninfected ones and that it increased considerably with the increase in progression of infection (Figures 2, 3). Peroxidase activity in pathogen infected leaves of M. elengi was found to be significantly (P<0.05) higher (40.62 ± 1.60) on 14th day and after that it gradually declined but in normal ones, it remains unchanged during the periods of incubation (Figure 2).

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

120

Total phenol (mg catechol/g fresh tissue)

100 80 60 40 20 0 7 days 14 days Healthy 21 days Infected 28 days

Days of infection

Fig. 1. Total phenol content in healthy and Curvularia infected leaves of Mimusops elengi at different periods of infection

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Peroxidase activity (changes in absorbance / min/mg protein)

40 35 30 25 20 15 10 5 0 7 days 14 days Healthy 21 days Infected 28 days

Days of infection

Fig. 2 . Peroxidase activity in healthy and Curvularia infected leaves of Mimusops elengi at different periods of infection.

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

Peroxidase is a member of the PR-9 family (pathogenesis- related protein) responsible for plant defense response via formation of lignin (Ray et al., 1998). PO activity is associated with disease resistance (Lin and Kao, 2001) and increases in host plant following pathogen infection (Borden and Higgins, 2002; Zheng et al., 2004). PPO usually accumulates during pathogenic attack, in wounds or tissue senescence in plants. Plant cell- membrane disruption occurrence might initiate formation of quinines following an increase in accessibility of PPO to its substrate. The capability of adaptation to phytopathogenic stress was related to the increase or sustained ability to form defence barriers for reinforcing the cell structure by lignification- related enzymes (Zheng et al., 2005). Increase in the activity of peroxidase and polyphenol oxidase in host tissues in response to infection by the pathogen has been reported in many cases (Ojha et al., 2005; Khatun et al., 2009, Senthil et al., 2010; Khatun and Chatterjee, 2011). Peroxidase is a key enzyme in the biosynthesis of lignin and other oxidized phenols (Bruce and West, 1989). Peroxidase and polyphenol oxidase mediate the oxidation of phenols and oxidized phenols are highly toxic to the pathogen (Sequeira, 1983).

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Polyphenol oxidase activity (changes in absorbance / min/mg protein)

60 50 40 30 20 10 0 7 days 14 days Healthy 21 days Infected 28 days

Days of infection

Fig. 3. Polyphenol oxidase activity in healthy and Curvularia infected leaves of Mimusops elengi at different periods of infection Polyphenol oxidase activity seems to be gradually increases and showed significantly (P<0.05) higher activity (61.83 ± 0.092) on 21st day after C. lunata challenged M. elengi while uninfected M. elengi showed remains unchanged (Figure 3). PO and PPO catalyse the oxidation of phenolic compounds through a PPO-PO-H2O2 system (Srivastava, 1987). A number of studies have found a correlation between PPO and the resistance response (Velazhahan and Vidhyasekaran, 1994). PO itself was also found to inhibit the spore germination and mycelial growth of certain fungi (Joseph et al., 1998). Peroxidase may be rapidly involved in the peroxidation of substrate molecule, leading to the accumulation of highly toxic compounds (i.e., phenolic compounds), which may contribute to resistance via

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European Journal of Medicinal Plants, 1(3): 40-49, 2011

their antifungal potential (Ward, 1986).The role of phenol oxidases in resistance is based on the observations that the activity of these enzymes is increased in infected tissues and that the oxidized phenols i.e., quinones are more reactive and more toxic to microorganisms compared to their non-oxidized form.

4. CONCLUSION

Die-back disease of M.elengi is a serious disease and that too is a first report of its kind in India, posing thereby a serious threat to the timber industry. Therefore, the greater activity of PO and PPO, along with higher amount of total phenols enhancing host resistance is in compliance with the previous report. This might suggest that the induced PPO and PO activities along with phenols in Curvularia lunata-treated Mimusops elengi could be a defensive response against C. lunata.The phenolic substances along with their oxidizing enzymes such as PPO and PO are the key factors responsible for host defense after pathogenic attack. Hence the characterization of the phenolics as well as the isozyme pattern of these oxidizing enzymes would be the future scope of research in this direction.

ACKNOWLEDGEMENT

First author Selima Khatun acknowledges the UGC, Government of India, New Delhi for granting Meritorious Fellowship as financial assistance.

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© 2011 Khatun et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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