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Antifungal activity of phenolic acids against Ganoderma boninense and possible development of resistance.

INTRODUCTION

Malaysia currently accounts for 39% of world palm oil production and 44% of world exports [13]. In 2013, the oil palm plantation areas in Malaysia reached 5.2 Million hectares [12]. However, the oil palm industry is hampered by the devastating disease Basal Stem Rot (BSR). This incurable disease is caused by Ganoderma boninense. The oil palm industry is seriously damaged by BSR for more than 50 years and eventually causing large amount of losses in revenue [8,15]. Many researches have been conducted to search for potential control of this disease but to date no conclusive solution is reported. The more recent approach is investigating the role of phenolic acids in oil palm defense against Ganoderma infection [1,3,4,5,6]. Plants does have its own defense system which acts upon the infection. Phenolics are components of secondary metabolite which is a preformed antifungal produced in plant and activated in response to infection. Caffeic acid, syringic acid and 4-hydroxybenzoic acid (4-HBA) are phenolic acids which reported to have high antifungal activity against Ganoderma [3,5]. Although phenolic acids are produced in plant, the amount being produced might not be sufficient to encounter the infection if the accumulation of those phenolic acids is low. The information on the sufficient amount of phenolic acids to contribute to resistance of oil palm against Ganoderma is very little. Therefore, this experiment is designed to investigate the response of Ganoderma to the different concentration of caffeic acid, syringic acid and 4-HBA which was reported to be effective against G. boninense and the possible development of resistance of this pathogen to these phenolic acids.

MATERIALS AND METHODS

Preparation of Cell Assay Plates:

Cell assay plates with concentrations of 0.0, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg and 2.5 mg [mL.sup.-1] of each caffeic acid (CA), syringic acid (SA) and 4-hydroxybenzoic acid (4-HBA) were prepared by incorporating the respective phenolic acids into Potato Dextrose Agar (PDA), with the phenolic acids being first dissolved in acetone:water (50:50; v/v) before incorporated into the medium. PDA without phenolic acids served as control.

Antifungal Activity of Phenolic Acids against G. boninense:

The synergy effect of CA, SA and 4-HBA on the antimicrobial activity against G. boninense was investigated by measuring the diameter growth of mycelium in centimeter (cm) of G. boninense on cell assay plates incorporated with different concentrations of phenolic acids. Plugs (8 mm) of G. boninense were taken from the edge of seven (7) to eight (8) days old cultures, using a sterile micropipette tip and placed to the middle of the cell assay plates. The growth of G. boninense was observed daily for 14 days.

Possible Resistance of G. boninense to Phenolic Acids:

Plugs (8 mm) of G. boninense were placed to the middle of the cell assay plates containing 0.1 mg [mL.sup.-1] of individual phenolic acids. The growth of the pathogen was monitored for 14 days. Surviving mycelia of G. boninense were transferred to cell assay plates with higher concentration; 0.2 mg [mL.sup.-1] of each phenolic acids. The growth of the pathogen was further monitored for 14 days. The transferring and monitoring processes were repeated to other higher concentration such as 0.3 mg, 0.4 mg, 0.5 mg and 2.5 mg [mL.sup.-1] of cell assay plates with respective phenolic acids. The diameter growth (cm) of G. boninense on cell assay plates was measured throughout the experiment. The assays were repeated three times to closely monitor if any possible resistance of G. boninense derived from this incubation.

Results:

Antimicrobial Activity of Phenolic Acids against G. boninense:

Lower concentrations of phenolic acids (0.1 to 0.5 mg [mL.sup.-1]) failed to inhibit the growth of G. boninense completely (Figure 1). However, phenolics with concentrations of 0.4 and 0.5 mg [mL.sup.-1] gave a significant slower growth rate of this pathogen compared to control. However, the highest concentration of phenolic acids (2.5 mg [mL.sup.-1]) tested in this experiment had inhibited the growth of G. boninense completely.

Possible Resistance of G. boninense to Phenolic Acids:

Although the G. boninense was exposed and trained under different concentrations of phenolic acids (0.1 mg [mL.sup.-1] to 0.4 mg [mL.sup.-1]) in cell assay plates, G. boninense was incapable to develop resistance to higher concentrations of phenolic acids (0.5 mg [mL.sup.-1] and 2.5 mg [mL.sup.-1]) (Figure 2 to 5).

Discussion:

Antimicrobial Activity of Phenolic Acids against G. boninense:

The lag phase of G. boninense was affected by different concentrations of phenolic acids in cell assay plates. Lag phase is the condition where the fungi are trying to adapt the new environment when their cells had depleted various essential constituents and required time to restart the biosynthesis. The growth of G. boninense was slowly inhibited as the concentration of phenolic acids was increased. Extended of lag phase usually occurred when microbes were transferred from a rich cultured medium to a poorer one [11]. Rich culture medium possesses all nutrients needed by the subject of culture. In this case, G. boninense which was first cultured on PDA without phenolic acids (rich medium), was struggling when transferred to the media containing phenolic acids with different concentrations within the 14 days of incubation. Besides the extension period of lag phase, G. boninense in phenolic acids amended media was also unable grow to maximum (full plate) after 14 days. Combination of these three phenolic acids at the concentration of 2.5 mg [mL.sup.-1] was found to have the ability to inhibit G. boninense growth and hence gave a constant diameter size throughout the period of incubation. This is as accordance to the findings reported by Chong et al. 2009b [7] which 2.5 mg [mL.sup.-1] of these three phenolic acids were very fungitoxic to G. boninense.

Possible Resistance of G. boninense to Phenolic Acids:

Antifungal resistance is defined as a stable, inheritable adjustment by a fungal cell to an antifungal agent, which resulted in a less than normal sensitivity to that antifungal [2]. In addition, mutation in the regulatory gene can cause resistance of fungi towards the phenolic acids [16]. Caffeic acid and 4-hydroxybenzoic acid required a higher concentration (up to 2.0 mg [mL.sup.-1]) to inhibit G. boninense but only 0.5 mg [mL.sup.-1] for syringic acid [7]. Phenolics have the probability to be degraded in several ways. Fusarium flocciferum shows its capability to degrade some phenolic compounds namely, syringic, caffeic, ferulic acids, syringic aldehyde, gallic, and vanillic. After 24 hours of incubation, the fungus was found capable to reduce the phenolic concentration from 200 mg [L.sup.-1] to below detection limits. However, syringic acid and its aldehyde have the lowest degradation rates [14].

The capability of G. boninense to develop resistance against phenolic acids was also related to the enzymes secreted by the white rot fungi. G. neo-japonicum is a white rot fungi which was reported to have the ability to produce extracellular enzymes, including P-glucosidase, ligninase, cellulase, avicelase, pectinase, xylanase, protease, and amylase [9]. Among the enzymes, P-glucosidase and ligninase showed the highest activity in G. neo-japonicum. These enzymes were capable to catalyse the oxidation of phenols and their related compounds. Secretion of enzymes has led to the colour changes of the media surrounding Ganoderma culture. A formation of deeply coloured zone around the mycelium has also been reported as the reaction of extracellular oxidase [10]. The brown colour formation occurred as early as first day of the growth. In this experiment, G. boninense was trained in lower concentration of phenolic acids, during the transferring of G. boninense from cell assay plates with lower phenolic acids to higher, it was noticed that G. boninense which was cultured on cell assay plates (0.5 mg [mL.sup.-1] and 2.5 mg [mL.sup.-1]) started to form brown colour spot on the media few hours soon after transferred, which suggesting the degradation of phenolic acids (Figure 6). However, the mycelia of the pathogen turned to brown and dead indicating it failed to develop resistance and further degrade the high concentration of phenolic acids.

G. boninense was not successful to be re-isolated on fresh PDA suggesting this combination has fungicidal effect instead of fungistatic.

Conclusion:

The growth of G. boninense was inhibited as the concentration of phenolics acids (syringic acid, caffeic acid and 4-hydroxybenzoic acid) increased. Highest concentration tested in this experiment (2.5 mg [ml.sup.-1]) of the combination of these three phenolic acids successfully killed the pathogen. G. boninense failed to develop resistance against these phenolic acids.

ACKNOWLEDGMENT

The authors wish to thank Ministry of Education, Malaysia for providing financial assistance via grant PRGS0001-STWN-1/2012 and Sawit Kinabalu Sdn Bhd for their technical assistance. The authors also acknowledged their profound gratitude to the Sustainable Palm Oil Research Unit (SPOR) and Faculty of Science and Natural Resources, Universiti Malaysia Sabah for providing the facilities for the research work.

REFERENCES

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[15] Miller, R.N.G., M. Holderness, P.D. Bridge, R.R.M. Paterson, M. Sariah, M.Z. Hussin and E.J. Hilsley, 1995. A multidisciplinary approach to the characterization of Ganoderma in oil palm cropping systems. In: Ganoderma, Systematicsm Phytopathology and Pharmacology. Proceedings of the Fifth International Mycological Congress, 1994, pp: 57-66. P.K. Buchanan, R.S. Hseu and J.M. Moncalvo (eds.). Vancouver, Canada.

[16] Triyana, S.Y., 2009. Antibiotic resistance of pathogenic bacteria. Majalah Kesehatan Pharma Medika., 1(2): 92-94.

Jee, W.R. and Chong, K.P.

Sustainable Palm Oil Research Unit (SPOR), Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.

ARTICLE INFO

Article history:

Received 21 November 2014

Received in revised form 4 December 2014

Accepted 3 January 2015

Available online 28 January 2015

Corresponding Author: Khim-Phin Chong, Sustainable Palm Oil Research Unit (SPOR), Faculty of Science and Natural Resources, Universiti Malaysia Sabah.

Tel: +6088-320000 ext: 5571; Fax: +6088-435324; E-mail: chongkp@ums.edu.my
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Author:Jee, W.R.; Chong, K.P.
Publication:Advances in Environmental Biology
Article Type:Report
Date:Jan 15, 2015
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