Antimicrobial and Antioxidant properties of medicinal mushroom Ganoderma P. Karst

Ganoderma species have been known all over the world as highly medicinal mushrooms. Antimicrobial activity of it is an attractive approach which raises the global interests from the scientific community. In this study, the antimicrobial assay of ethanol and methanol extracts of Ganoderma were prepared by using the dried mycelial powder obtained from five different liquid media; was performed against seven plant pathogenic fungi viz., Alternaria macrospora, Aspergilus niger, A. flavus, Colletotrichum falcatum, Fusarium oxysporum, Pestalotiopsis mangiferae and Penicillium sp. and two plant pathogenic bacteria Xanthomonas oryzae and Ralstonia solanacearum. The extracts of mycelia obtained from Yeast Wine Media exhibited the highest inhibition percentage as compared to rest. At concentration 1000ppm, there was complete inhibition of mycelial growth for Alternaria macrospora, Aspergilus niger and A. flavus while for Fusarium oxysporum and Penicillum sp. complete inhibition was observed at 500ppm but for Colletotrichum falcatum and Pestalotiopsis mangiferae, more than 80% mycelial inhibition at concentration 1000ppm in both ethanol and methanol extracts. In the case of Xanthomonas oryzae and Ralstonia solanacearum, at concentration 1000ppm, methanol extract showed the highest inhibition zone (3.50mm, 3.75mm). Ganoderma exhibited antagonistic effect against plant pathogenic fungi could add to the interest of developing Ganoderma as a successful bioagent in the near future. Antioxidant activities were evaluated by using DPPH radical scavenging assay and Metal chelating activity on ferrous ions. The DPPH radical scavenging effect was detected in methanol extract (Inhibition% = 27.312%) was higher than that of the ethanol extract (Inhibition% = 24.79%) and also Ferrous ion chelating ability of methanol extract (Inhibition% =22.27%) was higher than the ethanol extract (Inhibition% =12.55%). It is clearly indicated that both methanol and ethanol extract of the Ganoderma show antioxidant properties and Ganoderma extracts act as an effective antioxidant agents.


Introduction
Ganoderma have attracted great attention all over the world because of their wide range of pharmacological values. It is an edible mushroom that has been used for centuries in Traditional Chinese Medicine for its health promoting properties. Ganoderma species are not listed among the group of edible mushrooms because the fruiting bodies are always thick, corky and tough and, do not have the fleshy texture characteristics of true edible mushroom [1], [2]. Although Ganoderma species could not be used for eating directly, they have been known all over the world as highly medicinal mushrooms [3], [4]. Its extracts are used worldwide as ingredients in health foods, herbal medicines and dietary supplements. Recent studies have proven that Ganoderma extract shows antimicrobial activity against various plant pathogens. It has inhibitory effect on growth and germination of Alternaria alternata; antimicrobial activities on Botrytis cinera, Colletotrichum gloeosporioides, and C. miyabeanus. Antimicrobial activity of it is an attractive approach which raises the global interests from the scientific community.
Oxidation processes are essential to many living organisms for the production of energy to fuel biological processes. The uncontrolled production of reactive oxygens species (ROS) and the unbalanced mechanism of antioxidant protection result in the onset of many diseases and accelerate ageing. Oxidative damage plays a significantly pathological role in human diseases. Cancer, emphysema, cirrhosis, atherosclerosis, and arthritis have all been correlated with oxidative damage. Oxygen-centered free radicals and other reactive oxygen species that are continuously produced in vivo, result in cell death and tissue damage. The antioxidants in human diets are of great interest as possible protective agents to help human body reduce oxidative. Ganoderma act to prevent lipid oxidation in food and to decrease the adverse effects of reactive species (both ROS and reactive nitrogen species) on normal physiological functions in humans.
Ganoderma has been well known for its antimicrobial and antioxidant activities. In this study, the antimicrobial activity of ethanol and methanol extracts of Ganoderma against some plant pathogens was evaluated and the contents of scavenging effects on radicals and chelating effects on ferrous ion were determined.

Methodology
This experiment was carried out in the laboratory of Department of Plant Pathology, N. M. College of Agriculture, Navsari Agricultural University, Navsari, and Gujarat. Survey for the collection of naturally growing Ganoderma was conducted at the different regions of Navsari viz., Navsari Agricultural University campus, Dandi and Gandevi. The sites of collection were soil, open lands, farm lands, roadside, research field etc. The data was analysed using CRD and the difference among mean value was tested by using critical differences (CD) values at 5% level of probability.

Ganoderma and test Pathogens
The fruiting bodies of Ganoderma were collected from the different locations of Navsari, Gujarat during June 2018 to September 2018. They were found to be growing wildly on dead stump of unknown trees and stem of Pink shower, Cassia grandis [5]. The test pathogens used for these studies were seven plant pathogenic fungi viz., Alternaria macrospora, Aspergilus niger, A. flavus, Colletotrichum falcatum, Fusarium oxysporum, Pestalotiopsis mangiferae and Penicillium sp. and two plant pathogenic bacteria Xanthomonas oryzae and Ralstonia solanacearum.

Preparation of the extracts
The collected specimens were isolated using tissue culture technique on sterilized Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) media in the petri plates [6]. Mycelial biomass production of Ganoderma sp. was carried out using five different liquid media viz., Glucose Aspargine Media, Hwang Liquid Media, Potato Dextrose Broth, Yeast Wine Media and Glucose Peptone Liquid Media [7]. The mycelial mats obtained from different five media were ground and the airdried powder was used for preparation of extracts. Ethanol extracts were prepared by adding 0.01g of dried mycelial powder in 10ml of methanol and wrapped with aluminium foil, leaving it for 6 hours. Then the extracts were centrifuged at 1000rpm for 15 minutes and the supernatant was used for further studies [8]. It was considered as pure 1000ppm solution and labelled as 'stock solution', from which 500ppm and 250ppm solutions were prepared by adding required amount of ethanol. Methanol extract was also prepared in the similar way but methanol was used instead of ethanol.

Antifungal activities
The antifungal activity of Ganoderma extracts was determined by poisoned food technique [9]. Three concentrations viz., 250ppm, 500ppm and 1000ppm of both methanol and ethanol extracts of all five dried mycelia powder obtained from five different liquid media were used against the test pathogens.

Antibacterial activities
The antibacerial activity of Ganoderma extracts was performed by Disc diffusion method using paper disc as reservoir against the test plant pathogens viz., Xanthomonas oryzae and Ralstonia solanacearum. Here, the filter paper discs of 6mm diameter were impregnated with 20μl of three concentrations viz., 250ppm, 500ppm and 1000ppm of both methanol and ethanol extracts of all five dried powder obtained from five different liquid media and placed on inoculated agar plates.

DPPH radical scavenging assay
Radical scavenging activity was determined by a spectrophotometric method based on the reduction of a methanol solution of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) using the method of Blois (1958) [10]. Both methanol and ethanol extract solutions were added to a 0.004% methanol solution of DPPH. The mixture was shaken vigorously and kept at room temperature for 30 minutes in the dark. Then the absorbance was measured at 517nm against a blank in spectrophotometer [11]. Butylated hydroxyanosole (BHA) and Butylated hydroxytoluene (BHT) were used as positive controls. The inhibition of DPPH free radical as a percentage (I%) was calculated according to the formula: Where, AControl is the absorbance of the blank at 517nm; ASample is the absorbance of the test compound at 517nm

Metal chelating activity on ferrous ions (Fe 2+ )
Metal chelating activity was determined according to the method proposed by Decker and Welch (1990) [12]. In brief, 1ml of the Ganoderma extract was mixed with 0.05ml of 2mM FeCl2 and 0.1ml of 5mM ferrozine. Then, the mixture was shaken vigorously and kept at room temperature for 10 minutes. Then, its absorbance was measured spectrophotometrically at 562nm. Ethylene diamine tetraacetic acid (EDTA) was used as positive controls. The percentage of inhibition of ferrozine-ferrous ion complex formation was calculated using the formula given below: Where, AControl is the absorbance of the blank at 562nm ASample is the absorbance of the test compound at 562nm

Statistical analysis
The data was analyzed using CRD and the difference among mean value was tested by using critical differences (CD) values at 5% level of probability. It was observed that Ganoderma extract demonstrated various degrees of antifungal activities against the seven test fungi (Fig. 1). For both ethanol and methanol extracts, the highest antifungal activity was demonstrated by extract of the dried mycelia powder obtained from the Yeast Wine Media. There was complete inhibition of mycelial growth for Alternaria macrospora, Aspergilus niger and A. flavus at concentration 1000ppm while for Fusarium oxysporum and Penicillum sp. complete inhibition was observed at 500ppm but minimum inhibition observed at 250ppm; whereas at concentration 1000ppm more than 80% inhibition of mycelia of Colletotrichum falcatum and Pestalotiopsis mangiferae was observed in both ethanol and methanol extracts. In control sets, however no fungal or mycelia inhibition was observed.

Antibacterial activities
In the case of Xanthomonas oryzae, at concentration 1000ppm, methanol extract of mycelia obtained from Yeast Wine Media showed the highest inhibition zone (3.50mm) than the ethanol extract (3.25mm). Whereas in Ralstonia solanacearum, methanol extract of mycelia obtained from Yeast Wine Media showed the highest inhibition zone (3.75mm) and no inhibition was recorded in ethanol extract (Table 1 and 2).

DPPH radical scavenging assay
The Table 3 showed that the percentage of inhibition of ethanol and methanol extracts of mycelia obtained from Hwang Liquid Media was significantly more (24.79% and 27.31%) as compared to the rest media and the DPPH radical scavenging effect in methanol extract (Inhibition% = 27.31%) was higher than the ethanol extract (Inhibition% = 24.79%). The Fig 2 showed that the scavenging abilities of Ganoderma extracts were in a concentration-dependent fashion. It increased with increasing of concentration.  The highest percentage of inhibition of ferrozine-Fe 2+ complex formation was noted in ethanol and methanol extracts of mycelia obtained from Hwang Liquid Media (Table 4) and Ferrous ion chelating ability of methanol extract (Inhibition% =22.27%) was higher as compared to the ethanol extract (Inhibition% =12.55%). The Fig 3 showed that the chelating ability of the Ganoderma extracts also were in a concentration-dependent fashion. It increased with increasing of concentration.

Discussion
The results of antimicrobial activities of Ganoderma extract were supported by the Singh et al. (2014) [13] who reported that the all extracts of Ganoderma possessed strong antifungal activity against five fungal strains which were tested by using poisoned food technique. Moreover, the antifungal activities of almost all the extracts were found to be concentration dependent. Fidler et al. (2015) [14] reported the antimicrobial activity of alcoholic extracts of mycelial biomass of Ganoderma obtained in medium prepared with yeast wine was higher as compared to mycelial biomass obtained in other medium. Prakash and Sharma (2016) [8] observed the maximum antimicrobial activity of both aqueous and ethanolic extract of G. lucidum at a concentration of 1000ppm.
The findings of DPPH radical scavenging assay of Ganoderma extract are in close enough with the Celik et al. (2014) [11] who reported that DPPH radical scavenging effect was higher in the methanol extract as compared to the ethanol extracts. Fan et al., (2012) [15] reported that the scavenging abilities of the polysaccharides of Ganoderma were in a concentration-dependent fashion. The results of metal chealting activity on Ferrous ion of Ganodema extract were in agreement with Celik et al. (2014) [11] who reported that methanol extract ferrous iron chelating ability was higher than the ethanol extract. In addition, they reported that the chelating effects of methanolic extracts from G. lucidum on ferrous ion increased with increasing concentrations. Mau et al. (2002) [16] found that the methanolic extracts of G. lucidum chelated ferrous ions.

Conclusion
It can be concluded that Ganoderma shows considerable antimicrobial potential. So, it may be an alternate ecofriendly option for plant disease management without having toxic residues in raw and processed food and can reduce the negative effect of chemical pesticides. Also some of the reports show that it possesses some specific characteristics like biodegradability and target specificity. In this study, it is clearly indicated that both methanol and ethanol extract of the Ganoderma show antioxidant properties and Ganoderma extracts act as an effective antioxidant agent. So, further work is needed towards the field evaluation of their antimicrobial potential, antioxidant property, identification of bioactive principles and elucidation of their mechanism of action.