Broad spectrum antimicrobial activity of ectomycorrhizal specie; Russula delcica

Broad spectrum antimicrobial activity of ectomycorrhizal specie; Russula delcica

Neha Jain1, Veena Pande2

1,2 Department of Biotechnology, Kumaun University, Nainital, Uttaarakhamnd, India.

1 neha.bt16@gmail.com

Abstract: Ectomycorrhizal species produces antibiotic substances found to be inhibitory for various pathogenic microorganisms. The scientific community has reported antimicrobial activity of several mushrooms. Russula delica Fr. is a well known macro fungi which is used as a food in many parts of world. Antimicrobial activity of R.delica was tested against disease causing fungal and bacterial pathogens using disc-diffusion method. Three antibiotics; amoxicillin, phnexin and hexamox were used as positive control and DMSO (Dimethyl sulfoxide) was used as negative control. Ethanol extract of R. delica showed significant antimicrobial activity against one fungal and three bacterial pathogens. Positive fractions were further tested for minimum inhibitory concentration (MIC). Study showed that ethanol extract of ectomycorrhiza can be used as better alternative therapeutic agent than commercially used synthetic drugs which also have many side effects.

Keywords: Antimicrobial, Ectomycorrhiza, Minimum inhibitory concentration, Russula delica

1. INTRODUCTION

   The ectomycorrhizal symbiosis represents one of the most prominent and ecologically crucial mutualistic associations in terrestrial habitats. It involves several species of fungi grouped within the phyla Bassidobycota, Ascomycota, Zygomycota and hundreds of mostly woody plant species worldwide. Ectomycorrhizas occur in most of the temperate and boreal ecosystem and in large forested area of tropical and subtropical regions (Smith and Read, 1997; Caimey and Chambers, 1999; Verbeken and Buyck, 2001; Comandini et al., 2006; Wang and Qiu.2006). Mycorrhizal plants are often more resistant to diseases, such as those caused by microbial soil borne pathogens. This may be due to the improved water and mineral uptake. The

   resistance may also be due to the mechanical barrier afforded by mycorrhizal fungal mantle. However, species of certain ectomycorrhizal fungal genera such as Lactarius, Hygrophorus and Cortinarius produce antibiotic substances. Some of these antibiotics are found to be antifungal on Rhizoctonia solani, Pythium debaryanum and Fusarium oxysporium (Liu et al. 2007).

   Natural products with antimicrobial activity are used to aid the endogenous protective system, increasing interest in the antimicrobial role of nutraceutic products. Mushrooms have also become attractive as a functional food and as source for the development of drugs and nutraceuticals (Barros et al., 2007). Medicinal mushrooms have an established history of use in traditional oriental therapies. The scientific community has found various therapeutic activities such as anti-inflammatory, immunosuppressor, anticarcinogenic and antibiotic and researchers have showed antioxidant and antimicrobial activities of several mushrooms (Dulger et.al. 2002, Gao et.al. 2005, Karaman et.al. 2003, Mercan et.al. 2006 and Turkoglu et.al. 2007). Chloroform and ethyl acetate extracts of dried mushrooms have antimicrobial effect against some bacterial and fungal species. MDR (Multiple drug resistance) in human pathogenic microorganisms has developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases. According to some toxicologists and nutritionists, the side effects of some commercially used antimicrobial drugs have already been documented like allergy. All these situations forced scientists for searching new antimicrobial chemotherapeutic agents. The chloroform and ethyl acetate extracts of the dried mushroom have been reported to have antibacterial activity against Streptococcus mutans and Prevotella intermedia (Hirasawa et.al.1999). Fruiting bodies and the mycelium of both the species contain compounds with wide-ranging antimicrobial activity (Jong et.al.1993).

   To the best of our knowledge, not much work has been reported on antimicrobial activities of the extract of Russula delica and in the light of facts mentioned above this study was planned with the objective to evaluate the antimicrobial potential of Russula delica.

2. MATERIAL AND METHODS

   Source of plant material

   Fruiting bodies of ectomycorrhizal species Russula delica was obtained from forest of Nainital region and identified on the bases of various phenotypic characters.

   Determination of antimicrobial activity

   For the evaluation of broad spectrum antimicrobial activity of R.delica, ethanol extract of fruiting body was tested against the four fungal (Rhizactonia solani, Tricoderma atroviride, Alternaria solani and Fusarium oxysporium) and four bacterial pathogens (E. coli, Erwinia herbicola, Staphlococcus aureus and Agrobacterium rhizogens) which are known to cause various diseases in plants and animals using Disc-diffusion method. Three most commonly used antibiotics (Amoxicillin, Phnexin and Hexamox) were used as positive control and DMSO (Dimethyl sulfoxide) was used as negative control. All the fractions which showed significant antimicrobial (antifungal and/or antibacterial) activity were further tested to evaluate minimum inhibitory concentration (MIC).

   Preparation of fungal extract

   Fresh mushrooms (450g) were air dried in hot air oven at 400c before the analysis. Dried mushroom samples (50g) were extracted by stirring with 500 ml of ethanol at 300c at 150 rpm for 24 hrs and filtered through Whatman no. 4 filter paper (Turkoglu et. al. 2007). The residue was then extracted with additional 500 ml of ethanol as described above. The combined ethanol extract were then rotary evaporated at 400c to dryness and dissolved in ethanol to a concentration of 10 mg/ml and stored at 400c for further use.

   Culture and maintenance of pathogenic microorganisms

   Different media were used to grow pathogenic fungal and bacterial species. All the pathogenic microorganisms tested for the antimicrobial activities were spread on their respective solidified agar media

   in petri plates and incubated for two days at appropriate temperature (table 1).

   Table 1: Media and appropriate growth temperatures used for different pathogens

   S.NO.	Microorganism	Media used	Growth temperature
   1.	E. herbicola	L.B. medium	370C
   2.	E. coli	Nurient agar medium	370C
   3.	S. aureus	Nurient agar medium	370C
   4.	A. rhizogenes	Yeast Extract Mannitol Agar	250C
   5.	R. solani	Czapek-Dox Agar medium	250C
   6.	T. atroviride	Czapek-Dox Agar medium	250C
   7.	A. solani	Czapek-Dox Agar medium	250C
   8.	F. oxysporium	Czapek-Dox Agar medium	250C

   Application of discs

   Whatman filter paper no.4 discs of approximately 5 mm diameter were placed in petridish and autoclaved. These discs were impregnated with ectomycorrhizal extract, 3 antibiotics and DMSO separately. Once plates were inoculated with pathogenic microorganisms, five filter paper discs; one impregnated with ethanol extract of R. delica, another impregnated with DMSO (negative control) and three impregnated with standard antibiotics; Amoxicillin (10 mg/ml), Phnexin (10 mg/ml) and Hexamox (10 mg/ml) (positive control) were applied under aseptic conditions. The plates were inverted and placed in incubator set at appropriate temperature for the growth of the inoculated pathogens just after the discs were applied (table 1).

   Reading of plates

   After 24 to 48 hrs of incubation, each plate was examined. The diameter of complete inhibition zone was measured, including the diameter of disc using a ruler. The zone margin was taken as the area showing no obvious, visible growth of any tiny colonies.

   MIC evaluation

   Fourfold serial dilution of ethanol extract of R. delica was prepared by diluting 10% DMSO to achieve a decreasing concentration range from 2000 µg/ml to

   500 µg/ml. Autoclaved filter paper discs were impregnated with the ethanol extract of R. delica of different concentration and then applied in the petriplates inoculated with pathogenic microorganisms for all the positive fractions. After that plates were incubated at appropriate temperature for the growth of the inoculated pathogenic microorganisms for 24 to 48 hrs (table 1). The least concentration of each fugal extract showing the inhibition zone was taken as MIC.

3. RESULTS AND DISCUSSION

   Antimicrobial screening

   Antimicrobial screening reveals that the ethanol extract of R. delica have a broad antimicrobial spectrum as found effective against one fungal and three bacterial pathogens (table 2). When antimicrobial activities of ethanol extract of R.delica was compared with those of commonly used antibiotics against pathogenic microorganisms it was found that in some cases, fungal extracts were even more active than the commercially used antibiotics as shown in table 2. Antimicrobial activity of ethanol extract of R. delica against various pathogenic microbes has been reported (Turkoglu et.al. 2007 and Jain & Pande 2013). R. delica is easily available at high altitude of eastern zone of India. The extracts of ectomycorrhizal fungus can be used as better alternative therapeutic agent than commercially used synthetic drugs which also have many side effects.

   Table 2. Antimicrobial activity of R. delica against different pathogens.

   S.NO.	Microorganisms	R.delica	Antibiotics
   			A#	P#	H#
   1.	E. herbicola	+	–	–	–
   2.	E. coli	–	+	+	+
   3.	S. aureus	+	+	–	–
   4.	A. rhizogenes	+	+	+	+
   5.	R. solani	–	–	–	–
   6.	T. atroviride	–	–	–	–
   7.	A. solani	–	–	–	–
   8.	F. oxysporium	+	+	–	–

   # (A- Amoxicillin, P- Phnexin, H- Hexamox, + – Effective, – – Not effective)

   Most interesting result was obtained in case of bacterial pathogen E. herbicola as it was found to be

   resistance against all the three antibiotics and no inhibition zone was obtained but extract of R. delica was found inhibitory as an inhibition zone of 7mm was observed. Extract of R. delica also showed inhibition against S. aureus, A. rhizogense, F. oxysporium with inhibition zones of 9.5, 6 mm and

   1. mm respectively. Out of three antibiotics used only Amoxicillin was found effective against all three pathogens. R. solani, T. atroviride and A. solani were found to be resistant against R. delica and all the three antibiotics as no inhibition zones were observed (table 3).

      Table 3. Inhibition Zone (mm) formed by ethanol extract of R. delica and three antibiotics.

      S.NO.	Microorganisms	R.delic a	Antibiotics
      			A#	P#	H#
      1.	E. herbicola	7	–	–	–
      2.	E. coli	–	7	5.8	6
      3.	S. aureus	9.5	8	–	–
      4.	A. rhizogenes	6	11	6	5.5
      5.	R. solani	–	–	–	–
      6.	T. atroviride	–	–	–	–
      7.	A. solani	–	–	–	–
      8.	F. oxysporium	5.5	6	–	–

      # (A- Amoxicillin, P- Phnexin, H- Hexamox, + – Effective, – – Not effective)

      MIC evaluation

      All the fractions of ethanol extract of R. delica found significantly active in disc diffusion analysis at 2000

      µg/ml concentration were further analyzed to determine MIC. It was observed that to inhibit the growth of A. rhizogens and F. oxysporium a minimum concentration of 1.0 mg/ml of R. delica extract was required whereas in case of E. herbicola minimum 1.5 mg/ml of extract was required for inhibition. In case of S. aureus only 0.5 mg/ml of R. delica extract was sufficient for inhibition (table 4).

      Test microorganism	MIC (mg/ml) of R. delica
      F. oxysporium	1.0
      E. herbicola	1.5
      S. aureus	0.5
      A. rhizogenes	1.0

      Table 4. Minimum concentration required to inhibit the growth of test pathogenic microorganism.

      Acknowledgements

      Authors are thankful to the Department of Biotechnology, Kumaun University, Nainital for providing necessary facilities to execute this work.

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