JOURNAL OF PURE AND APPLIED MICROBIOLOGY, December 2012. Vol. 6(4), p. 1997-2001

* To whom all correspondence should be addressed.Mob.: +91-9817208432, 09418356611E-mail: mehta.sheetal1@gmail.com

In vitro Comparative Evaluation of Antibacterial Activity ofFruiting Body and Mycelial Extracts of Ganoderma lucidum

against Pathogenic Bacteria

Sheetal Mehta* and Savita Jandaik

Faculty of Biotechnology, Shoolini University of Biotechnologyand Management Sciences, Solan, Himachal Pradesh - 173 212, India.

(Received: 08 April 2012; accepted: 14 May 2012)

A wide variety of organisms are emerging as resistant to antibiotics, and multipledrug resistant organisms pose a serious threat to the treatment of infectious diseases.Hence, mushroom derived antimicrobial substances have received considerable attentionin recent years. In this study antagonistic effects of the methanol, acetone and waterextracts of mycelia and fruiting body of Ganoderma lucidum were tested against sevenbacterial species: Escherichia coli, Staphylococcus aureus, Bacillus subtilis,Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae, andSalmonella typhimurium. All the extracts exhibited various degree of inhibition againstall test bacteria. Widest inhibitory zone (33mm) was obtained with mycelial acetoneextract of Ganoderma lucidum against Pseudomonas aeruginosa. Lowest zone ofinhibition (7mm) was observed with fruiting body aqueous extract against Staphylococcusaureus and Klebsiella pneumoniae. Minimum inhibitory concentration (MIC) of acetoneextract of fruiting body and mycelial extract was determined, for mycelial extract itranged between 4 to 12mg/ml and for fruiting body extract it ranged between 15 to 35 mg/ml for test bacteria.

Key words: Ganoderma lucidum, Antibacterial activity, Bioactive molecules, Extraction.

Mushrooms are defined as “macrofungi”with distinctive fruiting bodies that are largeenough to be seen by the naked eye and to bepicked by hand. In recent years, more varieties ofmushrooms have been isolated and identified, andthe number of mushrooms being cultivated for foodor medicinal purpose has been increasing rapidly1.Development of bacterial resistance to currentlyavailable antibiotics has made it necessary tosearch for new antibacterial agents; natural plantproducts are being investigated because medicinalplants have been widely used for treatment of manytypes of acute and chronic diseases and many

plants with antimicrobial activity have beenreported2,3. Several mushroom species belongingto the Polyporaceae family are now being regardedas the next candidate producers of valuablemedicines4. G.lucidum (Curtis: Fr) Karst is abasidiomyceteous fungus used as a traditionalmedicine for more than 2000 years5. It’s a popularremedy to treat many diseases like chronichepatitis, nephritis, hypertension, hyperlipemia,arthritis, neurasthenia, insomnia, bronchitis,asthma, gastric ulcer, arteriosclerosis, leucopenia,diabetes, anorexia6,4. The fruiting body, mycelia andspores of G.lucidum contain approximately 400different bioactive compounds, which mainlyinclude polysaccharides, triterpenoids, fatty acids,nucleotides, protein/ peptides, sterols, vitamins,minerals etc7, 8, 4, 9, 10. These metabolites are reportedto be responsible for the medicinal properties of

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this mushroom. Many natural products are activeas anti- HIV agents11, these compounds belong toa wide range of different structural classes viz,terpenes, polysaccharides, coumarins andflavenoids etc.12. Because of presence of some ofthese compounds (triterpenoids) in this mushroom,it has potent inhibitory activity against HIV. Someother constituents such as ganomycin,triterpenoids and aqueous extracts fromGanoderma species have a broad spectrum of in-vitro antibacterial activity against gram positiveand gram negative bacteria13, 14, 8, 15, 16, 17, 18. Thismushroom also has some antifungal compoundsin it 19. Ganoderma lucidum and other Ganodermaspecies are used to treat various bacterial diseasesin combination with other therapeutic agents also15.The present study, for the first time reports thecomparison of antimicrobial activity of fruitingbody and mycelial extracts of Ganoderma lucidum.

MATERIALS AND METHODS

Fungal sampleThe fruit bodies of Ganoderma lucidum

used in this study were collected from differentareas of Himachal Pradesh, and were identified onthe basis of microscopic and macroscopicmorphological traits with the standard descriptionof Stamet 19936. Mycelial biomass was obtainedby inoculating 200ml of potato dextrose broth (HiMedia) with 6mm disc of inoculum from 7 days oldplate of G. lucidum culture. Flasks were incubatedat 28°C for 15 days and biomass was separated byfiltration and was dried.Test Organisms

In vitro antimicrobial susceptible studieswere performed using seven human pathogenicbacteria (procured from MTCC Chandigarh):Escherichia coli (MTCC 739), Staphylococcusaureus (MTCC 737), Bacillus subtilis (MTCC 441),Pseudomonas aeruginosa (MTCC741),Enterobacter aerogenes (MTCC 111), Klebsiellapneumoniae (MTCC 109), and S. typhimurium(MTCC 98). Antibacterial activity of extracts wasscreened by filter paper disc diffusion method andactivity was measured in terms of zone of inhibitionsize (mm) obtained after 24 h incubation at 37°C.Preparation of bacterial inoculum

Test bacterial colonies were transferredin nutrient broth and were incubated at 37°C. In

order to standardize the inoculums density for test,a BaSO

4 turbidity standard (equivalent to 0.5 Mc

Farland std. = turbidity equals to 0.5 =1 to 2 x10 8

cfu /ml) was used.Preparation of fungal extract

The fruit bodies were cut into bits anddried at 40 ºC. These dried fruit bodies and driedmycelial biomass were pulverized in a blender. Theextraction of the mushroom fruit bodies andmycelial biomass was carried out using threesolvents (water, methanol and acetone). For waterextraction, 1 litre of sterile distilled water wasdispensed into conical flasks containing 100g ofpowdered mushroom fruit bodies and mycelialsample. These were allowed to stand for 72 h withintermittent agitation. For methanol and acetoneextraction, 100g of the pulverized fruit bodies andmycelial biomass was separately soaked in 1 litreof absolute methanol and acetone in conical flasks.These were covered with aluminum foil and allowedto stand for 7 days for extraction. The mixtureswere filtered using Whatman filter paper No. 1 andthe filtrate was concentrated under a reducedpressure in a rotator evaporator until a semi solidsubstance was obtained. These were dried insidethe crucible under a controlled temperature (45ºC)to obtain solid extract. The left residues were keptin refrigerator until used20.Antimicrobial activity of extracts

The concentration of the extracts usedwas 60 mg/ml and sterile distilled water was usedto reconstitute the extract residues. Fordetermination of antimicrobial activity sterile filterpaper discs (6mm diameter) were soaked with thetest extracts (60mg/ml) and dried at 40 ºC for 1 h.The discs were placed on bacteria seeded MullerHinton Agar (Hi Media) plates and placed in therefrigerator for 12 h to allow the diffusion of theextracts into the growing medium. The plates wereincubated for 24 h at 37ºC after which the zone ofinhibition was observed and measured20.Minimum Inhibitory Concentration (MIC)

This study aimed in finding out the lowestconcentration of acetone extract that will inhibitthe growth of the test microorganisms. It wascarried out by following the method described byHirasawa et al (1999) 21. Different concentrations(1- 40mg/ml) were prepared using sterile distilledwater as the diluent. Filter paper disc diffusionmethod was used22.

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1999MEHTA & JANDAIK: STUDY OF Ganoderma lucidum AGAINST PATHOGENS

RESULTS

In this study antibacterial activity ofmethanol, acetone and aqueous extracts ofG.lucidum was determined by disc diffusionmethod. The antimicrobial activity of samplesvaried according to the solvent. Results presentedin Table I and Table II shows the antibacterialactivity of different extracts of fruiting body andmycelial biomass respectively. Table III shows MICvalue of acetone extract of fruit body and mycelialbiomass. It is clear from the data presented inTable 1 and Table 2 that the acetone extract ofGanoderma lucidum showed maximumantibacterial activity followed by methanol andaqueous extract. Mycelial extract of the G.lucidumshowed high antibacterial activity as compared tofruit body extract. Maximum inhibitor activity ofacetone extract of mycelial biomass (Table 2) wasshown against P.aeruginosa (33mm), followed byE. coli with zone size of 30mm. This extract showedequal inhibitory effect against K. pneuminae andE. aerogenes (24mm). A 14mm zone was shown forB.subtilis and least zone was shown againstS.aureus (12mm) at the same concentration. In caseof methanol extract, maximum activity was foundagainst K. pneuminae (19mm) followed by E. coli(18mm). Aqueous extracts were found to becomparatively less effective against all bacterialstrains. MIC of only acetone extract wasdetermined because it exhibited maximumantagonistic activity. MIC value of mycelialbiomass was found to be 4mg/ml againstP.aeruginosa followed by E.coli with a value of 6mg/ml. In case of fruit body MIC was 15 mg/ml forP.aeruginosa and E.coli.

DISCUSSION

Resistant bacteria are emerging worldwide as a serious threat to the outcome of commoninfections in community and hospitals. Therefore,novel antimicrobial agents from different biologicalsources are continuously sought. Rosa et al. (2003)detected 14 mushroom isolates with highantimicrobial activity against target micro-organisms23. Zjawiony (2004) observed that 75%of polypore fungi that have been tested showstrong antibacterial activity24. Ganoderma lucidumwas reported to be best among other Ganoderma

Table 1. Antibacterial activity of various extractsof Ganoderma lucidum (fruiting body)

Test bacteria Zone of inhibition (mm)Fruit body(60mg/ml)

Methanol Acetone Aqueous

S.aureus 14 10 7E.coli 18 11 11K.pneuminae 19 8 7B.subtilis 17 13 9Enterobacter 9 13 11P.aeruginosa 11 16 9S.typhimurium 17 19 10

Table 2. Antibacterial activity of various extractsof Ganoderma lucidum (mycelial biomass)

Test bacteria Zone of inhibition (mm)Mycelial biomass(60mg/ml)

Methanol Acetone Aqueous

S.aureus 16 12 12E.coli 22 30 10K.pneuminae 16 24 8B.subtilis 11 14 9Enterobacter 15 24 12P.aeruginosa 24 33 12S.typhimurium 10 14 9

Table 3. Showing MIC values for acetone extract

Test Bacteria MIC value (mg/ml)

Mycelial biomass Fruit body

S.aureus 11 30E.coli 6 15K.pneuminae 8 25B.subtilis 6 20Enterobacter 12 35P.aeruginosa 4 15S.typhimurium 7 20

species that generally exhibited high antagonisticactivity against test bacteria25. Recently, morestudies demonstrated that G.lucidum containantibacterial constituents that are able toinhibit gram-positive and /or gram-negativebacteria18, 17, 14, 15, 13. It is evident from the results of

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2000 MEHTA & JANDAIK: STUDY OF Ganoderma lucidum AGAINST PATHOGENS

present investigation that G.lucidum extracts hadinhibitory activity against both Gram positive andGram negative bacteria. These results also affirmthe claims of traditional herbalists in the southwestern Nigeria that Ganoderma species could beused as feed supplement to resist microbialinfections in human being.

All the extracts were different in theirantimicrobial effectiveness depending on theirextractive solvent used. Our results agree favorablywith the suggestions of Oloke and Kolawole (1988)that bioactive components may differ in theirsolubility depending on the extractive solventsused26. Kawagishi et al., (1988), observed thatsome of the active phytochemicals are soluble inalcohol but insoluble in water as in case of Agaricusblezei27. Cowan (1999) reported that the most activecomponents are generally water insoluble, henceit is expected that low polarity organic solventswould yield more active extracts2, our findings ofpresent study are consistent with theseobservations, the organic extracts exhibited highantimicrobial activity than aqueous extract.

Yamac and Bilgili (2006) investigatedantimicrobial activity of chloroform anddichloromethane extracts of fruit body/mycelialcultures of some (20 isolates) mushrooms against7 bacterial strains and observed good antimicrobialactivity with all the extracts for all tested organisms,in their findings mycelial extract of many fungipossess high inhibitory activity against Gramnegative bacteria28, our results are in collaborationwith this finding, mycelial extract had greaterinhibitory effect against E.coli and P. aeruginosain the present study. Mycelial biomass extractsexhibited high inhibitory activity as compared tofruit body extracts possibly because of varyingcontent of bioactive molecule in fruit body andmycelia. It is essential to carry out further researchregarding this difference in antagonistic activitypattern of fruit body and mycelia; unfortunatelywe were unable to perform this due to limitedfacilities.

CONCLUSION

In conclusion, this study has shown thatdifferent extracts (aqueous, methanol and acetone)have been used in vitro to inhibit the growth ofsome pathogenic bacteria. It can therefore be

suggested that, Ganoderma lucidum is promisingantimicrobial fungus and could be employed tocombat several bacterial diseases.

ACKNOWLEDGMENTS

The authors wish to thank IMTECH,Chandigarh, for providing bacterial cultures andShoolini University of Biotechnology andManagement Sciences, Solan (HP), for supportingresearch.

REFERENCES

1. Chang, S.T. Ganoderma The leader in productionand technology of mushroom nutriceuticals. InProc 6th Int symp Recent Adv. Ganodermalucidum Res (Ed. B. K. Kim, I.H. Kim and Y.S.Kim), 1995; pp 43-52. The pharmaceuticalSociety of Korea, Seoul, Korea.

2. Cowan, M.M. Plant products as antimicrobialagents. Clinical microbiology review, 1999; 12:564-582.

3. Dulger, B., Gonus, A. Antimicrobial activity ofcertain plants used in Turkish traditionalmedicine. Asian Journal of Plant Science, 2004;3: 104-107.

4. Mizuno, T. Reishi, Ganoderma lucidum andGanoderma tsugae: bioactive substances andmedicinal effects. Food Rev. Int., 1995; 11:151-166.

5. Fang, Q.H. and Zhong, J.J. Submergedfermentation of higher fungus Ganodermalucidum for production of valuable bioactivemetabolites- ganoderic acid and polysaccharides.Biochem. Eng. J., 2002; 10: 61-65.

6. Stamet, P. Growing Gourmet & MedicinalMushrooms, Ten Speed Press, Berkeley, 1993.

7. Yoon, S.Y., Eo, S.K., Lee, C.K., Han, S.S.Antimicrobial activity of Ganoderma lucidumextract alone and in combination with someantibiotics. Archieves of Pharmacal Research,1994; 17: 438-442.

8. McKenna, D.J., Jones Hughes, K. ReishiBotanical Medicines. The desk refrence forMajor Herbal Supplements, 2nd Ed.; TheHaworth Herbal Press: New York, Londan,Oxford, 2002; 825-855.

9. Kim, H.W., Kim, B.K. Biomedical triterpenoidsof Ganoderma lucidum (Curt.: Fr.) P. Karst.(Aphyllophoromycetideae). Int. J. Med.Mushrooms, 1999; 1: 121-138.

10. Gao, Y., Zhou, Sh., Chen, G., Dai, X., Ye, J. Aphase I/II study of Ganoderma lucidum (Curt.:

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Fr.) P. Karst. Extract (ganopoly) in patients withadvanced cancer. Int. J. Med. Mushrooms, 2002;4: 207- 214.

11. Kartikeyan, S., Bharmal, R.N., Tiwari, R.P. andBisen, P.S. HIV and AIDS Basic Elements andPriorities, 2007; Springer Verlag: Dordrecht, TheNetherlands.

12. Vermani, K. and Garg, S. Herbal medicines forsexually transmitted diseases and AIDS.J. Ethnopharmacol, 2002; 80: 49-66.

13. Wasser, S.P., Weis, A.L. Medicinal Mushrooms.Ganoderma lucidum (Curtis: Fr.), P. Karst;Nevo, E., Eds.; 1997; Peledfus Publ House:Haifa, Israel 39.

14. Hobbs, Ch. Medicinal Mushrooms: AnExploration of Tradition, Healing, and Culture,2nd Ed.; Botanica Press, Inc., 1995; Santa Cruz,CA, USA.

15. Gao, Y., Zhou, S., Huang, M., Xu, A.Antibacterial and antiviral value of the genusGanoderma P. Karst. Species(Aphyllophoromycetideae): A review.International journal of medicinal mushrooms,2003; 5: 235-246.

16. Smith, J., Rowan, N., Sullivan, R. MedicinalMushrooms. Their Therapeutic Properties andCurrent Medical usage with Special Emphasison Cancer Treatment; Special ReportCommissioned by Cancer Research UK, 2002;The University of Strathclyde in Glasgow 256.

17. Stamet, P. Growing Gourmet & MedicinalMushrooms, Ten Speed Press, 2000; Berkeley.

18. Suay, I., Asensio, F.J., Basilio, A., Cabello, M.A.,Diez, M.T., Garcia, J.B., del Val, A.G.,Corrochategui, J., Hernandez, P., Pelaez, F.,Vicente, M.F. Screening of Basidiomycetes forantimicrobial activities. Antonie vanLeewenhoek, 2000; 78:129-139.

19. Wang, H. and Ng, T.B. Ganodermin an antifungalprotein from fruiting bodies of the medicinal

mushroom Ganoderma lucidum. Peptides, 2006;27: 27-30.

20. Jonathan, S.G. and Fasidi, I.O., Antimicrobialactivities of two edible macrofungi-Lycoperdonpusilum (Bat.ex) and Lycoperdon giganteum(Pers). African Journal of Biomedical Reseach,2003; 85-90.

21. Hirasawa, M., Shouji, N., Neta, T., Fukushima,K. & Takada, K. Three kinds of antibacterialsubstances from Lentinus edodes (Berk.) Sing.(Shiitake, an edible mushroom). Int J AntimicrobAgents, 1999; 11: 151-157.

22. Jonathan, S.G. and Fasidi, I.O. Antimicrobialactivities of some selected Nigerian Mushrooms.African Journal of Biomedical Reaserch, 2005;8: 83-87.

23. Rosa, L.E., Machado, KMG., Jacob, C.C.,Capelari, M., Rosa, C.A., Zani, C.L. Screeningof Brazilian Basidiomycetes for antimicrobialactivity. Memorias do Instituto Oswaldo Cruz,2003; 98:967-974.

24. Zjawiony, J.K. Biologically active compoundsfrom Aphyllophorales (polypore) fungi. Journalof Natural Products, 2004; 67: 300-310.

25. Jonathan, S.G. and Awotona, F.E. Studies onantimicrobial potentials of three Ganodermaspecies. Afr. J. Biomed. Res., 2010; 133-139.

26. Oloke, J.O. and Kolawole, D.O. TheAntibacterial and Antifungal Activities of Certaincomponents of Aframomum meleguetea fruits.Fitoterapia, 1998; 59(5): 384 388.

27. Kawagishi, H.A., Nomura, T., Yumen, T.,Mizumo,T., Hgwara, A., and Nakamuna, T.Isolation and properties of lecitin from thefruiting bodies of Agaricus blazei.CarbohydrateResearch,1988; 15 183(1): 150-154.

28. Yamac, M., Bilgili, F. Antimicrobial activities offruit bodies and /or mycelial cultures of somemushroom isolates. Pharmaceutical Biology,2006; 44: 660-667.

International Journal of Medicinal Mushrooms, 16(6): 585–591 (2014)

5851045-4403/14/$35.00 © 2014 Begell House, Inc. www.begellhouse.com

Effect of Cost-Effective Substrates on Growth Cycle and Yield of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes) from Northwestern Himalaya (India)Sheetal Mehta,1,* Savita Jandaik,1 & Dharmesh Gupta2

1Shoolini University, Faculty of Biotechnology, Solan, HP, India; 2Dr. Y.S. Parmar University of Horticulture and Forestry Nauni, HP, India

*Address all correspondence to: Sheetal Mehta, Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Bajhol, Solan, HP 173212, India; Tel.: +91-941-835-6611; Email: mehta.sheetal1@gmail.com.

ABSTRACT: To find a cost-effective alternative substrate, the medicinal mushroom Ganoderma lucidum was grown on sawdusts of sheesham, mango, and poplar. Optimum spawn level was determined by spawning in substrates at various levels (1, 2, 3, and 4%). To determine the effect of supplementation, substrates were supplemented with wheat bran, rice bran and corn flour at different concentrations (10, 20, and 30%). Duration of growth cycle, mushroom yield, and biological efficiency data were recorded. Among substrates, mango sawdust was superior, with 1.5-fold higher yields than poplar sawdust, which was the least suitable. However with respect to fructification, mango sawdust produced the first primordia earlier (21±1 days) compared with the other investigated substrates. 3% spawn level was found to be optimal irrespective of the substrate. Yield and biological efficiency (BE) were maximally enhanced by supplementation with wheat bran, whereas rice bran was the least suitable supplement among those tested. Growth cycle shortened and mushroom yield increased to a maximum at the 20% level of supplements. Mango sawdust in combination with 20% wheat bran, if spawned at the 3% level, resulted in a high yield (BE = 58.57%).

KEY WORDS: medicinal mushrooms, Ganoderma lucidum, sawdust, supplement, yield, biological efficiency

ABBREVIATIONS: CF, corn flour; ITS, internal transcribed spacer; MS, mango sawdust; PS, poplar sawdust; PVC, polyvinyl chloride; RB, rice bran; SS, sheesham sawdust; WB, wheat bran.

I. INTRODUCTION

Lingzhi or Reishi medicinal mushroom, Ganoder-ma lucidum (W.Curt.:Fr.) P. Karst. (Ganodermata-ceae, higher Basidiomycetes) has become the object of intensive research because of its multiple health benefits with apparent absence of side effects.1,2 All pharmacological properties have been correlated to large pool of bioactive compounds produced by its fruiting bodies, mycelia and spores.3 G. lucidum is rare in nature; consequently, the supply of wild mushrooms is insufficient for commercial exploi-tation, as more than 100 brands of Ganoderma are sold on the market.4 Chang and Wasser reported the total global production of this mushrooms in 2012.5 In India, the market for Ganoderma-based nutri-

ceuticals is growing very rapidly and was estimated to be approximately US $20 million in 2012. Even though the cultivation technology has advanced in many Asian countries, very little is known about the cultivation of this mushroom in India, where Gano-derma products are imported from Asian countries, even though Ganoderma mushrooms are indigenous to India. Considering the increased demand for this mushroom in domestic and export markets, its cul-tivation has become an essential focus of research.

G. lucidum can be grown with large variety of substrates, including lignocellulosic components (cellulose, hemicellulose, and lignin) that provide soluble inorganic and organic materials. In recent years, successful artificial cultivation of mushrooms has been reported on solid substrates containing

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agricultural residues such as rice, banana, wheat, sorghum, varagu, sugarcane, poplar, oak, corn and beech, nepal alder, Indian horse chestnut, red ce-dar, black jack oak, sunflower seed hulls, and whey permeate.6–11 One strategy for improving yield may include supplementation of the substrate with nitrogen-based supplements such as millet, gram, rye, corn, etc.12–14 Some of these raw materials may not be available or are available at relatively higher prices in some parts of India. Thus, growers are perpetually searching for alternative substrates that may be more readily available and economical and that provide higher yield and better quality.

Cost-effective production of mushrooms de-pends on the reliability, availability, and cost of substrate ingredients. Mango, sheesham, and pop-lar sawdusts are easily available agro-wastes in In-dia, and as hardwoods, they are likely to support mushroom growth. Wheat, corn, and rice are also among the top crops of India, offering easy avail-ability and inexpensive and nutrient-rich supple-ments.

In the present mushroom cultivation study, we specifically aimed (1) to determine optimum spawn level, (2) to investigate the suitability of various sawdusts as growth media, and (3) to de-termine the effect of various supplements at differ-ent spawn levels.

II. MATERIAL AND METHODS

A. Substrate and Supplement Materials

For this study, sawdusts of sheesham (Dalbergia sissoo), mango (Mangifera) and poplar (Populus); brans of wheat (Triticum), rice (Oryza sativa); and corn flour (Zea mays) were collected from different regions of Punjab and Himachal Pradesh in India.

B. Mushroom Cultures

During the mycobiotic survey of different regions of Punjab and Himachal Pradesh, G. lucidum fruit bodies were collected. Polysaccharide content and antimicrobial activities of all isolates were evaluated, and one isolate (strain no. GL04) with

maximum polysaccharide content and antimicro-bial activity was chosen for cultivation studies at a mushroom farm. Identity of the culture was confirmed by amplifying and sequencing the In-ternal Transcribed Spacer (ITS) DNA, according to a previous report (GenBank accession number: KF648564). A pure culture was grown on malt extract agar slants. These slants were incubated at 28°C until substantial mycelial growth was ob-served. This mycelium (raised from single fruit body of the GL04 strain) was used as the inoculum for the mother culture and for spawn preparation on wheat grains (Triticum durum).

C. Experimental Design

In the first part of the experiment, the substrates were spawned at various levels. Each treatment had three replicates, resulting in 36 experimental units. The second part of the experiment investi-gated the most suitable substrate by spawning all test substrates at the optimal level. Each treatment had three replicates, resulting in nine experimental units. The third part of the experiment consisted of supplementing various substrates with three differ-ent supplements at variable concentrations. Each treatment had three replicates, resulting in 81 ex-perimental units. The effects of these experimental conditions were evaluated by their effect on the complete spawn run period, the time needed for primordia formation, yield, and the biological ef-ficiency (BE) of G. lucidum.

D. Substrate Preparation and Inoculation

Substrate preparation includes setting pH, estab-lishing moisture content, and substrate steriliza-tion. All sawdusts were stacked on a cemented floor and wetted thoroughly for 24 h to raise the moisture content to approximately 65%. Calcium sulphate (gypsum) and calcium carbonate (chalk powder) were added to achieve a pH of 5.5. The test substrates were loaded into polypropylene bags (20×35 cm, without filter) each weighing 1 kg (350 g dry weight). Then the bags were labeled and closed with PVC (polyvinyl chloride) rings (3 cm

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diam.) and plugged with cotton to prevent contam-ination by airborne organisms while allowing air exchange. Bags were autoclaved at 121°C for 2 h, were allowed to cool, and were then spawned.

E. Analysis of Effect of Spawn Level

This experiment was performed on all substrates (MS, SS, and PS). To determine the optimal spawn level, different doses of spawn were inoculated at 1, 2, 3 and 4% dry weight of all substrates. Spawn was mixed thoroughly in the substrate material by shaking the bags properly (i.e., ‘thorough spawn-ing’) under aseptic conditions.

F. Analysis of Best Substrate

To determine best growth substrate, all test sub-strates were spawned at the 3% level.

G. Analysis of Effect of Supplementation

To find a superior combination of media or to assess effect of supplementation, sawdusts and test supplements were mixed in different ratios (sawdust:supplements, 90:10, 80:20, and 70:30), spawned at the 3% level, and were then exposed to cultivation conditions.

H. Cropping and Harvesting

Inoculated bags were arranged on pest-resistant shelves in a cropping room to attain the five stages of mushroom growth: spawn run, primordial ini-tiation, stalk formation, cap differentiation, and maturation. The temperature and relative humidity were controlled at 28 ± 2°C and 65%, respectively, during the first stage (spawn run period was com-pleted without artificial lighting). The temperature and relative humidity were controlled at 28 ± 2°C and 90–95% during the second stage with light ex-posure (10 h, provided by white fluorescent bulbs).

FIG. A: Growth of G. lucidum on MS (without supplements). B: Growth of G. lucidum on MS supplemented with WB. C: Growth of G. lucidum on MS spawned at the 1% level. D: Growth of G. lucidum growth on MS spawned at the 3% level.

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The temperature and relative humidity were con-trolled at 28± 2°C and 70–80% during the third stage with light exposure. The temperature and relative humidity were controlled at 25°C and 85–90% during the fourth stage with light exposure and at 28±2°C and 60% relative humidity during the last stage. All inoculated bags remained closed until the substrate in the bags was fully colonized. Completely colonized bag tops were cut at the level of the substrate to allow the development of fruiting bodies. Days needed for the complete spawn run and primordia initiation were recorded. Fruit bodies were harvested when they had fully matured, and total yield (g) was measured. The BE percentage [(fresh weight of harvested basidi-omata/dry weight of the substrate) × 100] was cal-culated.

I. Statistical Analysis

One-way ANOVA followed by Tukey- Kramer multiple comparison test was performed using GraphPad Prism, version 5.02 (GraphPad Soft-ware, Inc.), to determine the significance of differ-ences between different study groups.

III. RESULTS AND DISCUSSION

A. Effect of Different Spawn Levels

Spawn level is defined as the weight ratio of spawn to substrate on a dry weight basis. This factor re-lates to the production cost of mushroom cultiva-tion, as spawn is a relatively expensive item. An attempt was made to determine the optimal spawn level so that minimal inoculum could be used with-out sacrificing mushroom yield to achieve the best economics. Among the tested spawn levels (1, 2, 3, and 4%), the 3% level was optimal, resulting in the shortest growth cycle and maximum yield (Table 1) with all substrates, and was statistically similar to the 4% spawn level. However, the 1% spawn level was least efficient in all aspects, with the longest spawn run period and a negligible yield with all substrates, which proves that a spawn level that is too low results in a longer growth cycle and

lower yield. No similar study has been reported so far; thus, this study is the first of its kind to our knowledge. In our study, an increase in the spawn level from 2 to 4% demonstrated an increase in yield and shortened the growth cycle duration in G. lucidum. This result may be explained by the fact that increased spawn level accelerates colonization and narrows the gap of opportunity for competi-tor invasion. Deviation in optimum spawn levels in various studies may be attributed to the type of substrate, mushroom species, spawn quality, and cultivation conditions.

B. Effect of Different Sawdusts

To determine the best substrate, test sawdusts were inoculated with 3% spawn and exposed to spawn-ing and cropping conditions. The prerequisite cri-teria for selecting these agro-wastes as substrates were their suitability as growth media, continuous support to growth, and the related activity of my-celium of some other mushrooms. Initiation of my-celial growth occurred on day 8 post inoculation in all substrates. This uniformity in the initiation of mycelial growth in all of the substrates may have been due to the low enzyme activity during sub-strate colonization. The implication of this result is that the level of breakdown of substrate materials for the release of nutrients is low or nonexistent within this short period of 8 days. Hence, nutri-ents in the substrate may have not been released and consequently were not available to the mycelia and thus had no influence on the initial growth of the mycelia. Spawn run refers to the propagation of mycelia in the colonized substrate bags dur-ing the vegetative growth stage before fruit body formation. In this study, this period ranged from 15 to 20 days as shown in Table 2. The minimum time for initiation of pin heads occured with MS followed by SS and PS, respectively. The highest yield (weight of fresh mushrooms) was obtained with MS, which was 1.5-fold higher than the yield on PS; however, the yield on SS was between that of MS and PS (Table 2). The lowest yield with PS could be due to the carbon:nitrogen (C:N) imbal-ance in the sawdust, which is in agreement with the

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Effect of Cost-Effective Substrates on Growth Cycle and Yield of Ganoderma lucidum from India 589

previous observations of Stamets.14

C. Effect of Combinations of Sawdust and Supplements

The experiment was conducted to assess the best combination of media or the role of different or-ganic supplements in stimulating the growth of

G. lucidum so that productivity can be enhanced. Sawdusts and brans were mixed in different ratios (90:10, 80:20, or 70:30); inoculated with the 3% spawn level, and exposed to proper spawning and fructification conditions. Our results (Table 3) re-veal that supplementation enhanced the yield on all substrates at all concentrations, which can be attrib-uted to the change in decomposition rate and the se-

TABLE 1: Effect of Different Doses of Spawn on Growth Cycle and Yield of Ganoderma lucidum

Substrate Spawn rate (%)

Complete spawn run (Days)

Primordia initiation (Days from spawning) Yield (g/kg) BE (%)

SS 1 22 ± 1.0 29 ± 0 2.33 ± 1.45B,C,D 0.66 2 20 ± 1.0 27 ± 0.58 25 ± 5.00533A,C,D 7.14 3 17 ± 0.58 24 ± 0.58 120 ± 4.93A,B 34.28 4 17 ± 1.0 25 ± 0 120 ± 5.51A,B 34.28

MS 1 17 ± 0 24 ± 0 7 ± 2.08B,C,D 2.0 2 16 ± 0.58 24 ± 0 65 ± 4.04A,C,D 18.57 3 15 ± 0.58 20 ± 1.0 150 ± 2.64A,B 42.86 4 16 ± 1.0 24 ± 0 146 ± 4.58A,B 41.71

PS 1 22 ± 1.0 31 ± 1.0 2 ± 2C,D 0.57 2 22 ± 0.0 30 ± 0.58 1 ± 1C,D 0.28 3 20 ± 0.58 28 ± 1.0 100 ± 4.93 A,B,D 28.57 4 21 ± 0.58 29 ± 0.0 80 ± 4.36A,B,C 22.86

All substrates were inoculated with various spawn levels i.e., 1, 2, 3, and 4%, in order to determine optimum spawn level on basis of total yield at the end of harvest. Results represent the mean ± SEM of the three independent experiments followed by different letters indicate significant differences according to Tukey-Kramer HSD (P < 0.05), n=3. SS, sheesham sawdust; MS, mango sawdust; PS, poplar sawdust; BE, biological efficiency.

TABLE 2: Effect of Different Sawdusts on Growth Cycle and Yields of Ganoderma lucidum

Substrates Complete spawn run (Days)

Primordia initia-tion (Days from spawning) Yield (g/kg) BE (%)

SS 17 ± 0.58 24 ± 0.58 120 ± 4.93B,C 34.28

MS 15 ± 0.58 21 ± 1.0 150 ± 2.64A,C 42.86

PS 20 ± 0.58 29 ± 1.0 100 ± 4.93A,B 28.57

Spawn was added to different substrates at the 3% level, exposed to spawning and cropping con-ditions. After maturation, fruit bodies were harvested, total mushroom yield in grams per kg was recorded. Results represent the mean ± SEM of the three independent experiments followed by different letters indicating significant differences according to Tukey-Kramer HSD (P < 0.05), n=3. SS, sheesham sawdust; MS, mango sawdust; PS, poplar sawdust; BE, biological efficiency.

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Mehta, Jandaik, & Gupta590

quence of decomposition of substrate components by supplements.15,16 Of three different supplements (WB, CF, and RB) at variable concentrations (10, 20, and 30%), 20% of all supplements supported higher reduction in growth cycle duration and en-

hanced yield and BE on various substrates, though there was no statistical difference compared with 10% and 30% supplementation. Maximum yield was obtained with a mixture of MS and WB (205 g at 20%), followed by a mixture of SS and WB

TABLE 3: Effect of Combinations of Various Sawdusts and Supplement Doses on Growth Cycle and Yield of Ganoderma lucidum

Substrate Supplement Supplement Ratio (%)

Spawn run (Days )

Primordia initiation (Days from spawning) Yield (g/kg) BE (%)

SS WB 10 17 ± 0.58 24 ± 0.58 152.33 ± 3.18 43.52

20 15 ± 1.0 20 ± 0.58 171.66 ± 3.84 49.04

30 17 ± 1.0 25 ± 0.0 159.33 ± 2.03 45.52 CF 10 18 ± 1.0 21 ± 0.0 136.66 ± 3.48 39.04

20 17 ± 0.0 21 ± 0.58 156.66 ± 2.60 44.76

30 17 ± 1.0 22 ± 0.58 145.33 ± 3.93 41.52

RB 10 17 ± 1.0 23 ± 0.0 134.66 ± 3.84 38.47

20 17 ± 1.0 23 ± 0.0 148 ± 7.64 42.28 30 17 ± 0.0 24 ± 0.0 140.66 ± 7.75 40.19

MS WB 10 13 ± 0.0 18 ± 0.58 177 ± 4.04 50.57 20 13 ± 0.58 17 ± 0.0 205 ± 4.04 58.57 30 13 ± 0.58 18 ± 1.0 170.66 ± 4.98 48.76

CF 10 14 ± 1.0 20 ± 0.58 170 ± 6.08 48.57 20 14 ± 0.0 19 ± 0.0 194 ± 1 55.43 30 14 ± 1.0 20 ± 0.0 165 ± 5.86 47.14

RB 10 15 ± 0.58 21 ± 1.0 168 ± 4.62 48.0 20 15 ± 0.0 20 ± 0.58 185 ± 3.78 52.86 30 16 ± 0.0 20 ± 1.0 163 ± 8.89 46.57

PS WB 10 20 ± 0.0 28 ± 0.58 139.33 ± 1.85 39.80 20 18 ± 0.58 25 ± 1.0 156 ± 4.72 44.57 30 19 ± 0.0 27 ± 0.58 142.33 ± 3.76 40.66

CF 10 20 ± 0.0 28 ± 0.58 131.66 ± 2.90 37.52

20 21 ± 0.58 29 ± 1.0 147 ± 5.51 42 30 21 ± 0.58 29 ± 0.0 135 ± 7.37 38.57

RB 10 20 ± 0.58 29 ± 0.58 122.66 ± 4.70 35.04

20 20 ± 0.58 30 ± 1.0 144 ± 3.78 41.14 30 21 ± 1.0 30 ± 0.0 136.66 ± 3.84 39.04

Sawdusts and supplements were mixed in different proportions (90:10, 80:20, and 70:30) for cultivation trials. SS, sheesham sawdust; MS, mango sawdust; PS, poplar sawdust; WB, wheat bran; CF, corn flour, RB, rice bran; BE, biological efficiency. Results represent the mean ± SEM of the three independent experiments followed by different letters indicate significant differences according to Tukey-Kramer HSD (P < 0.05), n=3.

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Effect of Cost-Effective Substrates on Growth Cycle and Yield of Ganoderma lucidum from India 591

(194 g at 20%) and a mixture of MS and RB (185 g/kg at 20%). However, the mixture of PS and RB gave the lowest yield and BE, irrespective of the supplement ratio. Yield and BE of rice bran (RB) with all combinations was lower than with the other treatments, while WB gave the highest yield, irrespective of the substrate (Table 3). Supple-mentation with wheat bran enhanced the yield by 13.77–36.66%. This range was 10–29.33% with corn flour, followed by rice bran at 8.66–23.33% on the best substrate (MS). Thus, the current study indicates that wheat bran is a superior supplement among all investigated supplements. This recom-mendation is supported by the finding that a high production rate is achieved when wheat bran is used to supplement Lentinus edodes mushroom substrates.15 Information regarding combinations of various sawdusts and brans in the cultivation of G. lucidum is very scanty, but the efficiencies of various brans strongly agree with Erkel,8 who reported wheat bran supplemented sawdust to be the best substrate for G. lucidum cultivation. Our present findings also confirm the recent study by Pani,16 who reported higher mushroom yield in Calocybe indica supplemented with wheat bran compared with rice bran.

The major findings of the current study are that shorter growth cycle and higher yield and BE in G. lucidum can be obtained with mango sawdust supplemented with 20% wheat bran spawned at the 3% level. Cultivation of G. lucidum may be-come one of the most inexpensive and profitable agri-businesses, producing medicinal and food products with agro-waste materials, and helping dispose of agricultural residues in an environmen-tally friendly manner. Thus, these findings may be useful in many mycoremediation applications.

ACKNOWLEDGMENTS

The authors wish to thank the Department of Sci-ence and Technology, Govt. of India, for financial support (SR/FT/LS-888/2009 dated 04-09-2009) and Shoolini University of Biotechnology and Management Sciences, Solan (HP), for providing facilities.

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