Research Paper Isolation and Taxonomic Characterization Of

Research Paper

Isolation and taxonomic characterization of medicinal mushroom Ganoderma spp.

Accepted 25th September, 2014

ABSTRACT

The present study deals with isolation, taxonomic characterization and biological activity of medicinal mushroom Ganoderma spp. The results of the present study Ganoderma sp. DKR1 and Ganoderma sp. DKR2 were isolated from two hard woods of Casurina equiestifolia and Morinda tinctoria respectively in Cuddalore district, Tamil Nadu, India. The Ganoderma isolate were characterized for macro and micro morphological properties. The colony, mycelial, basidiospore K. Rajesh, D. Dhanasekaran and A. morphology of fungal isolate was evidence of Basidiomycetes family. 18S rRNA Panneerselvam gene of Ganoderma sp. DKR1 was sequenced by automated gene sequencer.

1. Department of Microbiology, School of Sequence were submitted NCBI and in accession number is GQ495094. The Life Sciences, Bharathidasan University, phylogenetic analysis of 18S rRNA gene of Ganoderma was compared with other Tiruchirapalli, 620 024, Tamilnadu, species of Ganoderma and identified as Ganoderma sp. DKR1. The secondary India. structure and restriction sites present in the 18S RNA gene were predicted using 2. P.G. & Research Department of Botany & Microbiology, A.V.V.M. Sri Pushpam bioinformatics tools. The medicinal mushroom was isolated from hard woods of C. College, (Autonomous), Poondi, 613 503 equiestifolia and M. tinctoria. Further, Ganoderma sp. DKR1 was subjected to Thanjavur District, Tamil Nadu, India. antibacterial activity which showed inhibition against Staphylococcus aureus and Micrococcus sp. *Corresponding author Email: [email protected] Phone: +914312407082 Key words: Ganoderma sp., phylogenetic tree, 18S rRNA, fruiting body. Mobile: +919486258493

INTRODUCTION

Ganoderma species belong to the kingdom of Fungi, the are available on the market including ground fruiting division of Basidiomycota, the class of bodies or mycelium processed into capsule or tablet form; Homobasidiomycetes, the order of Aphyllophorales, the extracts from fruiting body or mycelium dried and family of Polyporaceae, (Ganodermataceae) and the genus processed into capsule or tablet form, Ganoderma beer and of Ganoderma (Chang, 1995; Wasser and Weis, 1999). They Ganoderma hair tonics (Jong and Birmingham, 1992). have a woody or leathery feel and the presence of these Within the genus Ganoderma, over 250 taxonomic names pores are obvious characteristics that distinguish polypores have been reported worldwide including G. adspersum, G. from other common type of mushrooms. They degrade the applanatum, G. australe, G. boninense, G. cupreum, G. wood overtime and produce a fruiting body (or conk) on incrassatum, G. lipsience, G. lobatum, G. lucidum, G. oerstedii, the surface of the wood. Ganoderma species are among G. oregonense, G. pfeifferi, G. platense, G. resinaceum, G. those fungi that can thrive under hot and humid conditions sessile, G. sinense, G. tornatum, G. tsugae and G. webrianum. are usually found in sub tropical and tropical regions (Moncalvo et al., 1995) (Moncalvo and Ryvarden, 1998). In recent years more variety of mushrooms have been Ganoderma species are not classified as edible isolated and identified, and the number of mushrooms mushroom, as the fruiting bodies are always thick, corky being cultivated for food or medicinal purposes has been and tough and do not have the fleshy texture characteristic increasing rapidly. Mushroom “nutraceuticals” are of true edible mushroom such as the common white button bioactive compounds that are extractable from mushrooms, mushroom Agaricus bisporus. Although they are not and they have nutritional and medicinal features that may classified as edible, several types of Ganoderma products be used in the prevention and treatment of disease (Chang Academia Journal of Microbiology Research; Rajesh et al. 062

and Buswell, 1996). Several nutraceutical products have biological activity of Indian Ganoderma species from hard been isolated from medicinal mushrooms and three of wood of Casurina equiestifolia and Morinda tinctoria, these, which are carcinostatic polysaccharides drugs, have Cuddalore district, Tamil Nadu, India. been developed from mushroom in Japan. These are “Krestin” from the cultured mycelium of Kawaratake (Trametes vercicolor), “Lentinan” from the fruiting bodies of MATERIALS AND METHODS Shiitake (Lentinus edodes) and “Schizophyllan” from the cultured fluid of Suehirotake (Schizophyllum commune) Collection of Ganoderma spp. (Mizuno, 1995). Lentinan and Schizophyllan are pure Beta - glucans is a protein bound polysaccharide. Particular focus The two wood rotting fungal species of Ganoderma were on Ganoderma species, it is apparent that most of the collected from hard wood tree such as C. equiestifolia and M. available data on active extracts and compounds relates to tinctoria Cuddalore district, Tamil Nadu. The fungal fruiting the pharmacological effects on tumor cells, which appear to body was harvested and transferred to sterile polythene be based on the enhancement of the host’s immune system. bag. The specimen was brought to the laboratory for Early work in the 1950s found that a number of further investigation. Basidiomycetous fungi had antibacterial activities. More recently, several studies have demonstrated the antibacterial activity of Basidiomycetes (Coletto et al., Isolation of Ganoderma spp. 1994; Bianco and Striano, 2000). The mycelial extracts of Basidiomycetes exhibited antibacterial activity against The potato dextrose agar (peeled Potato 20 g, Dextrose 2.0 Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, g, Agar 2.0 g, Distilled water 100 ml) medium was prepared Escherichia coli, Pseudomonas aeruginosa, Salmonella sp. and sterilized at 121°C for 15 min, 15 lbs in autoclave. The and Candida albicans. Recently, more studies demonstrated sterile medium was transferred to sterile Petri plate and that G. lucidum and other Ganoderma species showed allowed for solidification, the small piece of fungal fruiting antibacterial constituents those are able to inhibit Gram- body was inoculated in PDA medium under aseptic positive and Gram-negative bacteria (Yoon et al., 1994; Li et condition. The plates were incubated at room temperature al., 1999). The aqueous extract from the carpophores of G. for 48 to 72 h. lucidum inhibited 15 types of Gram-positive and Gram- negative bacteria at different extracts using the serial broth dilution method, with the most potent inhibition against Characterization of Ganoderma spp. Micrococcus luteus [minimum inhibitory concentration (MIC) 0.75 mg/ml)]. Macro morphological characterization For a long time past, classification of Ganoderma mushroom is based on morphological characteristics, which The Ganoderma species were collected from hard wood is often highly subjective, with unambiguous identification tree and the following property of fungi were observed to the species level often being extremely difficult. such as size, surface colour, attachment of stipes to pileus, Additionally, the morphological differences observed may margin pattern and surface pattern in naked eyes. be the product of simple mutations or media, cultivation effects and thus are not always reliable characters. It limited the accuracy of identification, and the genetic Micro morphological characterization variety of different strains cannot be objectively described. In production, some strains identification and Lacto phenol cotton blue staining circumscription were often unclear and taxonomic segregation of the genus remained controversial, and even A drop of lacto phenol cotton blue was placed at the centre a number of Ganderma strains have been misnamed. of the glass slide and a portion of fungal colony was The limitations of traditional identification techniques transferred to the stain. Then a clean cover slip was kept indicate that alternative methods need to be developed for over the specimen. The micro morphology of the fungi was the identification of these fungi. With the development of observed under microscope (Labomed). molecular biology, some new techniques have been applied to fungal classical taxonomy. DNA fingerprinting techniques, however, would be allowed to identify the Thin section procedure Ganoderma species and cultivars, indicating that it is a useful tool for the valid protection of newly bred cultivars. The fruiting body of Ganoderma sp. was taken and small The review on medicinal mushroom Ganoderma research in thin sections were taken using sharp blade. The thin section Tamil Nadu, India is very scanty, the present research is was cleaned with distilled water and placed on a clean glass planned with isolation, taxonomic characterization and slide. Then a clean cover slip was kept over the specimen Academia Journal of Microbiology Research; Rajesh et al. 063

and the micro morphology of the polypore was observed (1985) with modifications as described by Moncalvo et al. under phase contrast microscope (Labomed). (1995). Fungal biomasses were harvested by filtration. A mass of 50 mg of biomass was suspended in 500 μl of extraction buffer (200 mm Tris HCl, pH 8.5, 250 mm NaCl, Measurement of fungal cell using micrometer 25 mm EDTA, 0.5% SDS) and vortexed. The resulting suspension was extracted with 350 μl of ice cold phenol: An ocular micrometer was inserted in the eyepiece. The chloroform: isoamyl alcohol (25:24:1) by vortexing for 1 stage micrometer was placed on the stage of the min and followed by centrifugation at 15000 rpm for 45 microscope with low power objective in position and min in a microcentrifuge. The upper phase was transferred graduation of the stage micrometer was brought into focus. to a fresh 1.5 ml eppendorf tube. Then 350 μl of phenol: The eyepiece was rotated until the graduation of the ocular chloroform: isoamyl alcohol (25: 24:1) was added to this micrometer line parallel to the lines of the stage eppendorf tube, mixed thoroughly and spun at 15000 rpm micrometer. The calibration was done with low power for 10 min. The genomic DNA was precipitated from the objective lens. After the completion of calibration, stage supernatant by adding 250 μl of cold isopropanol. The micrometer was removed from the stage and Ganoderma nucleic acid pellet was washed using 70% ethanol, air dried sp. specimen slide was placed in stage. The ocular and resuspended in 50 μl of TE buffer (10 mm Tris HCl, pH micrometer lines were superimposed on the fungal cells. 8.0 and 1 mm EDTA). The length of fungal cell was determined by counting the number of ocular divisions. The size of fungal cell was calculated by following formula Separation genomic DNA by agarose gel electrophoresis Size of the fungal cell = Number of ocular divisions × calibration factor TBE buffer was prepared. One percent agarose was dissolved in TBE buffer and melted. Then one µl of ethidium bromide stain was incorporated in to the gel. The gel In vitro evaluation of antimicrobial activity by agar casting tray was sealed on both sides with tape and agarose diffusion assay was poured into the tray. The comb was placed in the gel and allowed for solidification. After solidification the comb Agar diffusion assay carried out the antimicrobial activity of and the tape was removed. The gel tray was placed in the Ganoderma sp. was evaluated by agar diffusion method electrophoresis tank and TBE buffer was pored over to both for aqueous and solvent extracts using Mueller Hinton cover the gel. A volume of 3 µl of bromophenol blue agar for bacteria and Sabouraud’s agar for yeast and mold. (tracking dye) and 7 µl of the DNA sample were mixed well. The 24 h broth cultures of test organisms were used for the Then the samples were loaded into the wells using assay. A sterile cotton swab was dipped into the micropipette. The power was switched on and the gel was standardized bacterial culture. The swab was then evenly run at 50 V. The power was switched off when the tracking inoculated in three directions over the entire surface of the dye reached three fourth of the gel. The DNA bands were agar plate to obtain the uniform inoculums. Wells were observed on gel doc imaging system (UVP). made on the seeded plates using a sterile borer. The plates were allowed to dry for 3-5 min after which 50 μl of the extracts (aqueous and solvent extracts) and the controls PCR amplification of 18S rRNA were dispensed into each well using micropipette. The concentration of the test samples were 1 g/10 ml of The fungal 18S rRNA was amplified by PCR using universal distilled water (10%). The plates were incubated at 37°C primer pair of 1.0 µl forward primer (5’- for 24 h for bacteria and room temperature for 48- 72 h for CATCCATTTTCAGGGCTAGT-3’) and 1.0 µl reverse primer fungi. The zone of inhibition surrounding the well was (5’-CGCTGAACTTAAGCATATCA-3’). The 50 µl reaction measured compared with control. mixture contained the following components: 5.0 µl template DNA, 5.0 µl of 10X buffer, 2.0 µl of Taq DNA polymerase, 2.0 µl of dNTP mixture and sterile water 34.0 Molecular characterization Ganoderma sp. µl. The 50 µl reaction mixture was taken in 0.5 ml of micro centrifuge tube. The total 50 µl mixture in the tube was Isolation of genomic DNA gently spun for 10 s and allowed to settle the contents. The samples were kept in PCR thermal cycler (Applied G. lucidum was cultivated in 500 ml flasks containing 100 biosystem). The PCR program was carried out in the ml Potato dextrose broth at 27°C under continuous following manner of 30 cycles with an initial denaturation agitation (150 rpm) for 5 days. Total DNA was extracted step at 94°C for 2 min, followed by denaturation step at according to the methods described in Raeder and Broda 94°C for 45 s, annealing at 52°C for 30 s, extension at 72°C Academia Journal of Microbiology Research; Rajesh et al. 064

Figure 1. Macro morphological characteristics of Ganoderma sp. DKR1 and Ganoderma sp. DKR2.

for 1 min and final extention at 72°C for 2 min. A volume of online www.genebee.msu.su/services/rna 2-reduced.html 10 µl of PCR product with 2 µl of loading dye was mixed and loaded on a 1% agarose gel and analyzed electrophoretically at 60 V for 45 min. The gel was Restriction sites analysis observed on gel doc imaging system (UVP). The restriction sites present on the 18S rRNA of Ganoderma sp. were analyzed using the NEB Cutter program version 18S rRNA gene sequencing 2.0 tools in online http://tools.neb.com/NEBCutter2/index.php Amplified PCR products were sequenced for 18S rRNA gene MWG Biotech Pvt. Ltd, Bangalore. Each reaction was carried out in a 12 μl volume containing 120 ng DNA and 12.8 pM RESULTS AND DISCUSSION sequencing primers. The Ganoderma sp. was collected from hard wood tree such as C. equiestifolia and M. tinctoria in Cuddalore district, Phylogenetic analysis Tamil Nadu, India. The two fungi were identified as Ganodermataceae family because of typical characteristics The 18S rRNA nucleotide sequence was obtained from features. The two isolates have been designated Ganoderma sequencing the PCR product. A BLAST of obtained sequence sp. DKR 1 and Ganoderma sp. DKR 2. was performed with that of available EMBL database using the site http://www.ncbi.nlm.nih.gov/genebank and the 18S rRNA nucleotide sequence was subjected to Isolation and morphological characterization of Phylogenetic analysis using Bioinformatics tool available in Ganoderma sp. online using software Mega clustral W and neighbor joining method. The Ganoderma sp. DKR1 and Ganoderma sp. DKR2 were observed such as the size, shape, colour of pore surface, margin pattern, attachment of stipes and surface pattern by Prediction of 18S rRNA secondary structure naked eye (Figure 1 and Table 1). The present findings of Ganoderma were similar to the findings of Steyaert (1972). The secondary structure of the Ganoderma sp.18S rRNA The growth of fungal isolate Ganoderma sp. DKR1 in PDA was predicted using the bioinformatics tool available in observed white-pale orange colour, size of the colony for Academia Journal of Microbiology Research; Rajesh et al. 065

Table 1. Macro morphological description of Ganoderma sp.

S. No Characters Ganoderma sp.DKR1 Ganoderma sp.DKR2 Size 1. Pileus Up to 20 cm 38cm Stipes 12 cm 8.5cm

2. Shape Round Kidney shape Colour of pore surface 3. 1. stipes Dark brown Dark brown 2. pileus Yellowish brown Reddish brown 3.contex Yellow Reddish brown

Attachment of stipes 4. Lateral attachment Lateral attachment to pileus 5. Margin Pattern Smooth Smooth Well-developed concentric zone, 6. Surface Patten Well-developed concentric zone leathery in nature

4th day is 5 cm. The colony appearance was even, fatty. The much as a search for a correct tree (Brekhman and Leipe, Ganoderma DKR2 is white in colour; size of the colony for 2001). 4th day is 6 cm appearance was felty to floccous and The increasing application of modern biochemical cottony (Figure 2 and Table 2). The present results were techniques in fungal taxonomy has brought species similar to the findings of many researcher Adaskaveg and concepts under more critical scrutiny. In particular, there Gilbertson (1986). has been an upsurge in the use of nucleic acid characters to The microscopic observations, such as the size and differentiate and characterize fungal taxa (Zhao Zhu, 1999; morphology of basidiospores, have been adopted as the Zangh, 2002). As genetic variations are discovered and criteria for the taxonomy of Ganoderma. Mycelial evaluated, it is becoming apparent that thorough morphology of Ganoderma sp. DKR1 and Ganoderma sp. characterization of species and strains is imperative if the DKR2 was observed as hyphae with clamp connection, fiber Ganoderma industry is to realize the potential of certain or skeletal hyphae by lactophenol cotton blue staining. The taxa. The gene currently used most extensively in basidiospore morphology was observed and identified as phylogenetic studies is that encoding the ribosomal RNA ovoid and size of the spore 8.5-11× 6.5-8.5 µm. The molecule present in the small subunit of the ribosome. The Ganoderma sp. DKR2 was observed the spore size 8.5- nuclear-encoded homologue of eukaryotes typically 13×5.5-7 µm and round shape by micrometry method comprises about 1800 nucleotides, and sediments at 18S. (Table 3). Sequences of 18S rRNA gene (rDNA) have been used widely The thin cross section of Ganoderma sp. DKR1 and to infer relationships among higher taxa of organisms, from Ganoderma sp. DKR2 fruiting body were observed and families upwards; little has been reported on its variability recorded the polypore morphological structure using phase within species or among congeners of fungi. contrast microscope. In the present study, DNA was extracted from the fungal mycelium of Ganoderma sp. DKR1 was separated by agarose gel electrophoresis, a sharp band was observed by Molecular characterization Ganoderma sp. using UVP Gel doc imaging system (Figure 3a). The amplification of 18S rRNA gene of the fungal isolate Taxonomy aims to reflect a natural classification of taxa and DKR1 was performed by PCR technique using universal molecular data offer a set of objective characters on which primers. PCR product was analyzed in 1% agarose gel. A to base taxonomic decisions (Buchanan, 2001). The use of sharp band was observed which confirmed the presence phylogenetic program to analyze such molecular data has amplified PCR product (Figure 3b). rapidly become popular and it’s the resulting phylograms The amplified 18S rRNA sample was sent to MWG Biotech (Genetic Evolutionary Trees) that display Pvt. Ltd, Bangalore for partial 18S rDNA nucleotide monophylogenetic groups, the member of which share a sequencing. The nucleotide sequence was obtained from common ancestor. The practice of phylogenetic analysis sequencing the PCR product. A BLAST of the obtained should be conceived as a search for a correct model, as nucleotide 18S rRNA sequence for the fungal isolate DKR1 Academia Journal of Microbiology Research; Rajesh et al. 066

the input data (Brinkman and Leipe, 2001), careful consideration was taken in choosing appropriate taxa to include in the study. Initial analyses were performed on a large number of closely related taxa (> 45). Secondary structure of the 18S rRNA sequence of the Ganoderma sp. DKR1 was predicted using the genebee tool and the structure showed 29 loops and 42 stems with an overall free energy of -216.1 kcal/mol (Figure 5). The restriction sites present on the 18S rRNA of Ganoderma sp. DKR1 was analyzed using the NEB (New England Biolabs) cutter program and it showed the site for various commercial enzymes such as Cla, Bsu36, and Hae etc. The restriction sites present in 18S rRNA of Ganoderma sp. DKR1 was around 47 in number. It showed the GC and AC contents to be 50 and 50% respectively (Figure 6).

Antimicrobial activity against clinically important pathogens

The screening of antibacterial activity was performed using a well diffusion assay for aqueous extract from Ganoderma sp. DKR1 and inhibitory effect was measured against a numbers of Gram positive and Gram negative bacteria. The amount of extract used for each well was 50 µl. Ganoderma sp. DKR1 exhibited complete inhibition against the Gram positive bacteria, S. aureus and Micrococcus spp. There was no activity observed for the Gram negative bacteria and fungi (Table 5). The Antibacterial activity has been observed against Gram-positive bacteria from the fruiting body extracts of G. lucidum (Kim et al., 1993)

Conclusion

The present study deals with isolation, taxonomic

Figure 2. Colony morphology of Ganodema sp. DKR1 and Ganoderma characterization and biological activity of medicinal sp. DKR2. mushroom Ganoderma sp. Ganoderma sp. DKR1 and Ganoderma sp. DKR2 was isolated from two hard woods of C. equiestifolia and M.tinctoria respectively in Cuddalore was performed. The 18S rRNA gene sequences of district, Tamil Nadu, India. The Ganoderma isolate were Ganoderma sp. DKR1 is deposited in NCBI and its Accession characterized for macro and micro morphological number is GQ495094. characteristics. The colony, mycelial, basidiospore Phylogenetic analysis is all about understanding the morphology of fungal isolate was evidence of evolutionary relationship between the microorganisms. Basidiomycetece family. 18S rRNA gene of Ganoderma sp. The easiest way to depict any evolutionary relationship DKR1 was sequenced by automated gene sequencer. between groups of organisms is building up a phylogenetic Sequence were submitted NCBI and accession number is tree. A phylogenetic tree of the isolate DKR1 was GQ495094. The phylogenetic analysis of 18S rRNA gene of constructed using its 18S rRNA sequence with that of the Ganoderma was compared with other Ganoderma sp and other Ganoderma sp., from the NCBI data base (Table 4). identified as Ganoderma sp. DKR1. The bioactive The resultant phylogenetic tree showed similarity of the compounds in Ganoderma sp. DKR1 was extracted by isolate DKR1 only at the genus level. A Phylogenetic tree aqueous and solvent extract. The extract was evaluated for was constructed based on the 18S rRNA sequences of antimicrobial activity. It is concluded from the present Ganoderma sp, and closely related fungi (Figure 4). As the study that the Ganoderma sp. from enormous bioactive outcome of an evolutionary study can vary depending on potential in particular provides the source for antimicrobial Academia Journal of Microbiology Research; Rajesh et al. 067

Table 2. Cultural characteristics of Ganoderma sp.

S.No Characters Ganoderma sp.DKR1 Ganoderma sp.DKR2 1 Colony morphology colour White – pale orange White

2 Colony size 1st day No growth No growth 3rd day 4 cm 5 cm 4th day 5 cm 6 cm

3 Colony appearance Even, fatty Felty to floccous and cottony

Table 3. Microscopic Basidiospore morphology of Ganoderma sp.

S.No Characters Ganoderma sp. DKR 1 Ganoderma sp. DKR2 1 Shape Ovoid Round 2 Basidiospore wall Thick Thin 3 Size of the spore 8.5-11× 6.5-8.5µm 8.5-13×5.5-7 µm 4 Spore length 1.6 µm 1.32 µm 5 Spore width 1.5 µm 1.58 µm 6. Mycelium Hyphae with clamp connection Fiber or skeletal hyphae

(a) Separation of Genomic DNA

(b) PCR amplification of 18S rRNA

Lane 1: Molecular Marker Lane 5: Amplified product of DKR1

Figure 3. Separation of Genomic DNA and amplified PCR product of 18S rRNA from Ganoderma sp. DKR1. Academia Journal of Microbiology Research; Rajesh et al. 068

Table 4. Nucleotide sequence of the 18S r RNA region from Ganoderma sp. obtained from Genbank.

S.No Name of the organism Genbank Accession No Host/Source Country 1 Ganoderma carnosum EU486458 From soil/root Canada 2 Ganoderma lucidum GQ249886 Hard wood India 3 Ganoderma sp AY332525 Unknown Indonesia 4 Ganoderma tsunodae AB368127 Hard wood Japan Ganoderma resinaceum 5 FJ805250 Dead wood France BRFM 753 6 Ganoderma tsunodae WD2034 AB368069 Soft wood Japan 7 Ganoderma lucidum WD565 AB368068 Hard wood Japan 8 Ganoderma lipsiense FJ627253 Dead wood China 9 Ganoderma gibbosum XSD08067 FJ582638 Unknown China 10 Ganoderma lucidum FJ501559 From soil/root China 11 Ganoderma tropicum EU021458 Litter Asia 12 Ganoderma sinense GS175 DQ425014 Dead wood China 13 Ganoderma sp. DKR1 GQ495094 Dead wood of Casuarinas sp India

Figure 4. Phylogenetic tree derived from partial 18S rRNA gene sequence of Ganoderma sp. DKR1 using neighbor joining method.

Figure 5. Secondary structure of 18S rRNA of Ganoderma sp. DKR1. Academia Journal of Microbiology Research; Rajesh et al. 069

Figure 6. Restriction site analysis and GC content of 18S rRNA of Ganoderma sp. DKR1.

Table 5. Antimicrobial activity of aqueous extract of Ganoderma sp. DKR1 against the pathogens.

S.No Organisms Zone of inhibition (mm) 1 Staphylococcus aureus 20 2 Staphylococcus epidermidis - 3 Micrococcus sp 25 4 Escherichia coli - 5 Proteus sp - 6 Pseudomonas sp - 7 Klebsiella sp - 8 Enterococci faecalis - 9 Enterobacter sp - 10 Candida sp - 11 Aspergillus niger - 12 Fussarium sp -

- No zone formation

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