Invention Journal of Research Technology in Engineering & Management (IJRTEM) www.ijrtem.com ǁ Volume 1 ǁ Issue 9 ǁ
ISSN: 2455-3689
Growth Pattern, Molecular Identification and Bio molecules Analysis of FOMITOPSIS FEEI 1, 1,2,
S.V.S.S.S.L.Hima bindu N, 2,M. A. Singara Charya
Department of Microbiology, Kakatiya University, Warangal – 506 009, Telangana, India.
Abstract : Fomitopsis feei, a brown rot fungus is identified tentatively using morphological characteristics and confirmed phylogenetically by 28S rDNA analysis and sequence was submitted in EMBL Nucleotide Sequence Database. Its growth pattern was studied on eight different solid media and found to be good on Malt extract agar medium. Biomolecules such as proteins and lipid were screened qualitatively and estimated quantitatively. Aminoacid analysis by chromatography and fatty acid analysis by FAME were also done and revealed that tryptophan (20.53%), valine (20.51%) and cis-linoleic acid (43.38%) and palmetic acid (17.88%) were in high percentage.
Key words : Fomitopsis feei, growth, molecular identification and biomolecules INTRODUCTION Geologically, mushrooms existed on the earth even before man appeared on it, as evidenced from the fossil records of the lower cretaceous period. Thus, anthropologically speaking, there is every possibility that man used the mushrooms as food when he was still a food gatherer and hunter on the chronology of cultural evolution. They represent as one of the world’s greatest untapped resources of nutrition and palatable food of the future. Mushrooms are low in calories, high in fiber, and contain many important vitamins and minerals. Due to high amount of proteins, they can be used to bridge the protein malnutrition gap. Due to low starch content and low cholesterol, they suit diabetic and heart patients. One third of the iron in the mushrooms is in available form. The sources of these bioactive compounds include fruiting body, mycelia, cultivation broth, submerged cultivation mycelia and fermentation derivatives (Cheung 1996; Fiore and Kakkar 2003; Wu et al. 2010). Mushrooms have been found effective against cancer, cholesterol reduction, stress, insomnia, asthma, allergies and diabetes (Bahl 1983). The major biomolecules from mushrooms are carbohydrates, proteins and lipids or fats. The human body gets its energy from these three main classes of food. The carbohydrate content of mushrooms represents the bulk of fruiting bodies accounting for 50 to 65% on dry weight basis. Protein is an important constituent of dry matter of mushrooms (Chang and Buswell 1996). Mushrooms are very useful for vegetarian because they contain some essential amino acids which are found in animal proteins (Verma et al. 1987). The protein conversion efficiency of edible mushrooms per unit of land and per unit time is far more superior compared to animal sources of protein and mushrooms in general have higher protein content than most other vegetables (Bano and Rajarathnam 1988) and most of the wild plants (Kallman 1991). In mushrooms, the fat content is very low as compared to carbohydrates and proteins. Pedneault et al. (2006) reported that fat fraction in mushrooms is mainly composed of unsaturated fatty acids. Wright and Deschamps (1975) gave detailed macroscopic, microscopic and cultural descriptions of F.feei. F.feei was included in Trametes feei because of its annual basidiocarps and trimitic hyphal systems in the preliminary flora of East African Polypores (Ryvarden and Johansen 1980). F.feei was included in F.rosea complex which comprise a group of fungi distributed worldwide. A heterothallic bipolar mating system was determined for F.feei with some species more adapted to temperate regions and others to tropical ones (Carranza and Gilbertson 1986). Phylogenetic relationships of Antrodia species and related taxa based on analyses of nuclear large subunit ribosomal DNA sequences were done and based on these results it is revealed that the nine species of Fomitopsis in clade A in which F.feei is one of the species, grouped with Antrodia species (Zhi et al. 2010). Growth characteristics and mycelium production of F.feei was investigated on both solid media and in submerged conditions along with twenty other shelf fungi and two commercial edible mushrooms (Nipaporn and Niwat 2005). Fomitopsis feei grown in medium containing 50% of leachate with adjusted pH but did not grow on unadjusted pH of the medium during the study of the effect of adjusted and non-adjusted pH of the media for the bioremediation of leachates (Wan et al. 2011). Hence, the aim of the present study is to identify this fungus at molecular level and its growth pattern on different media and isolation of biomolecules from it.
MATERIAL AND METHODS Isolation of F.feei : The wood decaying fungus F. feei was collected from the wood logs at Pakhal forest (latitude 18°1'18.82"N and longitude 80°9'22.16"E), Warangal district, Telangana, India, during rainy season. The fruit bodies were sealed in polythene airtight sample covers, noted and preserved in our departmental macro fungal herbarium. A small piece of fruiting body was dipped in 0.01% mercuric chloride to remove surface contamination and washed several times with distilled water to remove the traces of mercuric chloride and with 70% ethanol and transferred aseptically on to 3% malt extract agar slants and were incubated for 5–7 days. The mycelium collected from the growing edge of those slant was sub cultured on to new malt extract agar slant every fortnight.
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Growth Pattern, Molecular Identification and Bio molecules Analysis… Growth pattern on different solid media : Eight types of different solid media such as Malt Extract Agar (MEA), Potato Dextrose agar (PDA), Czapek’s Dox Agar, Sabourauds Agar (SDA), Mushroom Complete Medium (MCM), Asthana Hawkers medium (AH), Yeast extract Malt extract Peptone Glucose medium (YMPG) and Nutrient Agar (NA), were used for testing the morphological growth appearance such as mycelium texture, mycelium color, border, border color, reverse color, medium coloration and growth pattern with time after 7 days of incubation at 28 oC. Identification of Fomitopsis feei Classical identification :Macromorphological characters like color, shape, size, odor of sporocarps and spore print were observed for classical identification (Krieger 1967; Laessoe 1998; Suhirman 2005). Molecular identification : The tentatively identified fungus was phylogenetically confirmed by molecular analysis of D2 region large subunit. DNA (deoxyribonucleic acid) of Fomitopsis feei which was labeled as NB was isolated from the slant culture. Its quality was evaluated on 1.2% agarose gel. Fragment of D2 region of LSU (Large subunit 28S rDNA) was amplified by PCR from the above isolated DNA (Christine et al. 1999). The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with DF and DR primers (DF:5'ACCCCGCTGAACTTAAGC-3' and DR: 5'- GGTCCGTGTTTCAAGACGG-3') using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 654bp of D2 region of LSU (Large subunit 28S rDNA) gene was generated from forward and reverse sequence data using aligner software. The D2 region of LSU (Large subunit 28S rDNA) gene sequence was used to carry out BLAST with the database of NCBI Genbank. Based on maximum identity score first ten sequences were selected and the phylogenetic tree was constructed using MEGA 4 software. Biomolecule analysis Proteins: Protein was measured according to the Biuret method (Burtis and Ashwood 2006) using 7 days old culture filtrate of both still and shake cultures. Protein from F. feei, was estimated using bovine serum albumin (100 μg/mL) as a protein standard (Lowry et al. 1951). Fomitopsis feei mycelium (10 mm) was taken from 7 days old culture plate and inoculated in 50 mL production broth medium and incubated for 20 days in still condition and after incubation, culture filtrate was centrifuged at 10,000 rpm for 20 minutes at 4 oC to remove suspended particles. The centrifuged culture filtrate was taken and saturated with different concentrations of ammonium sulphate under chilled conditions and kept for 2 hr. Percent saturation of ammonium sulphate used was 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100. Addition of ammonium sulphate was carried out with continuous stirring in an ice water bath, and then it was kept at 4 oC for overnight. The precipitated protein was removed by centrifugation at 10,000 rpm for 20 minutes at 4oC. Ammonium sulphate was again added to get the required saturation. The precipitated protein was again separated by centrifugation at 10,000 rpm for 30 minutes at 4 oC. Table 1 indicates the grams of solid ammonium sulphate (at 250C) to be added to one liter of solution to produce a desired change in the presence saturation of ammonium sulphate. Saturated solution is 4.1 M and required 767 grams of salt per liter (Green and Hughes 1955). The protein precipitate formed was lyophilized and was used for aminoacid analysis. Amino acid analysis : Amino acids of protein from Fomitopsis feei were quantified by HPLC. Protein solution was dissolved in the mobile phase (15.2 g of triethylamine was dissolved in 800 mL of water, adjusted to pH 3.0 with phosphoric acid and diluted to 1000 mL with water. 850 mL of this solution was added to 150 mL of a mixture of 2 volumes of propanol and 3 volumes of acetonitrile to obtain a concentration of 1.0 mg/mL). Reference solution was prepared by dissolving the mixed amino acids CRS in the mobile phase to obtain a concentration of 1.0 mg/mL. Column size: l = 0.10 m, Ø = 4.6 mm Stationary phase: Octadecylsilyl silica gel for chromatography R (3 µm). Mobile phase flow rate: 1.0-1.5 mL/min. Detection: Spectrophotometer at 220 nm. Injection: 20 µL of test solution /Standard solution. Run time: 90 min. Lipids : Lipids were screened qualitatively by emulsion test. This test was improved by adding the dye Sudan III, which stains lipids red (Glenn and Toole 2002). Lipids were estimated quantitatively (Folch et al. 1957). The mycelium of Fomitopsis feei (1g) was homogenized using pestle and mortar with 2:1 chloroform-methanol mixture (v/v). The residue was re-extracted with a new portion of solvent mixture and the suspension was filtered. The crude extract was mixed thoroughly with chloroform, methanol, and water in the proportions of 8:4:3 by volume during washing and the twice extracted residue collected and dried to constant weight. FAME analysis : Fomitopsis feei was grown in sabourauds dextrose broth medium at 28°C for 2-5 days in shaking condition at 150 rpm (revolutions per minute) using shaking incubator. Four steps were followed for cleaving the fatty acids from lipids. One mL of saponification reagent was added to the mycelium. The tubes were securely sealed with Teflon lined caps, vortexed briefly and heated in a boiling water bath for 5 minutes, at which time the tubes were vigorously vortexed for 5-10 seconds and returned to the water bath to complete the 30 minute heating. During methylation step, they were uncapped; 2 mL of methylation reagent was added. The tubes were capped and briefly vortexed. After vortexing, the tubes were heated for 10±1 minutes at 80±1°C.
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Growth Pattern, Molecular Identification and Bio molecules Analysis… After methylation, extraction was done by the addition of 1.25 mL of extraction reagent to the cooled tubes and recapped, gently tumbled on a clinical rotator for about 10 minutes. The tubes were uncapped and the aqueous (lower) phase is pipetted out and discarded. The last step i.e. base wash was done by adding 3 mL of base wash reagent to the organic phase remaining in the tubes, the tubes were recapped and tumbled for 5 minutes. After uncapping, about 2/3 of the organic phase was pipetted into a Gas Chromatographic vial which is capped and ready for analysis. The Sherlock MIS Software was used for the analysis of Fatty Acid Methyl Esters by gas chromatography (MIDI, Inc. 2001).
RESULTS AND DISCUSSION Growth pattern of F.feei : Morphlogical observations were presented in Table 2. The purpose of this study was to signify a medium for the best growth of F.feei and to reduce the duration of the growth period. The mycelium was white and cottony, the borders were white and diffuse, and the reverse color was also white on all media. Besides of slow growth on YMPG, mycelial density was also somewhat thin on Czapek Dox, NA and AH media. From this study it was found that F.feei showed good mycelial growth on MEA medium compared to other media within short time (Figure 1) and was superior to MCM, PDA and SDA. The density of mycelium on MEA was higher than other culture media. Hence it was selected for maintaining by sub culturing for further processes. The similar reports were obtained with Phellinus spp. in MEA, glucose peptone, and MCM whereas mycelial growth was poor in Czapek dox, Leonian, Hennerberg and Hoppkins medium (Woo et al. 2006). These findings were very much in line with the observation of Ghazala et al. 2001, who worked on Oyster and Chinese mushrooms for their growth on MEA, MS (Murashige and Skoog) medium and PDA and reported that the mycelial growth rate of both types of mushrooms was high on MEA medium as compared to MS and PDA media. Other reports also revealed the similar result with MEA medium (Kausar 1988; Suharban et al. 1996). MEA supported an excellent mycelial growth rate and density of P. pulmonarius among the other six media tested for mycelial growth (Stanley and Nyenke 2011). Nipaporn and Niwat (2005) reported that F.feei grown 25.50±1.80 mm on PDA (Potato Dextrose Agar) medium, 24.66±2.75 mm on PMP (Potato Dextrose Malt Peptone Agar) medium after 3 days of incubation and 1.60±0.26 on PMPB (Potato Dextrose Malt Peptone broth medium after 15 days of incubation. It may be due to availability of required nutrients for mushrooms in the medium plates of MEA. Earlier research also reported that medium composition greatly influenced the growth of mycelium (Fasidi and Olorunmaiye 1994; Eswaran and Ramabadran 2000). In our research this medium (YMPG) was not supported the growth of F.feei because the mycelium growth and appearance not only varies with the culture medium but also changes with the mushroom species such as Fasola et al. 2007 reported that potato dextrose agar (PDA) is the best culture media for mycelial growth of Volvariella speciosa. Classical and molecular level identification of F.feei : Based on morphological characters such as wholly bright pink basidiocarp, the cherry red reaction in KOH and the small pores and pink color spore print, this fungus was tentatively identified as Fomitopsis sp. Identification of the Fomitopsis feei was performed using modern molecular techniques including PCR and sequencing of DNA fragment and D2 region of the largest sub unit of 28S rDNA genes amplified using specific primers. During the evaluation of the quality of isolated DNA, a single band of high molecular weight DNA has been observed on 1.2% agarose gel. A single discrete PCR amplicon band of 700 bp was observed when resolved on agarose gel (Figure 2). After sequencing with forward and reverse primers, the D2 region of LSU (Large subunit 28S rDNA) gene sequence was used to carry out BLAST with the database of NCBI genbank and was depicted in Figure 3. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The optimal tree with the sum of branch length = 0.04440773 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980) and were in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 634 positions in the final dataset. Phylogenetic analyses (Figure 4) were conducted in MEGA4 (Tamura et al. 2007) software. Using this technique a number of researchers have began to examine the biodiversity of fungi (Sheffield et al. 1989). Based on nucleotide homology and phylogenetic analysis Fomitopsis sp. culture which was labeled as NB was found to be Fomitopsis feei (GenBank Accession Number: AY515327.1). Information regarding other close homologs for this culture was also given in the Table 3. The ITS region of the nuclear rDNA is known to exhibit a high degree of polymorphism between species but is often highly conserved within the species. Hence, they contain valuable genetic markers for species identification (Bruns et al. 1991). In past, wood decaying fungi were identified by hybridization of immobilized sequence specific oligonucleotide probes with PCR amplified fungal rDNA ITS (Oh et al. 2003). A reliable genomic DNA extraction protocol for white rot fungi was developed based on the direct sequence of the rDNA (ITS 1, 5.8S, ITS 2) region of the L. tuberregium (Manjunathan et al. 2011). F.feei partial 28S rRNA gene sequence was submitted in EMBL Nucleotide Sequence Database (Accession #:HE806299).
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Growth Pattern, Molecular Identification and Bio molecules Analysis… Biomolecules of Fomitopsis feei : The presence of biomolecules was screened qualitatively and the amount of biomolecules was estimated quantitatively both in still and shake conditions after 14 days of incubation (Table 4). Final pH and dry weight were also measured after the incubation period of 14 days. pH remains almost same in both the conditions but dry weight is almost two times higher in shake culture compared to still culture. The chemical composition of mushrooms determine their nutritive value and this nutritive value differs from one species of mushrooms to another species and that depends upon the nature of substrate, atmospheric conditions, stage of development of the mushrooms and part of fruiting body used besides the conditions of storage after harvest (Manzi et. al. 2001; Adejumo and Awesanya 2005). Proteins : Violet color was observed in biuret method which confirmed the presence of proteins. Compounds with two or more peptide bonds give a violet color with alkaline copper sulphate solution. Protein was quantitatively estimated by Lowry method. Protein content was same in both still and shake cultures (384 μg/mL and 381 μg/mL respectively). After the isolation of protein by ammonium sulphate precipitation it was analyzed for aminoacids. The percentage composition of amino acids in F.feei is shown in Table 5. The results showed that a total of 21 known amino acids were recorded in this mushroom from this study. Based on previous reports, in general, the kinds of amino acids that contained in mushrooms are in the range of 7–17 known amino acids (Mdachi et al. 2004; Beluhan and Ranogajec 2011). Human and animal bodies cannot produce any essential amino acids and should receive through food stock. From gas chromatography (Figure 5) it is revealed that Fomitopsis feei has essential aminoacids viz., Threonine (7.27%), Valine (20.51%), Methionine (0.07%), Isoleucine (1.67%), Leucine (0.14%), Phenylalanine (1.87%), Histidine (0.12%), Lysine (2.58%), and Arginine (18.68%), each of which plays a major role in growth, repair of the damaged tissues and correct performance of the immune system. The essential amino acid profiles of mushrooms reveal that the proteins are deficient in sulfur-containing amino acids, including methionine and cysteine. However, the edible mushrooms are comparatively rich in threonine and valine. Our present study also supported this. Although it is non-edible mushroom, it is showing 20.51% valine which is second highest aminoacid of isolated protein from F.feei. It has been reported that lysine, leucine, isoleucine, and tryptophan are the limiting amino acids in some edible mushroom proteins (Cheung 1997; Diez and Alvarez 2001; Barros et al. 2008). But surprisingly, tryptophan, the first most abundant aminoacid (20.53%) of F.feei is observed from this research. Arginine is one of the essential amino acids, which has an important role in the activity of macrophages for phagocytosis, which kills bacteria and factor of intercellular pathogens. The role of sulfur amino acids is currently considerable interest in human and animal health (Ball et al. 2006). Valine is an aliphatic and hydrophobic amino acid which will cause the proteins to be held beside each other, produced from the pyruvic acid and by the microorganisms (Dozier et al. 2008). In addition, F.feei contained all eight essential amino acids, particularly, isoleucine an essential amino acids and was not detected in most of the mushrooms (Mdachi et al. 2004); Beluhan and Ranogajec 2011). To our knowledge, this is the first study on composition of amino acids in F.feei. Lipids : In qualitative screening, a layer of cloudy white suspension formed at the top of the solution. Upon close inspection the tiny globules of fat suspended (emulsion) in the solution could be observed. Since lipids are undialyzable, the amount of undialyzable substances in the upper phase would represent the maximal amount of lipids lost, and the dialyzable substances would, of necessity, represent non-lipid contaminants. The solutes in the undialyzable fractions were completely soluble in chloroform-methanol, indicating that they were all lipids. 0.05 mg/g and 0.03 mg/g lipid content was estimated from F.feei in both still and shake culture conditions respectively. Fatty acid methyl ester analysis was carried out to know the fatty acids of F.feei. Methylation drops the pH of the solution below 1.5 and causes methylation (for the increased volatility in a partially polar column) of the fatty acid. The fatty acid methyl ester is poorly soluble in the aqueous phase at this point. Extraction will extract the fatty acid methyl esters into the organic phase for use with the gas chromatograph. Base wash reduces contamination of the injection port liner, the column and the detector. Gas chromatographic (Figure 6) analysis of methyl esters of fatty acids obtained from saponification and methylation revealed that C18:2 Cis linoleic acid, C16 Palmitic acid, C18:1 Cis Oleic acid and C13:3OH Tridecanoic acid were predominant in this species. The presence of C13 Tridecanoic acid, C15 Pentadecanoic acid, C17 Margaric acid in low quantity was also present. C18.2 Cis linoleic acid was dominant fatty acid in F.feei that accounts for 43.38 % of total fatty acid (Table 6). It was observed that unsaturated fatty acids were more dominant in wild edible mushrooms. Oils rich with unsaturated fatty acids are considered as health oil for human nutrition. Oils with high levels of linoleic and oleic acids are very important human health, as they reduce atherosclerosis in blood. The concentration of unsaturated fatty acids in these mushrooms is very important from nutritional standpoint. The most important essential fatty acid in human nutrition is linolenic acid. Linoleic acid was the dominant fatty acid identified (Yilmaz et al. 2006; Panagiota et al. 2012). Palmetic acid (17.88%) is the second highest fatty acid (saturated fatty acid) of F.feei and there are also reports in agreement with this (Ruess et al. 2002; Kwang et al. 2011). The lipid extracted from F.feei contained more amounts of unsaturated fatty acids than the saturated fatty acids. The results are in close agreement with the earlier reported result (Yilmaz et al. 2006). Oleic acid is found in olive oil and is known for its effectiveness in reducing cholesterol levels, which promotes the decrease of cardiovascular diseases (Ribeiro et al. 2009). Filipa et al. 2011) reported that F.pinicola showed the presence of palmitic acid, Cis oleic acid, Cis linoleic acid. From our research it is also revealed that these fatty acids might be common in Fomitopsis genus.
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Growth Pattern, Molecular Identification and Bio molecules Analysis… CONCLUSION Morphological and molecular level identification studies confirmed that it is a brown rot fungus and found to be Fomitopsis feei (AY515327.1). Malt extract agar medium is best for the sub culturing of this fungus. Biomolecules such as protein and lipid were estimated. Essential amino acids which have an important role in the activity of macrophages for phagocytosis such as arginine and considerable quantity of linolenic acid, which is regarded as very essential for human health, are present in this fungus. However, studies on lipid and fatty acids at the physiological level may provide information for their large-scale production and utilization.
ACKNOWLEDGEMENTS The authors are thankful to the Head, department Microbiology, Kakatiya University, Warangal for providing laboratory facilities and also acknowledge the support given by Xcelris labs, Ahmedabad, AtoZ pharmaceuticals, Chennai and Royal Life Sciences, Secunderabad.
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Mol Biol Evol.24:1596-1599. 50. Verma RN, Singh G, Bilgrami KS. 1987. Fleshy fungal flora of N. E. H. India- I. Manipur and Meghalaya. Indian Mushroom Sci.2:414- 421. 51. Wan RWAR, Zalina MN, Abdullah N.2011. Screening method for selecting the potential fungi for use in the bioremediation of leachate. 2nd International Conference on Environmental Science and Technology IPCBEE vol.6) IACSIT Press, Singapore. pp. 191-194. 52. Woo SJ, Rew YH, Choi SG, Seo GS, Sung JM, Uhm JY. 2006. The culture conditions for the mycelial growth of Phellinus spp. Mycobiol.34:200-205. 53. Wright JE, Deschamps JR. 1975. Basidiomicetos xilofilos de la Region Mesopotamica II. Los generos Daedalea, Fomitopsis, Heteroporus, Laetiporus, Nigroporus, Rigidoporus, Perenniporia, Vanderbylia. Rev. Inv. Agrop. INTA, Argentina. Serie V Pat Veg.12:127-204. 54. Wu DM, Duan WQ, Liu Y, Y. Cen Y. 2010. Anti-inflammatory effect of the polysaccharides of golden needle mushroom in burned rats. Int J Biol Macromol.46:100-103. 55. Yilmaz N, Solmaz M, Turkekul I, Elmastas M, 2006. Fatty acid composition in some wild edible mushrooms growing in the middle black sea region of Turkey. Food Chem.99:168.
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Growth Pattern, Molecular Identification and Bio molecules Analysis‌ 56. Zhi HY, Wu SH, Wang DM, Chen CT, 2010. Phylogenetic relationships of Antrodia species and related taxa based on analyses of nuclear large subunit ribosomal DNA sequences. Bot Stud.51:53-60. Figure 1 Mycelial growth of Fomitopsis feei on eight types of media
Figure 2 Gel image of PCR amplicon
Figure 3 BLAST data of F. feei (Alignment view of NCBI GenBank)
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Growth Pattern, Molecular Identification and Bio molecules Analysis… Figure 4 Phylogenetic Tree and evolutionary relationships of 11 taxa of F. feei
Figure 5 HPLC of the protein of F.feei
Figure 6 FAME analysis of F.feei
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Growth Pattern, Molecular Identification and Bio molecules Analysis‌ Table 1: Initial concentration of ammonium sulphate in grams A = Initial concentration of the solution A % 0 1 0 1 5 2 0 2 5 3 0 3 3 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5
Final concentration needed 1 0 5 6
1 5 8 4 2 8
20
25
30
33
35
40
45
50
55
60
65
70
75
80
85
90
95
11 4 57
14 4 86
28
57
17 6 11 8 88
29
59
19 6 13 7 10 7 78
20 9 19 0 12 0 91
30
49
61
24 3 18 3 15 3 12 3 93
19
30
62
27 7 21 6 18 5 15 5 12 5 94
12
43
74
31
63
31 3 25 1 22 0 18 9 15 8 12 7 10 7 94
31
63
35 1 28 8 25 6 22 5 19 3 16 2 14 2 12 9 97
32
65
39 0 32 6 29 4 26 2 23 0 19 8 17 7 16 4 13 2 99
33
66
43 0 36 5 33 3 30 0 26 7 23 5 21 4 20 0 16 8 13 4 10 1 67
47 2 40 6 37 3 34 0 30 7 27 3 25 2 23 8 20 5 17 1 13 7 10 3 69
51 6 44 9 41 5 38 2 34 8 31 4 29 2 27 8 24 5 21 0 17 6 14 1 10 5 70
56 1 49 4 45 9 42 4 39 0 35 6 33 3 31 9 28 5 25 0 21 4 17 9 14 3 10 7 72
61 0 54 0 50 6 47 1 43 6 40 1 37 8 36 4 32 8 29 3 25 6 22 0 18 3 14 7 11 0 74
66 2 59 2 55 6 52 0 48 5 44 9 42 6 41 1 37 5 33 9 30 2 26 4 22 7 19 0 15 3 11 5 77
71 3 64 0 60 5 56 9 53 3 49 6 47 2 45 7 42 0 38 3 34 5 30 7 26 9 23 2 19 4 15 5 11 7 77
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33
34
34
35
36
38
39
38
10 0 76 7 69 4 65 7 61 9 58 3 54 6 52 2 50 6 46 9 43 1 39 2 35 3 31 4 27 5 23 7 19 8 15 7 11 8 77 39
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Growth Pattern, Molecular Identification and Bio molecules Analysis… Table 2: Morphological observations of Fomitopsis feei on eight types of solid media Type of culture medium
Mycelium texture
Mycelium color
Border
Border color
Reverse color
Medium coloration
White White White
Growth pattern with time +++++ +++ +++
Malt Extract Agar Potato Dextrose Agar Sabouraud’s Agar
Cottony Cottony Cottony
White White White
Diffuse Diffuse Diffuse
White White White
Yeast extract, Malt extract, Peptone, Glucose medium
Cottony
White
Diffuse
White
White
++
-
Mushroom Complete Medium
Cottony
White
Diffuse
White
White
++++
-
Asthana Hawkers Medium
Cottony
White
Diffuse
White
White
++
-
Czapek’s Dox Agar
Cottony
White
Diffuse
White
White
++
-
Nutrient Agar
Cottony
White
Diffuse
White
White
+++
-
Pale White
Table 3: Sequences producing significant alignments of Fomitopsis feei Accession
Description
AY515327.1
Fomitopsis feei strain CBS546.50 28S ribosomal RNA gene Fomitopsis pinicola strain CBS221.39 28S ribosomal RNA gene Fomitopsis pinicola 5.8S ribosomal RNA gene, partial sequence Fomitopsis pinicola strain FP 105760-T 28S ribosomal RNA gene Fomitopsis pinicola strain FP 98533-T 28S ribosomal RNA gene Fomitopsis pinicola isolate AFTOL-ID 770 25S ribosomal RNA gene Antrodia juniperina strain FP 97452-T 28S ribosomal RNA gene Fomitopsis pinicola 25S large subunit ribosomal RNA gene Fomitopsis spraguei strain CBS365.34 28S ribosomal RNA gene Antrodia malicola strain MJL 1167SP 28S ribosomal RNA gene
AY515334.1 AF347106.1 AY333811.1 AY333810.1 AY684164.1 AY333839.1 AF287858.1 AY515335.1 AY333835.1
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Max Score 1164
Total Score 1164
Query Coverage 99%
E Value 0.0
Max. Identity 98%
1162
1162
99%
0.0
98%
1155
1155
99%
0.0
98%
1146
1146
98%
0.0
98%
1144
1144
98%
0.0
98%
1138
1138
97%
0.0
98%
1134
1134
98%
0.0
98%
1127
1127
99%
0.0
98%
1120
1120
99%
0.0
97%
1118
1118
98%
0.0
98%
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Growth Pattern, Molecular Identification and Bio molecules Analysis… Table 4: Chemical composition of Fomitopsis feei Property
Still culture 3.0 6.4 384 0.05
pH Dry weight (g/L) Proteins (μg/mL) Lipids (mg/g)
Shake culture 3.1 12.1 381 0.03
Table 5: Amino acid composition of Fomitopsis feei Amino acid
Retention time (min.)
Aspartic acid Glutamic acid Asparagine Serine Glutamine Glycine Threonine Arginine Alanine Cystine Tyrosine Histidine Valine Methionine Iso-leucine Phenyl alanine Leucine Lysine Proline Tryptophan Taurine
-
4.06 6.41 9.20 10.21 11.56 13.3 14.96 18.06 19.5 21.7 24.99 27.21 29.91 31.44 36.33 37.92 39.33 43.45 45.2 46.2 17.33
% of quantity 5.3660 0.2064 0.0078 0.0040 0.1250 1.3680 7.2770 18.6860 0.0020 19.3220 0.1020 0.1210 20.5130 0.0790 1.6720 1.8790 0.1450 2.5890 0.0020 20.5320 0.0020
Table 6: Fatty acid composition of crude lipids in Fomitopsis feei Fatty acid
Retention time
C13:tridecanoic acid C13:tridecanoic acid C15:pentadecanoic acid C16:palmitic acid C17:margaric acid C18.1:Cis oleic acid C18.2:Cis linoleic acid
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– – – – – – –
% of quantity
5.672 7.731 8.704 10.840 11.954 13.975 13.887
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3.93 12.46 3.70 17.88 1.57 17.08 43.38
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