Anda di halaman 1dari 11

Invention Journal of Research Technology in Engineering & Management (IJRTEM) ISSN: 2455-3689 ǁ Volume 1 ǁ Issue 9 ǁ

Growth Pattern, Molecular Identification and Bio molecules Analysis of


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

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.


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.

| Volume 1 | Issue 9 | | 16 |

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

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.

| Volume 1 | Issue 9 | | 17 |

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).


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).

| Volume 1 | Issue 9 | | 18 |

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.

| Volume 1 | Issue 9 | | 19 |

Growth Pattern, Molecular Identification and Bio molecules Analysis…

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

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,

1. Adejumo TO, Awesanya O. 2005. Proximate and mineral composition of four edible mushroom species from South Western
Nigeria. Afr J Biotechnol.4:1084-1088.
2. Bahl N. 1983. Medicinal value of edible fungi. In: Proceeding of the International conference on science and cultivation
technology of edible fungi. Ind Mushroom Sci. II, pp. 203-209.
3. Ball RO, Martin GC, Pencharz P. 2006. The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in
animals models and adult humans. J Nutrition. 136:1682s-1693s.
4. Bano Z, Rajarathanum S. 1988. Pleurotus mushroom part II. Chemical composition nutritional value, post-harvest
physiology, preservation and role as human food crit. Rev Food Sci Nutr. 27: 87-158.
5. Barros L, Cruz T, Baptista P, Estevinho LM, Ferreira IC. 2008. Wild and commercial mushrooms as source of nutrients and
nutraceuticals. Food Chem Toxicol. 46: 2742-2747.
6. Beluhan S, Ranogajec A. 2011. Chemical composition and non-volatile components of Croatian wild edible mushrooms.
Food Chem. 124:1076–1082.
7. Bruns TD, Taylor JW, White TJ. 1991. Fungal molecular systematic. Ann Rev Ecol Sys. 22:525–564.
8. Burtis CA, Ashwood ER, 2006. Teitz Fundamentals of Clinical Chemistry (5 th ed). Reed Elsevier India Private Limited, New
Delhi, India. pp:348-349.
9. Carranza MJ, Gilbertson RL, 1986. Taxonomy of the Fomitopsis rosea complex (Aphyllophorales; Polyporaceae). Mycotax.
10. Chang ST, Buswell JA, 1996. Mushroom Nutriceuticals. World J Microbiol Biotechnol. 12:473-476.
11. Cheung PCK. 1996. The hypocholesterolemic effect of two edible mushrooms: Auricularia auricula (tree-ear) and Tremella
fuciformis (white jelly-leaf) in hypercholesterolemic rats. Nutr Res.16:1721-1725.
12. Cheung PCK. 1997. Chemical evaluation of some lesserknown edible mushroom mycelia produced in submerged culture
from soymilk waste. Food Chem.60: 61-65.
13. Christine YT, Steven ES, Daryl JH, James AK, Amin MK. 1999. Rapid identification of fungi by using the ITS2 genetic
region and an automated fluorescent capillary electrophoresis system. J Clin Microbiol.37:1846-1851.
14. Diez VA, Alvarez A. 2001. Compositional and nutritional studies on two wild edible mushrooms from northwest Spain. Food
15. Dozier WA, Kidd MT, Corzo A, 2008. Dietary aminoacid responses of broiler chickens. J Appl Poult Rev.17:157-167.
16. Eswaran A, Ramabardan R, 2000. Studies on some physiological, cultural and post harvest aspects of Oyster mushroom
Pleurotus eous (Berk). Tropical Agric Res.12:360-374.
17. Fasidi IO, Olorunmaiye KS, 1994. Studies on the requirements for vegetative growth of Pleurotus tuberregium (Fr.) Singer,
a Nigerian mushroom. Food Chem.50:397-401.
18. Fasola TR, Gbolagade JS, Fasidi IO. 2007. Nutritional requirements of Volvariella speciosa (Fr. Ex. Fr.) Singer, a Nigerian
edible mushroom. Food Chem.100:904-908.
19. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evol.39:783-791.
20. Filipa SR, Pereira E, Barros L, Sousa MJ, Martins A, Ferreira ICFR. 2011. Biomolecule profiles in inedible wild mushrooms
with antioxidant value. Molecul.16:4328-4338.
21. Fiore MM and Kakkar VV. 2003. Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa
by preventing interaction(s) of enzyme with polysaccharide. Biochem Biophy Res Com.311:71-76.
22. Folch J, Lees, M, Stanley GHS. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J
Biol chem.226:497-509.
23. Ghazala N, Malik SH, Bajwa R, Afzal M, Mian SW. 2001. Effect of three different culture media on mycelial growth of
Oyster and Chinese musrhooms. Online J Biol Sci.1:1130-1133.
24. Glenn T, Toole SM. 2002. Essential as biology.pp.13 Nelson Thornes publishers, United Kingdom.
25. Green AA, Hughes WL. 1955. Protein solubility on the basis of solubility in aqueous solutions of salts and organic
solvents. Methods in Enzymol.1:67-90.
26. Kallman S. 1991. Nutritive value of Swedish wild plants. Svensk Bot Tidskr.85:397-406.

| Volume 1 | Issue 9 | | 20 |

Growth Pattern, Molecular Identification and Bio molecules Analysis…

27. Kausar T. 1988. Cultivation of musrhooms using crop residues as substrates. Ph.D. thesis, Bot. Univ of Punjab, Lahore,
28. Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of
nucleotide sequences. J Mol Evol.16:111-120.
29. Krieger LCC. 1967. The mushroom handbook. Dover Publications, Inc., New York, ISBN: 486-21861-9, pp 560.
30. Kwang JL, Yun IJ, Kim KH, Lim SH, Ham HJ, Eum WS, Joo JH. 2011. Amino acid and fatty acid compositions of Agrocybe
chaxingu, an edible mushroom. Journal of Food Compos Anal.24:175–178.
31. Laessoe T. 1998. Mushrooms. Dorling Kindersley Limited, London, ISBN-13: 9780789432865, pp. 303.
32. Lowry OH, Roseborough JN, Farr AL, Randall RJ. 1951. Protein measurements with Folin phenol reagent. J Biol
33. Manjunathan J, Kumar M, Kaviyarasan V. 2011. Taxonomic studies, rapid and efficient protocol for dna extraction,
purification, molecular characterstics of the basidiomycete lentinus tuberregium (fr) gq292711. Asian J Pharmaceut Clin
Res.4: 54-58.
34. Manzi PA, Agguzzi A, Pizzoferrato L. 2001. Nutritional mushrooms widely consumed in Italy. Food Chem.73:321-325.
35. Mdachi SJM, Nkunya MHM, Nyigo VA, Urasa IT. 2004. Amino acid composition of some Tanzanian wild mushrooms. Food
36. MIDI Inc.2001. Sherlock Microbial Identification System References.
37. Nipaporn A, Niwat Sanoamuang. 2005. Mycelium growth characteristics of shelf fungi on different synthetic media. K K U
Res J.10:311-321.
38. Oh S, Kamden DP, Keathley DE, Han KH. 2003. Detection and species identification of wooddecaying fungi by
hybridization of immobilized sequence specific oligonucleotide probes with PCR-amplified fungal ribosomal DNA internal
transcribed spacers. Holzforschung 57:346–352.
39. Panagiota D, Papanikolaou S, Katsarou E, Komaitis M, Aggelis G, Philippoussis A. 2012. Mushroom polysaccharides and
lipids synthesized in liquid agitated and static cultures. Part II: Study of Volvariella volvacea. Appl Biochem Biotechnol.167:
40. Pedneault KP, Gosselia A, Tweddell RJ. 2006. Fatty acid composition of lipids from mushrooms belonging to the family
Boletaceae. Mycol Res.110:1179-1183.
41. Ribeiro B, Guedes de Pinho P, Andrade PB, Baptista P, Valentão P. 2009. Fatty acid composition of wild edible mushrooms
species: a comparative study. Microchem J. 93:29–35.
42. Ruess L, Haggblom MM, Garcıa Zapata EJ, Digton J. 2002. Fatty acids of fungi and nematodes possible biomarkers in the
soil food chain? Soil Biology Biochem.34:745–756.
43. Ryvarden L, Johansen I. 1980. A preliminary polypore flora of East Africa. Fungiflora, Oslo, Norway. pp.636.
44. Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol
45. Sheffield VC, Cox RD, Lerman S, Myers RM. 1989. Attachment of a 40- base pair Ttc-rich sequence (Gc-Clamp) to
genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Nat
Acad Sci USA.86:232-236.
46. Stanley HO, Nyenke CU. 2011. Cultural studies on mycelia of Pleurotus pulmonarius (Oyster mushroom) in selected culture
media. Int J Sci Nat.2:183- 185.
47. Suharban M, Antony A, Kurup GT, Palaniswami S, Potty VP, Padmaja G, Kabeerathumma S, Pillai SV.1996. Suitability of
different tuber crops on the mycelial growth of sajar caju. In : Tropical tuber crops, problems, prospects and future strategies.
Science Publisher, Lebanan, USA, pp. 513-515.
48. Suhirman AM. 2005. Polypore fungi of Teras South Sumatra biodiversity and taxonomy. PT. Musi Hutan Persada, Jakarta,
pp. 98.
49. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version
4.0. 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.

| Volume 1 | Issue 9 | | 21 |

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)

| Volume 1 | Issue 9 | | 22 |

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

| Volume 1 | Issue 9 | | 23 |

Growth Pattern, Molecular Identification and Bio molecules Analysis…

Table 1: Initial concentration of ammonium sulphate in grams

A = Initial concentration of the solution

A Final concentration needed

% 1 1 20 25 30 33 35 40 45 50 55 60 65 70 75 80 85 90 95 10
0 5 0
0 5 8 11 14 17 19 20 24 27 31 35 39 43 47 51 56 61 66 71 76
6 4 4 4 6 6 9 3 7 3 1 0 0 2 6 1 0 2 3 7
1 2 57 86 11 13 19 18 21 25 28 32 36 40 44 49 54 59 64 69
0 8 8 7 0 3 6 1 8 6 5 6 9 4 0 2 0 4
1 28 57 88 10 12 15 18 22 25 29 33 37 41 45 50 55 60 65
5 7 0 3 5 0 6 4 3 3 5 9 6 6 5 7
2 29 59 78 91 12 15 18 22 26 30 34 38 42 47 52 56 61
0 3 5 9 5 2 0 0 2 4 1 0 9 9
2 30 49 61 93 12 15 19 23 26 30 34 39 43 48 53 58
5 5 8 3 0 7 7 8 0 6 5 3 3
3 19 30 62 94 12 16 19 23 27 31 35 40 44 49 54
0 7 2 8 5 3 4 6 1 9 6 6
3 12 43 74 10 14 17 21 25 29 33 37 42 47 52
3 7 2 7 4 2 2 3 8 6 2 2
3 31 63 94 12 16 20 23 27 31 36 41 45 50
5 9 4 0 8 8 9 4 1 7 6
4 31 63 97 13 16 20 24 28 32 37 42 46
0 2 8 5 5 5 8 5 0 9
4 32 65 99 13 17 21 25 29 33 38 43
5 4 1 0 0 3 9 3 1
5 33 66 10 13 17 21 25 30 34 39
0 1 7 6 4 6 2 5 2
5 33 67 10 14 17 22 26 30 35
5 3 1 9 0 4 7 3
6 34 69 10 14 18 22 26 31
0 5 3 3 7 9 4
6 34 70 10 14 19 23 27
5 7 7 0 2 5
7 35 72 11 15 19 23
0 0 3 4 7
7 36 74 11 15 19
5 5 5 8
8 38 77 11 15
0 7 7
8 39 77 11
5 8
9 38 77
9 39

| Volume 1 | Issue 9 | | 24 |

Growth Pattern, Molecular Identification and Bio molecules Analysis…

Table 2: Morphological observations of Fomitopsis feei on eight types of solid media

Type of culture Mycelium Mycelium Border Border Reverse Growth Medium

medium texture color color color pattern coloration
with time
Malt Extract Agar Cottony White Diffuse White White +++++ -
Potato Dextrose Agar Cottony White Diffuse White White +++ -
Sabouraud’s Agar Cottony White Diffuse White White +++ Pale

Yeast extract, Malt Cottony White Diffuse White White ++ -

extract, Peptone,
Glucose medium

Mushroom Complete Cottony White Diffuse White White ++++ -


Asthana Hawkers Cottony White Diffuse White White ++ -


Czapek’s Dox Agar Cottony White Diffuse White White ++ -

Nutrient Agar Cottony White Diffuse White White +++ -

Table 3: Sequences producing significant alignments of Fomitopsis feei

Accession Description Max Total Query E Max.

Score Score Coverage Value Identity
AY515327.1 Fomitopsis feei strain CBS546.50 28S 1164 1164 99% 0.0 98%
ribosomal RNA gene
AY515334.1 Fomitopsis pinicola strain CBS221.39 28S 1162 1162 99% 0.0 98%
ribosomal RNA gene
AF347106.1 Fomitopsis pinicola 5.8S ribosomal RNA 1155 1155 99% 0.0 98%
gene, partial sequence
AY333811.1 Fomitopsis pinicola strain FP 105760-T 1146 1146 98% 0.0 98%
28S ribosomal RNA gene
AY333810.1 Fomitopsis pinicola strain FP 98533-T 28S 1144 1144 98% 0.0 98%
ribosomal RNA gene
AY684164.1 Fomitopsis pinicola isolate AFTOL-ID 770 1138 1138 97% 0.0 98%
25S ribosomal RNA gene
AY333839.1 Antrodia juniperina strain FP 97452-T 28S 1134 1134 98% 0.0 98%
ribosomal RNA gene
AF287858.1 Fomitopsis pinicola 25S large subunit 1127 1127 99% 0.0 98%
ribosomal RNA gene
AY515335.1 Fomitopsis spraguei strain CBS365.34 28S 1120 1120 99% 0.0 97%
ribosomal RNA gene
AY333835.1 Antrodia malicola strain MJL 1167SP 28S 1118 1118 98% 0.0 98%
ribosomal RNA gene

| Volume 1 | Issue 9 | | 25 |

Growth Pattern, Molecular Identification and Bio molecules Analysis…

Table 4: Chemical composition of Fomitopsis feei

Property Still Shake culture

pH 3.0 3.1
Dry weight (g/L) 6.4 12.1
Proteins (μg/mL) 384 381
Lipids (mg/g) 0.05 0.03

Table 5: Amino acid composition of Fomitopsis feei

Amino acid Retention time (min.) % of quantity

Aspartic acid - 4.06 5.3660

Glutamic acid - 6.41 0.2064
Asparagine - 9.20 0.0078
Serine - 10.21 0.0040
Glutamine - 11.56 0.1250
Glycine - 13.3 1.3680
Threonine - 14.96 7.2770
Arginine - 18.06 18.6860
Alanine - 19.5 0.0020
Cystine - 21.7 19.3220
Tyrosine - 24.99 0.1020
Histidine - 27.21 0.1210
Valine - 29.91 20.5130
Methionine - 31.44 0.0790
Iso-leucine - 36.33 1.6720
Phenyl alanine - 37.92 1.8790
Leucine - 39.33 0.1450
Lysine - 43.45 2.5890
Proline - 45.2 0.0020
Tryptophan - 46.2 20.5320
Taurine - 17.33 0.0020

Table 6: Fatty acid composition of crude lipids in Fomitopsis feei

Fatty acid Retention time % of quantity

C13:tridecanoic acid – 5.672 3.93

C13:tridecanoic acid – 7.731 12.46
C15:pentadecanoic acid – 8.704 3.70
C16:palmitic acid – 10.840 17.88
C17:margaric acid – 11.954 1.57
C18.1:Cis oleic acid – 13.975 17.08
C18.2:Cis linoleic acid – 13.887 43.38

| Volume 1 | Issue 9 | | 26 |