RESULTSshodhganga.inflibnet.ac.in/bitstream/10603/9365/10/10... · 2015. 12. 4. · spores...
Transcript of RESULTSshodhganga.inflibnet.ac.in/bitstream/10603/9365/10/10... · 2015. 12. 4. · spores...
78
RESULTS
1. COLLECTION OF PLANT SAMPLES
T e t o selected edicinal plants ere collected fro Irula tribe o en’s elfare
society (ITWSS) Thandarai, Chengalpet at four different seasons (June - Aug, Sep - Nov, Dec
- Feb, Mar - May) and their taxonomic characters were noted.
2. ISOLATION OF ENDOPHYTES - SEASONAL RECURRENCE STUDIES
2.1 Surface sterilization of plant material
The healthy plant materials were chosen for surface sterilization and process was carried
out as described in materials and methods. Ten different methods of treatment were adopted
for surface sterilization. The number of endophytic strains isolated by adopting 10 different
methods has been compared using the bar chart (Fig: 3). Surface sterilization method VII
yielded the largest number of endophytes. Methods II and IX yielded the second largest
number of endophytes as well as more effective in isolating sterile mycelium and yeasts.
Methods VIII and X does not produce any endophytes indicates that the endophytes within the
plant tissue have been destroyed. However the other methods yielded few numbers of
endophytes comparatively. Also methods V and VI might have produced certain epiphytes too
(Fig: 4 and 5).
2.2 Isolation of endophytes
A total of 338 leaf segments were inoculated per sampling per plant. The incubated
plates were observed for the growth of endophytic fungal colonies from the second day. The
endophytic fungal colonies growing on the leaf segments were noted and subcultured further
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by hyphal tipping. Pure cultures of these fungal endophytes were later maintained on fresh
PDA plates and slants. These pure culture isolates were examined periodically for sporulation
and identified. Using Lactophenol cotton blue wet mounts these fungal isolates were
identified based on the morphology of the fungal culture, the mechanism of spore production
and characteristics of the spore following the standard mycological manuals. The
F
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3
I
s
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l
a
t
i
on of endophytes using different surface sterilization methods
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Fig: 4 Isolation of endophytes from Asparagus racemosus using different
surface sterilization methods on PDA plates
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Fig: 5 Isolation of endophytes from Hemidesmus indicus using different
surface sterilization methods on PDA plates
identification of molds was based on the shape, method of production and arrangement of
spores (conidial ontogeny). The fungal endophytes growing on the leaf segments were
observed and the data was recorded for statistical analysis.
Generally mitosporic fungi and ascomycetes as well as some sterile fungi were recorded
as endophytes from the host plants. A total of 5400 leaf segments were inoculated from the
host plants sampled in different seasons that harbored 2457 isolates totally comprising 1489
isolates belonging to 38 fungal species of 17 different genera represents around 60.6%, 12
miscellaneous mycelia sterilia of 968 isolates that represents around 39.4%, and 13 had yeast
– like growth (Fig: 6, 7 and 8).
2.3 Identification of fungal isolates
The endophytic isolates obtained were subcultured by hyphal tipping and their pure
cultures were prepared and maintained using fresh PDA plates and slants. The pure cultures
were examined periodically for sporulation and identified.
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Macroscopic appearance of the fungal colonies, their morphological appearance and the
mechanism of spore production and characteristics of the spores were noted using the standard
mycological manuals. The identification of molds was based on the shape, method of
production and arrangement of spores (conidial ontogeny). Microscopic appearance was
noted following wet mount preparation by lactophenol cotton blue staining and the
microscopic slides were mounted observing them under 4X, 10X and 40X objectives (Ellis,
1971; Sutton, 1980).
Following were the different fungal endophytes identified (Fig: 9) whose descriptive
morphological features are listed as follows:
Alternaria species
Colonies were fast growing, black to olivaceous-black or greyish, and were suede-like to
floccose. Microscopically branched acropetal chains (blastocatenate) of multicellular conidia
(dictyoconidia) were produced sympodially from simple, sometimes branched, short or
elongate conidiophores and were obclavate, obpyriform, sometimes ovoid and ellipsoidal,
often with a short conical or cylindrical beak, pale brown, smooth-walled or verrucose.
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Fig: 6 Isolation of endophytes from A. racemosus and H. indicus on PDA plates
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Fig: 7 Isolation of endophytes from A. racemosus on PDA plates
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Fig: 8 Isolation of endophytes from H. indicus on PDA plates
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Fig: 9 Fungal endophytes from A. racemosus and H. indicus
Aspergillus sp.
Curvularia sp.
Fusarium sp.
Acremonium sp.
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Fig: 9 Fungal endophytes from A. racemosus and H. indicus
Colletotrichum sp.
Penicillium sp.
Trichoderma sp.
Nigrospora sp.
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Fig: 9 Fungal endophytes from A. racemosus and H. indicus
Cladosporium sp.
Humicola sp.
Myrothecium sp.
Chaetomium sp.
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Alternaria alternata
Produced black colour olivaceous colonies on PDA. Reverse side of the colonies were dark
brown. Colourless hyphae, Smooth conidiophores, 0 μ long, 3–6 μ t ick in size arise singly
or in groups often branched, straight and flexuous. The conidia were formed in long chains,
obclavate, obpyriform and ovoid in shapes. Beaks of conidia were short, cylindrical and smooth.
The overall length of conidia was 20–63 μ and t e t ickness as 9–1 μ .
Alternaria helianthi
The colony grows with greyish–white on media. The length and breadth of the conidia
produced on PDA edia aried fro 117 to 173 μ and 1 to 33.7 μ , respecti ely.
Acremonium strictum
Colonies were white to pale pink in colour usually slow growing often compact and moist
at first became powdery, suede-like with age. Hyphae were fine and hyaline and produced
mostly simple awl-shaped erect phialides of 20–40 μ . Conidia ere usually one-celled
(ameroconidia) hyaline and pigmented, globose to cylindrical, and mostly aggregated in slimy
heads at the apex of each phialide.
Aspergillus flavus
It produced light greenish–yellow color colony. Reverse side of the colonies were
yellowish at first turned brownish in mature age. Conodiophores were 400-1000 μ × -1 μ
in size, pitted, rough and uncolored. The conodial heads were hemispherical to subglobose. The
vesicles were dome-like and 10-30 μ in dia eter. T e pri ary sterig ata ere 7-10 μ × 3-4
μ and t e secondary sterig ata ere 10-1 μ × 3- μ in size.3-4 μ . Colorless conodia
were seen pyriform to globose in shape.
Aspergillus niger
Black colour colonies grew rapidly on PDA. Smooth thick walled unseptate conidiophores
200-100 μ long ere seen colourless near t e esicle. Conidial eads ere fuscuous, black and
globose with a diameter upto 300- 00 μ . Conidial c ains ere present o er t e entire surface
of colorless, globose and thick walled vesicles with rough, globose conidia.
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Aspergillus fumigatus
The growth was more or less velvety, sporing tardily with heads bluish green. Columnar
conidial eads it arying lengt ere obser ed it 40 μ in breadt . Conidiop ores ere
smooth, short, greenish 2- μ in dia eter. P ialides ere borne directly on esicles ic ere
closely packed, lower ones deflected upwards 6-8 × 2-3 μ . Vesicles ere flask s aped and
fertile. Conidia were small globose smooth mostly 2.5-3 μ in dia eter.
Aspergillus flavipes
Silvery white or white colonies, reverse yellow to orange brown or reddish brown were
observed. Conidial heads columnar in size were observed on PDA media. Vesicles were globose
to ovate, metulae fertile over entire vesicle, conidial heads splitting over age. Conidia smooth,
globose, 2-3 μ in dia eter.
Cladosporium cladosporioides
Effuse, velvety olivaceous to dark brown colour colonies grew on PDA. Reverse sides were
blackis bro n. ong s oot conidiop ores pale to oli aceous bro n ere upto 3 0 μ long,
yet shorter, 2-6 μ t ick. Conidia ere o oid to cylindrical and irregular in size. T e conidia
were formed in long branched chains, smooth and 5-30 μ × 3-6 μ in size.
Curvularia species
Woolly, blackish brown colour colonies grew rapidly on PDA. Reverse was blackish
brown. Brown colored conidia 8-14 μ × 1–3 μ , cur ed it t ree transverse septa with
central cell dark and enlarged compared to end cells were observed.
Curvularia lunata
Dematiaceous hyphomycete produced sympodial mid or dark brown colonies, middle septa
was not median, 3 septate and smooth walled, 18-32 × 8-16 μ , Conidia were pale brown,
cylindrical or slightly curved with one of the central cells being larger and darker, germination
was bipolar and some species were having a prominent hilum.
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Colletotrichum species
Colonies were usually dark pigmented with white aerial mycelium consisting of numerous
black sclerotia and light brown coloured conidial masses, reverse was dark brown. Sclerotia were
usually abundant, spherical and were often confluent. Conidia were straight, fusiform, attenuated
at the ends 16-22 × 3-4 μ . Appressoria ere co on, cla ate, bro n, 11-16.5 × 6-9. μ ,
variable in shape.
Chaetomium species
Chaetomium produced darkly-pigmented, globose, ovoid, barrel to flask-shaped, ostiolate
ascocarps (perithecia) beset with dark-coloured terminal hairs (setae) which were straight,
branched or curved. Eight spored asci were clavate to cylindrical. Ascospores were one-celled,
darkly-pigmented, smooth-walled, of varying shape, mostly ovoid, ellipsoidal or lemon-shaped.
Fusarium moniliformae
White aerial mycelia at times tinged with purple reverse colorless turning purple, were
observed on PDA. Single celled, oval to club shaped microconidia 6 μ in size, ere for ed in
chains on monophialides. Sickle shaped thin walled macroconidia 23 – 4 μ long as
distinctly seen.
Fusarium oxysporum
Rapidly grown colonies of 4.5 diameter with white aerial mycelium, becoming purple, with
discrete orange sporodochia present in some strains, reverse hyaline to dark blue or dark purple
were just observed on the PDA media just after four days of inoculation. Conidiophores were
short, single, lateral monophialides in the aerial mycelium, later arranged in densely branched
clusters. Microconidia were abundant, never in chains, mostly non-septate, ellipsoidal to
cylindrical, straight and often curved, 5-12 × 2.3 - 3.5 μ . C la ydospores ere ter inal or
intercalary.
Fusarium solani
Fast grown colonies 4.5 cm in 4 days aerial mycelium white to cream, became bluish-
brown. Macroconidia were formed after 4-7 days from short multi-branched conidiophores.
Microconidia were abundant, cylindrical to oval one to two celled and formed from long lateral
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phialides 8-16 × 2-4. μ . Microconidia ere 3 to septate (usually 3 septate), fusifor ,
cylindrical, often moderately curved, with an indistinctly pedicellate foot cell and a short blunt
apical cell.
Myrothecium species
White aerial mycelia grows after 10 days produced yellow color on PDA. The sporodochia
were olivaceous-black, flattened and cushion like, 60-7 0 μ in dia eter and conodiop ores
were subhyaline and repeatedly branched. Rod shaped to ellipsoidal conodia were produced at
the terminal of the conidiophores.
Penicillium species
The colonies were velvety, orangish green in color with thin white margin. The reverse
sides of the colonies were pale orange where mature colonies were deeply, radiantly and
wrinkled spores were abundant with grey-green shades. The conidiophores were mostly 70-80 ×
2 μ in size and s oot alled. Fi e to ten parallel sterig ata occasionally 1 μ in lengt
were present on verticals. The spores were arranged in a chain and conidial chains were up to
100 μ in lengt . T e ature conidia ere elliptical, s oot and 4-5 × 3 μ in size.
Penicillium funiculosum
White-coloured colonies, sometimes of a pale green-grey, slightly slimy and shiny with
erected funiculi at the centre, filamentous and lighter towards the edges were seen growing on
PDA. The penicillia were biverticillate and symmetrical. Smooth-walled conidiophores were 25-
60 × 2-3 µm in dimensions. The phialides were verticillate (5-8 per metulae), in dense groups
and cylindrical (10 × 2 µm) and conidia were smooth walled.
Phoma species
Irregular olivaceous grey with darker patches colonies were produced by Phoma sp on
PDA. Reverse side of the colonies were blackish brown and mycelia were immerse, branched,
septate and brown in color. The conodiomata were pycnidial, dark brown in color, semi-
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immersed and thin walled. Pycnidia were developed separately or aggregately on media. The
conodia were straight, cylindrical, 5-6. μ in size.
Syncephalastrum species
Cottony to fluffy, white to light grey, became dark grey with the development of sporangia,
fast growing colonies were observed. Spore bearing sporangiophores were erect, stolon-like,
often producing adventitious rhizoids, and showed sympodial branching (racemose branching)
produced curved lateral branches. The main stalk bears finger-like merosporangia directly over
their entire surface. Merosporangia containing merospores which were thin-walled, evanescent
and contain 5-10 (18) globose to ovoid, smooth-walled spores.
Nigrospora species
Colonies were white, later brown to black during sporulation. Conidiophores
micronematous, branched, flexuous, colorless to brown and smooth. Conidia were solitary,
acregenous, simple, spherical and broadly ellipsoid, compressed dorsiventrally, black, shining,
smooth 10-16 μ in dia eter.
Humicola species
These colonies have grown hyaline, grey, greenish to dark brown and black in color.
Conidia were dark globose, smooth, producing singly at the end of the conidiogenous cells. The
latter cells were short hyaline, monoblastic, terminal. Spores were large, dark, spherical, single,
dry and smooth measuring 12-17 μ in dia eter.
Trichoderma species
Fast growing colonies with white and downy, latter developed yellowish-green to deep
green compact tufts, often only in small areas or in concentric ring-like zones on the agar
surface. Conidiophores were repeatedly branched, irregularly verticillate, bearing clusters of
divergent, often irregularly bent, flask-shaped phialides. Conidia were mostly green, sometimes
hyaline, with smooth or rough walls and were formed in slimy conidial heads (gloiospora)
clustered at the tips of the phialides.
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Geotrichum species
Geotrichum sp. typically produced chains of hyaline, smooth, onecelled, subglobose to
cylindrical arthroconidia. Hyphal elements were progressively compartmentalised by
fragmentation of septa. The arthroconidia, which were quite variable in size germinated at one
end giving the appearance of a bud which latter developed into a septate mycelium.
Blastoconidia production was not found.
Gloeosporium species
The mycelium was greenish-grey in color, velvety in appearance, produced black sclerotia
in culture. Appressorium-like cells were formed in terminal and intercalary positions from
sclerotial hyphae. Conidia were hyaline, single celled, having parallel sides with rounded ends,
measuring 13.4-20.1 × 4.5-6.7 μ .
2.4 Seasonal recurrence of endophytes
In a geoclimatic context endophytic assemblages appear to be richer in tropical than in
temperate or cold zones of the world (Fisher et al., 1995; Arnold and Lutzoni, 2007). The
diversity of endophyte assemblages in these tropical plants was highest comprising various
hyphomycete and coelomycete members. Higher number of endophytes was isolated during wet
periods rather than dry periods. Different endophytic isolates were harbored in different seasons
whose relative colonization densities are tabulated in Tables. 5 and 6. The foliar endophyte
assemblage of A. racemosus and H. indicus were made up of Acremonium strictum,
Colletotricum sp., Geotrichum sp., Phomopsis sp., Trichoderma sp., Humicola sp., etc.
In A. racemosus, Colletotrichum sp.1 and Phomopsis sp.1, were found to be dominant with
overall relative colonization densities (R.C.D) of 7.11% and 5.44% followed by A. strictum and
Trichoderma sp.1 of 4.83% and 4% respectively. However considering their seasonal recurrence,
A. strictum was recurring the most except in June-August, following Colletotrichum sp.1 but not
the Phomopsis sp. Among the mycelia sterilia, Morphotype sp.7
Table. 5 Relative Colonization Densities (%R.C.D) of fungal endophytes isolated
in different seasons from Asparagus racemosus
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S.No. Endophyte
Year I Year II Overall
%R.C.D June-
Aug
Sep -
Nov
Dec -
Feb
Mar -
May
June -
Aug
Sep -
Nov
Dec -
Feb
Mar -
May
1 Alternaria helianthi - 1.78 - - - 0.89 1.78 - 0.56
2 F. moniliformae 4.44 4.44 1.78 - 8.44 3.56 - - 2.83
3 F. oxysporum - - - - 7.11 3.11 4.00 - 1.78
4 P. funiculosum 2.67 - - 4.89 - - - - 0.94
5 Phomopsis sp.1 11.11 5.78 9.33 - 12.44 4.89 - - 5.44
6 Phomopsis sp.2 13.78 7.56 8.00 - - 5.78 - - 4.39
7 A. strictum - 4.89 4.00 9.33 - 4.89 7.11 8.44 4.83
8 A. niger 1.33 2.22 - 3.11 3.56 1.78 - 4.00 2.00
9 A. flavus - 0.89 - - - 0.44 - 2.22 0.44
10 Cladosporium sp.1 2.22 1.78 - 8.00 - - - - 1.50
11 Humicola sp. 0.44 4.00 1.33 - - - - - 0.72
12 Trichoderma sp.1 - 2.67 3.11 5.78 - 2.67 8.44 9.33 4.00
13 Myrothecium sp. - - - - 6.22 0.44 0.89 - 0.94
14 Geotrichum sp.1 - - - - - 4.44 5.33 6.22 2.00
15 Geotrichum sp.2 - - - - 4.00 2.22 - 0.78
16 Colletotrichum sp.1 3.56 6.67 6.67 12.00 - 5.33 10.67 12.00 7.11
17 Colletotrichum sp.3 7.56 8.89 4.89 - 11.11 6.67 - - 4.89
18 Morphotype sp.1 4.00 - - 3.56 4.00 - -- 2.22 1.72
19 Morphotype sp.2 4.89 5.33 4.44 - - 4.00 4.89 - 2.94
20 Morphotype sp.3 3.11 0.89 - 0.89 3.11 - - - 1.00
21 Morphotype sp.5 5.78 4.89 6.22 - 8.44 7.56 8.00 - 5.11
22 Morphotype sp.6 - 1.78 0.89 - - - - 0.33
23 Morphotype sp.7 12.89 13.33 - 7.56 12.00 12.44 - 7.56 8.22
24 Morphotype sp.8 - 6.67 7.56 - - - - 1.78
25 Morphotype sp.9 - 3.56 2.22 2.22 - 2.67 3.11 1.33 1.89
26 Morphotype sp.10 8.44 9.78 8.44 - 9.33 10.67 10.22 - 7.11
27 Morphotype sp.11 1.33 - - - - 6.22 - - 0.94
Table. 6 Relative Colonization Densities (%R.C.D) of fungal endophytes isolated
in different seasons from Hemidesmus indicus
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and Morphotype sp.10 were recurring in most seasons dominantly among other morphotypes
with a overall R.C.D of 8.22% and 7.11%, respectively (Fig. 10).
S.No. Endophyte
Year I Year II
Over all
% R.C.D June -
Aug
Sep -
Nov
Dec -
Feb
Mar -
May
June -
Aug
Sep -
Nov
Dec -
Feb
Mar -
May
1 A. alternata - 4.44 4.00 5.33 - 4.44 4.44 - 2.83
2 Alternaria sp.1 2.67 - 4.89 - - 4.00 - 1.44
3 F. moniliformae 5.78 4.89 4.44 - 5.78 4.89 - 4.89 3.83
4 F. solani 5.33 4.00 - - 5.33 4.44 - - 2.39
5 P. funiculosum - - - - - 0.44 0.89 - 0.17
6 Penicillium sp.1 - 0.89 0.44 - - - - - 0.17
7 Penicillium sp.2 - - - - 2.22 0.44 - 2.22 0.61
8 A. strictum 7.11 5.78 4.44 - 6.67 5.33 5.78 5.33 5.06
9 Acremonium sp.1 - 3.56 - 6.22 - - - - 1.22
10 A. flavipes - 0.44 3.56 - 0.44 0.44 1.78 0.83
11 A. fumigatus 2.22 0.44 0.44 - - - - - 0.39
12 Syncephalastrum sp. 5.33 2.67 2.67 - 4.00 2.67 - - 2.17
13 Cladosporium
cladosporioides - 2.67 0.44 4.89 - 2.22 3.11 - 1.67
14 Cladosporium sp.2 - - - - 3.11 1.78 - - 0.61
15 Nigrospora sp. - 2.67 3.11 - - 3.11 3.11 4.00 2.00
16 Trichoderma sp.2 3.56 0.89 0.89 - 3.56 0.89 - - 1.22
17 Gleoesporium sp.1 - 3.11 4.00 - 4.89 4.00 4.00 - 2.50
18 Gleoesporium sp.2 - - - - - 3.56 3.56 - 0.89
19 Curvularia lunata 4.89 1.78 1.78 - 4.00 1.78 2.22 - 2.06
20 Curvularia sp.1 - 0.89 1.78 - - - - - 0.33
21 Curvularia sp.2 4.00 - - 4.00 - - - 1.00
22 Curvularia sp.3 - - - - - 0.89 1.78 3.11 0.72
23 Chaetomium sp. - 1.33 1.33 - - 0.89 - - 0.44
24 Colletotrichum sp.2 - 6.67 5.33 - - 6.22 - 5.78 3.00
25 Morphotype sp.1 - 3.56 4.89 - 3.56 3.11 - - 1.89
26 Morphotype sp.2 6.22 6.67 - 5.78 - 5.33 - 2.22 3.28
27 Morphotype sp.4 5.78 5.33 - - 5.33 4.89 - - 2.67
28 Morphotype sp.5 - - 5.78 6.67 - - 4.00 4.89 2.67
29 Morphotype sp.6 - 4.89 7.11 - - 4.00 6.67 - 2.83
30 Morphotype sp.7 4.89 5.33 - - 4.00 - - 1.33 1.94
31 Morphotype sp.9 2.67 3.11 - - - 2.22 2.67 - 1.33
32 Morphotype sp.10 4.00 - - 4.89 6.22 6.22 - 4.00 3.17
33 Morphotype sp.12 4.44 4.00 5.33 - 4.89 - 4.89 - 2.94
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In H. indicus the dominant species was A. strictum having higher overall R.C.D of 5.06%
followed by F. moniliformae and Colletotrichum sp.2 of 3.83% and 3%, respectively (Fig: 10).
Further A. strictum was the most recurring isolate in almost all seasons followed by F.
moniliformae and Curvularia lunata having R.C.D of 2.06%. Among the sterile mycelia,
Morphotype sp. 2 and Morphotype sp. 10 were dominant recurring mostly among other
morphotypes with overall R.C.D of 3.28 and 3.17, respectively.
Other fungal endophytes isolated from both plants includes Syncephalastrum sp.
Nigrospora sp., Humicola sp., Cladosporium sp., Geotrichum sp., Gloesporium sp., etc. among
which A. flavus and Penicillium sp., were possessing least overall R.C.D of 0.44 and 0.17,
respectively.
2.5 Statistical analysis
Fungal occurrence was measured by calculating colonization density, colonization rates
and isolation rates. The data obtained was compared for all the four seasons between the host
plants represented graphically (Fig: 11). The mean over all colonization rates and isolation
rates of endophytes from A. racemosus were 73.89% and 76.22% and from H. indicus were
64.5% and 60.28%, respectively (Tables. 7 and 8). Further the overall colonization and
isolation rates were higher during the wet periods of Sep-Nov in both A. racemosus (92.22%
and 95.11%) and H. indicus (82% and 77.11%) compared to other seasons.
One way ANOVA was further performed to compare the isolation rates and colonization
rates of fungal endophytes of each plant occurring from four different seasons (Kumar and
Hyde, 2004). The test reveals significant differences that overall colonization rate and isolation
rate was significantly higher in A. racemosus in the month of Sep-Nov than that of H. indicus
(p < 0.05) as well as other seasons (Table. 9). Therefore it can be concluded that significant
differences in the total number of isolates and their means obtained in each season with their
corresponding colonization and isolation rates.
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Fig: 10 Overall % R.C.D of recurring fungal endophytes in A. racemosus
and H. indicus
F
i
g
:
1
1
O
v
e
r
a
l
l
colonization and isolation rates of endophytic fungi from
different seasons in A. racemosus and H. indicus
1 - Alternaria alternata 2 - F. moniliformae 3 - Phomopsis sp.1 4 - Acremonium strictum
5 - Aspergillus niger 6 - Trichoderma sp.1 7 - Trichoderma sp.2 8 – Cladosporium cladosporioides
9 - Syncephalastrum 10 - Gleoesporium sp.1 11 - Colletotrichum sp.1 12 - Colletotrichum sp.3
13 - Curvularia lunata
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Table 7 Over all colonization and isolation rates of endophytic fungi from different
seasons in A. racemosus
Colonization rate and
Isolation rate
June - Aug Sep - Nov Dec - Feb Mar - May
Year I Year
II
Year I Year
II
Year I Year
II
Year I Year II
Overall colonization rate (%)
85.33
84.00
95.11
89.33
66.22
63.56
56.00
51.56
Overall isolation rate (%) 87.56
85.78
97.78
92.44
68.89
66.6
57.33
53.33
Mean Overall colonization
rate (%) 84.6 92.2 64.8 53.7
Mean Overall isolation rate
(%) 86.6 95.1 67.7 55.3
Table. 8 Overall colonization and isolation rates of endophytic fungi from different
seasons in H. indicus
Colonization rate and
Isolation rate
June - Aug Sep - Nov Dec - Feb Mar - May
Year I Year
II
Year I Year
II
Year I Year
II
Year I Year II
Overall colonization rate (%) 72.00 67.56 86.22 77.78 62.22 56.89 48.89 44.44
Overall isolation rate (%) 68.89 63.56 80.00 74.22 58.22 51.56 46.22 39.56
Mean Overall colonization
rate (%) 69.78 82.00 59.56 46.67
Mean Overall isolation rate
(%) 66.22 77.11 54.89 42.89
100
. Table. 9 Statistical analysis – One way ANOVA analysis
ANOVA: Single Factor Analysis of colonization rates for Asparagus racemosus
SUMMARY
Groups Sum Average Variance
June - Aug 169.3333 84.66667 0.888889
Sep - Nov 184.4444 92.22222 16.69136 Dec - Feb 129.7778 64.88889 3.555556 Mar - May 107.5556 53.77778 9.876543
ANOVA
Source of
Variation
SS df MS F P-value F crit
Between Groups 1875.457 3 625.1523 80.6327 0.000493 6.591382
Within Groups 31.01235 4 7.753086
Total 1906.469 7
June - Aug Sep - Nov Dec - Feb Mar -
May
87.56 97.78 68.89 57.33
85.78 92.44 66.67 53.33
ANOVA: Single Factor analysis of isolation rates for Asparagus racemosus
SUMMARY
Groups Sum Average Variance
June - Aug 173.3333 86.66667 1.580247
Sep - Nov 190.2222 95.11111 14.22222
Dec - Feb 135.5556 67.77778 2.469136
Mar - May 110.6667 55.33333 8
ANOVA
Source of
Variation
SS df MS F P-value F crit
Between Groups 1947.062 3 649.0206 98.81704 0.000331 6.591382
Within Groups 26.2716 4 6.567901
Total 1973.333 7
June - Aug Sep - Nov Dec - Feb Mar -
May
72.00 86.22 62.22 48.89
67.56 77.78 56.89 44.44
101
ANOVA: Single Factor Analysis of colonization rates for Hemidesmus indicus
SUMMARY
Groups Sum Average Variance
June - Aug 139.5556 69.77778 9.876543
Sep - Nov 164 82 35.65432
Dec - Feb 119.1111 59.55556 14.22222
Mar - May 93.33333 46.66667 9.876543
ANOVA
Source of
Variation
SS df MS F P-value F crit
Between Groups 1353.16 3 451.0535 25.91158 0.004417 6.591382
Within Groups 69.62963 4 17.40741
Total 1422.79 7
June - Aug Sep - Nov Dec - Feb Mar -
May
68.89 80.00 58.22 46.22
63.56 74.22 51.56 39.56
ANOVA: Single Factor Analysis of isolation rates for Hemidesmus indicus
SUMMARY
Groups Sum Average Variance
June - Aug 132.4444 66.22222 14.22222
Sep - Nov 154.2222 77.11111 16.69136
Dec - Feb 109.7778 54.88889 22.22222
Mar - May 85.77778 42.88889 22.22222
ANOVA
Source of
Variation
SS df MS F P-value F crit
Between Groups 1300.222 3 433.4074 23.00524 0.005523 6.591382
Within Groups 75.35802 4 18.83951
Total 1375.58 7
102
3. SECONDARY METABOLITE PRODUCTION OF ACREMONIUM STRICTUM
A. strictum was the chosen endophytic fungi for further studies due to their common
prevalence in both plants recurring mostly. Prior to the extraction of secondary metabolites low
molecular weight metabolites preliminary growth studies were performed.
3.1 Preliminary growth studies of Acremonium strictum
The culture flasks inoculated with A. strictum was evaluated for culture turbidity by
recording the optical density (OD) from 0th
hour at 405 nm using UV-VIS spectrophotometer.
The portions (1 ml) of broth cultured were aseptically transferred into cuvette and their optical
density was recorded against the blank. Sequential OD measurements for every 24 h were
recorded until the decline phase was reached to generate growth curves.
The following phases in the growth curve (Fig: 12) could be characterized based on
changes in OD the lag phase in which no changes in OD were measured. A rapid increase in
OD was later observed that maximal slope indicating maximum growth at an OD of 3.26 was
reached on 18th
day during log phase. Following which the stationary phase is reached where
there is no change in the OD leading to decline phase.
Extraction and analysis of secondary metabolites
The inoculated flask cultures of A. strictum were harvested after 18 days when it has the
attained maximum growth. The culture filtrate and the mycelial mat separated were extracted
using different organic solvents that gave extracts amounted to varying yields as listed in
Table.10
The extracts were then subjected to preliminary studies like UV and IR
spectrophotometry. UV spectra 200–400 nm using UV-VIS spectrophotometer was recorded
for all the extracts obtained using different solvents (Table. 11). The Infra Red spectra (IR)
were also obtained on the Fourier Transform Infra Red Spectrometer (Nicolet, model 670,
USA) using pellets of KBr (Fig: 13). These results infer the physical properties of the active
compounds present in the biologically active extracts. The inference of 2954-2869 is prominent
in all the IR spectras indicating C-H stretching which denotes the presence of
103
Fig: 12 Growth curve of A. strictum
104
Table. 10 Yield of extracts from A. strictum
NAME OF THE
SOLVENT USED
YIELD (mg/L )
CULTURE
FILTRATE
MYCELIAL
MAT
Hexane
Dichloromethane
Diethyl ether
Acetone
350
479
282
314
1700
1540
1320
1490
105
Table. 11 UV Spectroscopic Absorbance of Different Solvent Extracts
S.NO NAME OF THE
SOLVENT
CULTURE FILTRATE MYCELIAL MAT
WAVELENGTH
(nm) ABSORBANCE
WAVELENGTH
(nm) ABSORBANCE
1.
Hexane
204
210
221
273
354
1.6978
2.7601
2.9316
2.4547
0.1512
204
210
221
273
354
2.3009
2.258
2.5120
0.9264
0.5752
2.
Dichloromethane
310
317
0.4071
0.3235
310
317
0.9412
0.3816
3.
Diethyl ether
285
233
227
222
217
212
210
203
0.5125
0.4396
0.2712
0.2806
0.3237
0.3799
0.4028
0.2148
304
232
288
236
225
215
210
203
2.1974
1.9976
2.0448
1.3761
0.4774
0.4969
0.5715
1.4349
4.
Acetone
300
354
1.4634
0.4005
300
354
0.5908
0.1563
Fig: 13 IR spectral analysis
106
a) IR spectrum of Diethyl ether extract of A. strictum – Culture filtrate
4000
.036
0032
0028
0024
0020
0018
0016
0014
0012
0010
0080
060
045
0.0
0.05101520253035404550556065707580859095
100.
0
cm-1
%T
A-E
THE
R
3395
3072
292528
55
1927
1688
1605
1521
1464
1349
1288
1210 11
91
1080
972
906
849 81
3772
706
645
107
b) IR spectrum of Dichloromethane extract of A. strictum – Culture filtrate
40
00
.03
60
03
20
02
80
02
40
02
00
01
80
01
60
01
40
01
20
01
00
08
00
60
04
50
.0
0.05
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
10
0.0
cm-1
%T
A-D
CM 3
26
5
29
2328
53
25
75 24
89
21
50
20
9820
27
19
40
18
66
17
79
16
84
15
201
46
4
13
78
13
47
13
13
12
80
10
631
01
197
3
90
0
81
5
74
370
5
65
1
108
c) IR spectrum of Hexane extract of A. strictum – Culture filtrate
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 0.0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100.0
cm-1
%T
A-HEXANE
3704
2956 2928
2873
2731 2673
2608 2383
2026
1462
1378
1294 1247
1136 1065
1038 984
957 903 889
815 757
725
659 522
109
d) IR spectrum of Acetone extract of A. strictum – Culture filtrate
110
e) IR spectrum of Diethyl ether extract of A. strictum – Fungal mat
111
f) IR spectrum of Dichloromethane extract of A. strictum – Fungal mat
112
g) IR spectrum of Hexane extract of A. strictum – Fungal mat
113
h) IR spectrum of Acetone extract of A. strictum – Fungal mat
114
alkanes. The following spectral data of 1712, 1606, 1462, 1377 & 1222, indicates the
presence of C=O stretching denoting ketones, asymmetric nitro group, O-CH3 stretching
denoting methyl/methylene groups, C-H stretching because of R-CH3 methyl groups
respectively to be present in the extracts. The existence of absorption peaks 1683, 1684, 1688
indicates C=C stretc ing ibration indicating t e presence of β Unsaturated a ines bonds,
can be related to the presence of unsaturated fatty acids in the organic extract being focused
in further part of the study.
Qualitative analysis revealed the presence of various secondary metabolites like
alkaloids, terpenoids, flavonoids etc. The quality of chemical compounds (secondary
metabolites) extracted however varied depending on the polarity of the organic solvent used
to obtain metabolites of hydrophilic and hydrophobic nature. Accordingly the results indicate
the presence of different metabolites extracted using different organic solvents among which
acetone and dichloromethane were better solvents in extraction of various metabolites
followed by hexane and diethyl ether (Table. 12) (Belofsky et al., 1998; Holler, 1999 and Lin
et al., 2000).
4. FATTY ACIDS PRODUCTION FROM A. STRICTUM
Growing the fungi in different media amended with varying carbon and nitrogen
sources optimization of process parameters like pH, temperature, light and dark conditions
etc. can affect their growth and the lipid accumulation. Hence media standardization for
strain improvement necessitates obtaining higher yield of fatty acids.
4.1 Standardization of media for strain improvement
The inoculated flask cultures of A. strictum grown in different media amended with
varying substrates as well as the cultures subjected to different conditions of varying pH and
temperature with 12:12 hours cycle of light and dark conditions were taken to study their
growth phases by taking optical density (OD) at 405 nm using UV-VIS spectrophotometer.
The sequential OD measurements were taken for every 24 h that was used to construct their
growth curves. The different phases in the growth curves could be characterized as initial lag
phase, the log phase showing maximal slope indicating maximum growth and the stationary
phase where there is no change in the OD leading to
115
Table. 12 Qualitative analysis of secondary metabolites
S.No.
CHEMICAL GROUP
& TEST
FILTRATE FUNGAL MAT
HEXANE DCM DE ACETONE HEXANE DCM DE ACETONE
1.
2.
3.
4.
5.
6.
Alkaloids (Mayer’s
test)
Carbohydrates
(Fe ling’s test)
Steroids (Lieberman
Burchand test)
Tannins(Ferric
chloride test)
Flavonoids (Shinoda
test)
Saponins (Frothing
test)
-
-
+
-
+
+
+
-
+
+
-
+
-
+
-
-
+
+
+
+
-
-
+
+
-
-
+
+
+
+
+
-
+
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
116
the decline phase.
As shown in Fig: 14, growth curves of the A. strictum culture obtained from different
media substrates were compared. The graph shows maximal slope reaching highest peak with
higher OD value of 3.35 for the Potato dextrose broth culture amended with yeast extract (PDB-
YE) compared to other media cultures.
Comparative studies among the growth curves shown in (Fig: 15 and 16) also reveals
highest peak for A. strictum culture grown in pH 6, room temperature with 12 hrs cycle of light
and dark with OD of 3.26 and 3.21 indicating the optimal conditions required to achieve
maximal growth of the A. strictum. These studies were evaluated along with their biomass yield
concurrently to standardize the optimal conditions for the growth of A. strictum for higher yield
of fatty acids.
4.2 Biomass production
The inoculated flask cultures of A. strictum grown in different media amended with varying
substrates as well as the cultures subjected to different conditions of varying pH and temperature
were harvested after 18 days of culture and their biomass was obtained (Yongmanitchai and
Ward, 1991). The biomass of the dried cultures was weighed and the results are represented in
(Fig: 17 and 18). Higher yield of nearly 0.87, 0.85 and 0.65 g of biomass per 100 ml of culture
media were obtained with the cultures grown in PDB-YE under conditions of pH 6 and room
temperature with 12 h cycle of light and dark conditions.
4.3 Extraction of fatty acid
Total lipids were extracted from the biomass obtained according to the Bligh and Dyer
method (Bligh and Dyer, 1959) with little modifications by using hexane and acetone instead of
chloroform and methanol (Fig: 19). The extracts obtained were subjected for TLC profiling.
4.4 TLC profiling
Using palm oil as standard, the lipid extracts obtained were subjected for TLC profiling on
precoated silica gel plates with the solvent system - diethyl ether: hexane: acetic acid (4:13:0.4).
After the run was complete the spots obtained were identified using potassium permanganate.
Fatty acid composition of palm oil constitutes: Palmitic acid,
117
Fig: 14 Effect of media on growth of A. strictum
Fig: 15 Effect of pH on growth of A. strictum
118
Fig: 16 Effect of temperature on growth of A. strictum
119
Fig: 17 Biomass production from different media under different conditions.
Fig: 18 Effect of media, pH and temperature on biomass of A. strictum
PSB PDB Richard’s Oat meal Corn meal PDB-YE
3 4 5 6 7 8
21oC 37oC 26oC
120
Fig: 19 Extraction of fatty acids
Tube A – 37ºC
Tube B – 26ºC
Tube O – 21ºC
Tube H – pH 3
Tube G – pH 4
Tube F – pH 5
Tube E – pH 6
Tube D – pH 7
Tube C – pH 8
Tube I – Ric ard’s
Tube J – PSB
Tube K – Oat meal
Tube L – PDB
Tube M – Corn meal
Tube N – PDB-YE
Lane O – pH 7
A B C D E F G H I J K L M N O
121
Stearic acid, Oleic acid and triglycerides (Rf: 0.54, 0.41, 0.32 and 0.7). The TLC profile
varied among the extracts obtained from biomasses of varying media, pH and revealed
nearly 3–4 spots with Rf values of 0.54, 0.41, 0.32 and 0.7 indicating the presence of
Palmitic acid, Stearic acid, Oleic acid and triglycerides compared with the palm oil
standard (Fig: 20).
4.4 Estimation of free fatty acids
Free Fatty acid content is known as the acid number/acid value. Based on the
methanolic KOH titration method (Cox and Pearson, 1962) using the titre value the amount
of free fatty acids present in the different extracts was calculated. The results indicate that
the extract obtained from the biomass harvested under the following culture conditions of
PDB-YE cultured under pH 6, room temperature produced higher amount of 36.3, 27.6 and
15.2 percentage of free fatty acids with an acid number of 72.23, 54.9 and 30.24 compared
to other cultures (Fig: 21, 22 and 23).
4.5 Preparation of FAMEs
Owing to increased acid value of 72.23, the fatty acid extract obtained from the PDB-
YE culture was subjected for the preparation of FAMEs. Once the FAMEs were prepared
they were analyzed using GC-FID.
4.6 Gas Chromatographic conditions
The FAMEs prepared were subjected for gas chromatography using GC–FID that the
FAME profiling revealed the presence of 14 fatty acids from A. strictum, with high
percentage of Tricosanoic acid of 28.6 g/100 g followed by Caprylic acid of 12 g/100 g and
Linoleic acid of 8.98 g/100 g of fat (Table. 13, Fig: 24 and 25).
Hence fatty acids extracted from various seed cultures being optimized for various
parameters were subjected for acid value estimation of fatty acids and quantified. The
culture harvested from PDB-YE optimized at pH 6 in room temperature showed increased
biomass of 0.87 g/100 ml with 36.3% of free fatty acids production was considered for
further studies.
122
* OA – Oleic Acid
,
Fig: 20 TLC profiling of fatty acids from different media under different
conditions of pH and temperature.
OA OA
A B C D E F G H I J K L M N O P
Lane A – Palm oil Std.
Lane B – PSB
Lane C – Corn meal
Lane D – PDB-YE
Lane E – Oat meal
Lane G – PDB
Lane H – Ric ard’s
Lane F – 21 ºC
Lane I – 26 ºC
Lane J – 37 ºC
Lane K – pH 3
Lane L – pH 4
Lane M – pH 5
Lane N – pH 6
Lane O – pH 7
Lane P – pH 8
123
Fig: 21 Effect of media on percentage of free fatty acids and acid value
Fig: 22 Effect of temperature on percentage of free fatty acids and acid value
124
Fig: 23 Effect of pH on percentage of free fatty acids and acid value
125
Fig: 24 GC chromatogram showing fatty acid profile of A. strictum
126
127
Fig:25 Fatty acid Profile of A.strictum
Fig: 25 Fatty acid profile of A. strictum
Table. 13 Fatty Acid Profile of A. strictum
S.No. Fatty acid Yield (g)/100 g
1. Caprylic acid 12
2. Lauric acid 1.23
3. Tridecanoic acid 3.34
4. Myristoleic acid 2.47
5. Pentadecanoic acid 3.99
6. Palmitic acid 2.37
7. Palmetoleic acid 5.47
8. Heptadecenoic acid 4.61
9. Elaidic acid 6.52
10. Oleic acid 2.63
11. Linoelaidic acid 8.06
12. Linoleic acid 8.98
13. Tricosanoic acid 28.6
14. Lignoceric acid 7.6
15. Saturated fat 0.202
16. Mono unsaturated fat 0.072
17. Poly unsaturated fat 0.06
18. Transfat 0.05
1 - Caprylic acid 2 - Lauric acid 3 - Tridecanoic acid 4 - Myristoleic acid
5 - Pentadecanoic acid 6 - Palmitic acid 7 - Palmetoleic acid 8 - Heptadecenoic acid
9 - Elaidic acid 10 - Oleic acid 11 - Linoelaidic acid 12 - Linoleic acid
13 - Tricosanoic acid 14 - Lignoceric acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14
128
5. ENHANCEMENT OF FATTY ACID PRODUCTION USING ELICITORS
Metallic salts are used as elicitors in inducing the production of metabolites in
microorganisms where these metal ions play as co-factors for key enzymes involved in
biosynthesis of lipids. Hence fatty acid production in the endophytic fungi, A. strictum was
enhanced using different metallic salts as elicitor compounds under the cultural conditions
optimized for the strain improvement.
5.1 Preparation of culture media and conditions
PDB-YE culture media amended with different metal ion salt concentrations as elicitors
ranging from 1 µg – 500 mg of Magnesium sulphate (MgSO4.7H2O), Manganese sulphate
(MnSO4.5H2O), Copper sulphate (CuSO4.5H2O), Zinc sulphate (ZnSO4.7H2O) and Ferric
chloride (FeCl3.6H2O) was prepared. Cultures grown in PDB-YE served as control. The
cultures were harvested after 18 days of inoculation being maintained at room temperature
with 12 h cycle of light and dark conditions.
5.2 Biomass production
The inoculated flask cultures of A. strictum grown in different media amended with
varying metal ion salt concentrations and the control were harvested after 18 days of culture,
and their biomass was obtained (Yongmanitchai and Ward, 1991). The biomass of the dried
cultures was weighed and the results were represented in Fig: 26. The biomass yield from the
control was 0.87 g/100 ml. Minimal increase in salt concentration fro 1 μg to 00 μg yielded
elevated levels of biomass that maximum yield of nearly 1.5 g of biomass was obtained with
the cultures grown in PDB-YE a ended it 00 μg of etallic salts (MgSO4.7H2O,
MnSO4.5H2O, CuSO4.5H2O, ZnSO4.7H2O and FeCl3.6H2O) under conditions of pH 6, room
te perature it 1 cycle of lig t and dark conditions. Ho e er ig er t an 00 μg of salt
concentration yielded lower levels of biomass interfering with their growth. The following
media composition for A. Strictum was standardized for higher biomass and fatty acid
production.
129
Fig: 26 Effect of metallic salt elicitors on biomass production
130
Potato dextrose broth with yeast extract (PDB-YE) and with added salt elicitors
Potato - 20 g
Dextrose - 2 g
Ammonium tartarate - 0.1 g
Pottassium dihydrogen phosphate - 0.7 g
Magnesium sulphate - 0.15 g
Disodium hydrogen phosphate - 0.2 g
Yeast extract - 0.15 g
Calcium chloride - 0.01 g
Cobalt nitrate - 0.00001 g
Magnesium sulphate - 500 µg
Manganese sulphate - 500 µg
Copper sulphate - 500 µg
Zinc sulphate - 500 µg
Ferric chloride - 500 µg
Distilled water - 100 ml
pH - 6
5.3 Extraction of fatty acid
Total lipids were extracted from the biomass obtained according to the Bligh and Dyer
method (Bligh and Dyer, 1959) with little modifications by using hexane and acetone instead
of chloroform and methanol. The extracts obtained were subjected for TLC profiling.
5.4 TLC profiling
Using palm oil as standard the lipid extracts obtained were subjected for TLC profiling
on precoated silica gel plates with the solvent system diethyl ether: hexane: acetic acid
(4:13:0.4). After the run was complete the spots obtained were identified using potassium
permanganate. Fatty acid composition of palm oil constitutes: Palmitic acid, Stearic acid,
Oleic acid and triglycerides (Rf: 0.54, 0.41, 0.32 and 0.7). The TLC profile varied among the
extracts obtained from biomasses of mother culture and the media amended with different
metal ion salt concentrations that revealed nearly 3–4 spots with Rf values of 0.54, 0.41, 0.32
131
and 0.7 indicating the presence of Palmitic acid, Stearic acid, Oleic acid and triglycerides
compared with the palm oil standard (Fig: 27).
5.5 Estimation of free fatty acids
Free Fatty acid content is known as the acid number/acid value. Based on the methanolic
KOH titration method (Cox and Pearson, 1962) using the titre value the amount of free fatty
acids present in the different extracts was calculated. The results indicate that the extract
obtained from the control produced 36.3% of free fatty acids with an acid number of 72.2
(Fig: 28). Further the extract harvested from the biomass of culture amended with metal ion
salt concentration of 00 μg produced ig est a ount of .34% of free fatty acids it an
acid number of 116.10 compared to other cultures (Fig: 28).
Owing to increased biomass production, higher percentage of free fatty acids and the
acid value, the strain grown in PDB-YE amended with 500 µg salt concentration is considered
to be the improved strain of A. strictum and thereafter referred to as ASIS01 strain.
5.6 Preparation of FAMEs
The fatty acid extracted from the biomass harvest from the ASIS01 strain grown in
PDB-YE amended it etal ion salt concentration of 00 μg as subjected for t e
preparation of FAMEs. Once the FAMEs were prepared they were analyzed using GC-FID.
5.7 Gas Chromatographic conditions
The FAMEs prepared were subjected for gas chromatography using GC–FID that the
FAME profiling revealed the presence of 7 fatty acids from the improved strain of A. strictum
ASIS01 with high percentage of Oleic acid of 22.99 g/100 g. (Table. 14, Fig: 29 and 30).
Comparing the results of fatty acid profiling of the control strain with ASIS01 it revealed the
production of 6 fatty acids in common, yielding higher percentage of Oleic acid of 22.99
g/100 g followed by pentadecenoic acid of 19.27 g/100 g of fat (Fig: 30). This reveals a 10
fold increase in the production of Oleic acid in the strain improved. Further the overall
percentage of total monounsaturated fat reveals 80% increase of 87.86 g/100g and
Polyunsaturated fat reveals 12% increase of 12.14 g/100 g in improved strain though the fatty
acid profile shows comparative qualitative difference in producing the following six
132
* OA – Oleic Acid
Fig: 27 TLC profiling of fatty acids from media containing different metallic
salt elicitor concentrations
S A B C S D E F S G H I S J K L
S M N O P Q S R T U V W X Y
Lane S – Palm oil Std.
Lane A – CNTRL
Lane B – 500 µg
Lane C – 5 mg
Lane D – 100 µg
Lane E – 1 mg
Lane F – 10 mg
Lane G – 25 µg
Lane H – 50 µg
Lane I – 75 µg
Lane J – 1 µg
Lane K – 5 µg
Lane L – 10 µg
Lane S – Palm oil Std.
Lane M – 15 mg
Lane N – 20 mg
Lane O – 25 mg
Lane P – 50 mg
Lane Q – 75 mg
Lane R – 100 mg
Lane T – 125 mg
Lane U – 150 mg
Lane V – 200 mg
Lane W – 300 mg
Lane X – 400 mg
Lane Y – 500 mg
OA
133
Fig: 28 Effect of salt concentration on percentage of free fatty acids and acid value
134
Fig: 29 GC chromatogram showing fatty acid profile of A. strictum ASIS01
135
136
Fig: 30 Fatty acid Profile of A. strictum (Control and ASIS01)
Table. 14 Fatty Acid Profile of A. strictum-ASIS01
S.No. Fatty acid Yield (g)/100 g
1 Caprylic acid 0
2 Lauric acid 0
3 Tridecanoic acid 0
4 Myristoleic acid 14.15
5 Pentadecanoic acid 19.27
6 Palmitic acid 0
7 Palmetoleic acid 17.94
8 Heptadecenoic acid 13.51
9 Elaidic acid 0
10 Oleic acid 22.99
11 Linoelaidic acid 0
12 Linoleic acid 12.14
13 Tricosanoic acid 0
14 Lignoceric acid 0
15 Saturated fat 0
16 Mono unsaturated fat 87.86
17 Poly unsaturated fat 12.14
18 Transfat 0
137
fatty acids: Myristoleic acid, Pentadecenoic acid, Heptadecenoic acid, Palmetoleic acid,
Oleic acid and Linoleic acid.
6. PURIFICATION OF OLEIC ACID
Fractions were collected via HPLC that the methyl ester of Oleic acid was eluted at
13.7 min and thus purified by HPLC under the conditions as described in materials and
methods (Fig: 31). The fraction that contained Oleic acid was thus collected and
concentrated. Following comparisons with the standard via GC-FID, the purified oleic acid
was checked further for its biological activity.
7. ANTICANCER STUDY OF OLEIC ACID AGAINST MCF-7 AND HEP-
G2 CELL LINES
The effect of Oleic acid on the viability of human breast cancer (MCF-7) and liver
cancer (Hep-G2) cell lines was evaluated to investigate the cytotoxicity and antiproliferative
activity. MTT assay, a sensitive simple and reliable practice which measure cell viability was
used to evaluate the cytotoxicity and antiproliferative activity of the Oleic acid. It induced a
dose dependent inhibitory effect against the cell lines tested. IC50, which represent the
concentration of oleic acid inhibiting 50% of the cells are presented in Table 15 and 16. The
IC50 values of Oleic acid were found to be 1.25 mg/ml against MCF-7 and 6 . μg/ l
against Hep–G2 cell lines respectively (Tab. 15 and 16, Fig: 32, 33, 34, 35 and 36).
8. ANTIOXIDANT ACTIVITY OF OLEIC ACID USING DPPH ASSAY
The Radical scavenging activity of Oleic acid fraction was determined using the α,α-
diphenyl-β-picrylhydrazyl (DPPH) assay. Free radical scavenging capacity of the Oleic acid
was noted to be increased in a concentration dependent manner. In the DPPH assay, the
ability of the examined fatty acid to act as donor of hydrogen atoms or electrons in
transformation of DPPH into its reduced form DPPH-H was investigated. The sample was
able to reduce the stable, purple-colored radical DPPH into yellow-colored DPPH-H. At a
concentration of 200 µg/ml the scavenging activity of Oleic acid reached 51.2%, which was
comparable to that of reference standard BHT (Table. 17, Fig: 37 and 38).
138
Fig: 31 HPLC chromatogram for the purification of Oleic Acid (OA)
OA
139
Table. 15 Anticancer activity of Oleic acid against MCF-7 cell lines
S.No Concentration
(mg/ml) Dilutions
Absorbance
(O.D)
Cell viability
(%)
1 10 Neat 0.04 10.52
2 5 1:1 0.1 26.31
3 2.5 1:2 0.16 42.1
4 1.25 1:4 0.19 50
5 0.625 1:8 0.24 63.15
6 0.312 1:16 0.28 73.68
7 0.156 1:32 0.33 86.84
8 Cell control - 0.38 100
Table. 16 Anticancer activity of Oleic acid against Hep-G2 cell lines
S.No Concentration (µg/ml) Dilutions Absorbance
(O.D)
Cell viability
(%)
1 1000 Neat 0.06 13
2 500 1:1 0.1 21.7
3 250 1:2 0.16 34.7
4 125 1:4 0.2 43.4
5 62.5 1:8 0.23 50
6 31.2 1:16 0.28 60.8
7 15.6 1:32 0.31 67.3
8 Cell Control - 0.46 100
140
Normal Vero cell line Cytotoxicity of Oleic acid
Fig: 32 Cytotoxicity effect of Oleic acid on Vero cell lines
141
Fig: 33 Anticancer activity of Oleic acid against MCF-7 cell lines
Normal MCF-7cell line Toxicity- 10 mg/ml Toxicity- 1.25 mg/ml
Toxicity- 0.625 mg/ml Toxicity- 0.312 mg/ml
Fig: 34 Anticancer activity of Oleic acid against MCF-7 cell lines
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Fig: 35 Anticancer activity of Oleic acid against Hep-G2 cell lines
Normal Hep-G2 cell line Toxicity- 1000 µg/ml Toxicity- 125 µg/ml
Toxicity- 62.5 µg/ml Toxicity- 31.2 µg/ml
Fig: 36 Anticancer activity of Oleic acid against Hep-G2 cell lines
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Table. 17 Antioxidant activity of Oleic acid using DPPH assay
S.No Concentration
(µg)
Sample
(µl)
DMSO
(µl)
DPPH
(ml)
Absorbance
(O.D)
(%) of
test
(%) of
BHT Std
1 0 0 0 0 0.614 0 0
2 20 2 38 2.96 0.596 6.5 12
3 40 4 36 2.96 0.581 9.2 18
4 60 6 34 2.96 0.577 11.3 22
5 80 8 32 2.96 0.576 12.1 27
6 100 10 30 2.96 0.555 13.3 34
7 120 12 28 2.96 0.528 15.4 41
8 140 14 26 2.96 0.494 19.6 45
9 160 16 24 2.96 0.415 24.8 50
10 180 18 22 2.96 0.336 36.8 53
11 200 20 20 2.96 0.656 51.2 57
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Fig: 37 Antioxidant activity of Oleic acid using DPPH assay
Fig: 38 Antioxidant activity of Oleic acid using DPPH assay
145
9. APOPTOTIC DNA FRAGMENTATION ASSAY
The cell death by DNA fragmentation was examined by conducting agarose gel
electrophoresis of DNA samples isolated from Hep – G2 and MCF – 7 cell lines treated
with Oleic acid sample. Healthy control cells maintained in culture medium alone did not
show DNA fragmentation. However the DNA isolated from Oleic acid treated cells
revealed DNA ladders consisting of fragments when stained with ethidium bromide.
Consistent with the MTT assay the cell lines treated with higher concentrations of Oleic
acid showed DNA smearing (slight DNA degradation) that the IC50 concentration of Oleic
acid - 1. g/ l and 6 . μg/ l as opti u enoug to induce frag entation in MCF –
7 and Hep – G2 cell lines respectively (Fig: 39 and 40).
10. MOLECULAR STUDY USING PCR
Initial olecular studies ere done to isolate t e δ-9 fatty acid desaturase gene
mediating Oleic acid production from A. strictum using PCR amplification technique.
10.1 Isolation of Genomic DNA
Genomic DNA was isolated from pure cultures of A. strictum – mother culture and
the improved strain ASIS01 using lysis buffer and the samples were electrophoresed in
0.8% agarose gel. The DNA bands were visualized using a UV transilluminator and the
images were captured using Gel documentation system. Intact fungal DNA bands were
isolated from the mother culture and the improved strain ASIS01 of A. strictum is shown in
Fig: 41, later e ployed for PCR a plification of δ -9 fatty acid desaturase gene.
10.2 Amplification using PCR
T o sets of degenerati e oligonucleotide pri ers ere generated for δ -9 fatty acid
desaturase gene responsible for Oleic acid production. Following PCR the products were
electrophoresed in 1% agarose gel and the amplified bands were observed after Ethidium
bromide staining. The bands were visualized using a UV transilluminator and the images
were captured using Gel documentation system. The amplification resulted in generation of
multiple bands indicative of binding of degenerative primers with the DNA apart from the
specified locus. A plification using first set of pri ers (δ -9, FAS Primer 1) revealed 3
146
Lane 1: 1 kb ladder Lane 2: 1 kb ladder
Lane 2: 10 mg/ml Lane 3: 1000 µg/ml
Lane 3: 1.25 mg/ml Lane 4: 125 µg/ml
Lane 4: 0.625 mg/ml Lane 5: 62.5 µg/ml
Lane 5: 0.312 mg/ml Lane 6: 31.2 µg/ml
Fig: 39 and 40 Apoptotic DNA fragmentation assay in MCF-7 cell lines and
Hep-G2 – cell lines
1 2 3 4 5 1 2 3 4 5 6 7
147
bands of nearly 350 bp, 470 bp and 600 bp in mother culture (Lane S1) and 4 bands of
nearly 350 bp, 470 bp, 560 bp and 650 bp (Lane S2) in improved strain when compared
with the molecular ladder respectively (Fig: 42, S1 and S2). Amplification using second set
of pri ers (δ -9, FAS Primer 2) revealed 2 bands of nearly 680 bp and 850 bp (Lane 1A) in
mother culture and 4 bands of nearly 590 bp, 810 bp and 900 bp (Lane 2A) in improved
strain when compared with the molecular ladder, respectively (Fig: 43, 1A and 2A).
A ong t e t o sets of pri ers t e first set of pri er (δ -9, FAS Primer 1), produced the
amplified products of expected size of approx. 469 bp and therefore can further be
employed in future for PCR optimization and gene sequencing studies.
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Fig: 41 Isolation of Genomic DNA from A. strictum
Lane MW: 1000 bp ladder Lane MW: 1000 bp ladder
Lane 1A: PCR bands – mother culture Lane 1A: PCR bands – mother culture
Lane 2A: PCR bands – ASIS01 Lane 2A: PCR bands – ASIS01
Fig: 42 Amplification of δ -9 fatty acid Fig: 43 Amplification of δ -9 fatty acid
desaturase using FAS Primer 1 desaturase using FAS Primer 2
Genomic DNA of
A. strictum
Molecular
Marker