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J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
Journal of Pharmaceutical, Chemical and Biological Sciences
ISSN: 2348-7658
CODEN: JPCBBG
Impact Factor (GIF): 0.701
Impact Factor (SJIF): 3.905
March - May 2018; 6(1):52-61
Published on: May 13, 2018
Diversity and Distribution of Endophytic Fungi Associated with Litsea
glutinosa (Lour.) C.B. Rob, an Ethno Medicinal Plant
M.Abhinesh, A.Aruna, J.Ramesh, V.Krishna Reddy*
Toxicology laboratory, Department of Botany, Kakatiya University, Warangal-506009, India
*Corresponding Author: V.Krishna Reddy, Toxicology laboratory, Department of Botany, Kakatiya
University, Warangal-506009.
Received: 22 January 2018 Revised: 08 March 2018 Accepted: 16 March 2018
INTRODUCTION
The Indian laurel (Litsea glutinosa (Lour.)
C.B.Rob.) is a rainforest tree in the laurel
family, Lauraceae. Vernacular names of this
plant include soft bollygum, bollygum, bolly
beech, brown bollygum, brown
bollywood, sycamore and brown beech. Litsea
glutinosa is native to India,
south China to Malaysia, Australia and the
western Pacific islands. It has been observed
throughout Asia, including China, India,
Bhutan, Myanmar, Nepal, Philippines, Thailand
and Vietnam [1, 2]. It has been introduced to La
Reunion, Mauritius and Mayotte [2] and in the
KwaZulu-Natal province in South Africa [3].
This plant, since ancient days, has been
identified as a high valued medicinal plant with
many therapeutic properties.
The Indian people use the bark and leaves of the
tree in the form of a demulcent as well as mild
astringent for treating conditions like dysentery
and diarrhea [4]. Different plant parts are also
used in relieving pain (gouty joints), bruises,
sprains, cut wounds, hemorrhages and helps in
arousing sexual power [5]. Roots of Litsea
glutinosa used as emollient and also useful in
treating trauma and fractural limbs. Oil from
the berries is used for rheumatism [6]. On the
other hand, people in China use the oil extracted
from the seeds for making soaps. Recent
pharmacognocological studies conducted
revealed that the plant is a source of
Research Article The work is licensed under
ABSTRACT
Endophytic fungi exhibit a great variation with respect to host, taxonomy, eco-physiological and
biochemical characteristics. In recent years, researchers all over the world are devoting their research
on these fungi, in view of their ability to produce a range of biochemicals of medicinal importance. In
the present investigations, endophytic fungi of Litsea glutinosa, a medicinal plant were explored. The
results revealed that a wide variety of fungi were associated with different plant parts such as leaf,
stem and root. However, the colonization frequency (CF), endophytic infection rate (EIR) varied with
the plant part. CF and EIR also varied with the seasons of year with maximum in rainy season and
least in summer. The relative percentage occurrence (RPO) was observed to more with respective
hyphomycetes. In light of the observations, it was concluded that further studies on endophytic fungi
are needed for their beneficial applications and significance to host.
Keyword: Litsea glutinosa; Endophytic fungi; colonization frequency; Endophytic infection rate;
seasonal variation ; relative percentage occurrence
Abhinesh et al 53
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
arabinoxylans, essential oils as well as other
compounds possessing antiseptic attributes.
Leaves and bark are rich in mucilage and
contain an alkaloid, laurotetanine, which causes
titanic spasm in animals. Leaves also contain
amino acids like cystine, glycine, L-alanine, ß-
alanine, valine, tyrosine, proline, phenylalanine
and leucine. A new flavonoid - naringerin along
with naringin, kaempferol-3- and 7-glucosides,
quercetin and its 3-rhamnoside, pelargonidin-3-
and 5-glucosides have also been isolated from
leaves. Seeds yield fatty oil which is a rich
source of lauric acid. The essential oil of the
fruits contains more than 40 compounds of
which ß-ocimene occurs in high proportion.
Other predominant biochemicals include
caryophyllene oxide and ß-caryophyllene. Two
new alkaloids - sebiferine and litseferine have
been isolated from trunk bark. Actinodaphnine
and its N-methyl derivatives, boldine and
norboldine have also been isolated from this
plant [7, 8].
The term endophytic fungi refers to an organism
which lives within photosynthetic plant tissue by
forming a symbiotic relationship with host and
without any harmful effect to the host plant [9,
10]. About a million endophytic fungal species
are reported to be present in all plants [11].
Mostly Ascomycetes, Deuteromycetes and
Basidiomycetes class fungi are reported as
endophytic fungi [12, 13]. Endophytic fungi
exhibit a wide diversity of microbial adaptations
that have evolved in special and unusual
environments, making them a great source of
study and research for new drugs for medical,
industrial and agriculture uses [14, 15, 16 and
17]. These fungi also represent an important and
quantifiable component of fungal biodiversity in
plants that impinge on plant community
diversity and structure [18, 19,20 and 21].
Endophytic fungi are well known to produce a
variety of bioactive secondary metabolites such
as alkaloids, terpenoids, steroids, quinones,
isocoumarins, lignans, phenylpropanoids,
phenols, and lactones [22, 23]. Medicinal plants
are reported to harbor endophytes which in turn
protect the host from infectious agents and
enable the plants to survive against adverse
conditions [24]. Most of the medicinal plants
were surveyed for the occurrence of endophytic
fungi from various parts and were exploited for
bioprospecting purpose [25, 26 and 27]. The
present work aimed at to understand the
diversity of endophytic fungi associated with
Litsea glutinosa. In order to get a complete
picture of associated endophytic fungi, plant
growing in different edaphic regions under
different seasons and different plant parts were
selected for isolation of endophytic fungi.
MATERIAL AND METHODS
Sample collection
In the present study endophytic fungal species
were isolated from different parts of the Litsea
glutinosa, (fig 01.a) growing in different forest
regions of Warangal district, Telangana (India).
Healthy and mature plant parts (root, stem and
leaf) were carefully chosen for sampling. The
plant parts were brought to the laboratory in
sterile polythene bags and processed for isolation
of endophytic fungi within 24 – 48 hours after
sampling.
Isolation of Endophytic Fungi
Endophytic fungi were isolated by following
methods employed by Petrini (1986) and
Hallman et al. (2007) [12, 28]. First, the plant
material was rinsed in tap water to remove the
dust and debris, then cut into small pieces by a
sterilized blade under aseptic conditions [29].
Each sample was surface sterilized by 70%
ethanol for 1 minute and after that immersed in
sodium hypochloride (NaOCl) / mercuric chloride
(HgCl2) for 30 seconds to 1 minute. The samples
were rinsed in sterile distilled water for 1
minute and then allowed to surface dry on filter
paper. After proper drying, 4 segments were
inoculated on PDA plate supplemented with
antibiotic (streptomycin) and incubated at 28 ±
2ºC for 5 to 7 days with 12 hours light and dark
cycle [30]. Pure colonies (like fig.01. b) were
transferred on to PDA slants. Pure cultures of
isolated fungal strains were preserved on potato
dextrose agar (PDA) slants at 4 to 5ºC with
proper labeling and were sub cultured from time
to time.
Identification of Endophytic Fungi
Lactophenol-cotton blue microscopic slides were
made from the isolated fungal cultures and
examined under light microscope and
fluorescent microscope then photomicrographs
were taken (fig 01.c & d) for the identification.
Abhinesh et al 54
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
Colony morphology, surface texture,
pigmentation and spore morphology were used
to identify the endophytic fungi at species level
using standard manuals [31]. Endophytic fungi
that did not produce the conidia even after
repeated subculturing on different media were
treated as "sterile form"[32, 33]. The endophytic
fungal isolates from host plant tissue segments
were analyzed in terms of Colonization
Frequency percentage (CF %), Relative
Percentage Occurrence (RPO) and Endophytic
Infection Rate (EIR) which were calculated by
using the following formulae [34].
(a) Litsea glutinosa (b) Trichoderma viride colony
(C) Aspergillus flavus (d) Alternaria alternate
Fig. 01: Endophytic fungi isolated from Litsea glutinosa
Abhinesh et al 55
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
Colonization Frequency (CF %)
Number of species isolated
CF %= --------------------------------------- ×100
Number of segments screened
Endophytic Infection Rate (EIR)
Number of infected segments
EIR (%) = -------------------------------------------- ×100
Total number of segments screened
Relative Percentage Occurrence (RPO)
Density of colonization of one group
RPO (%) = ----------------------------------------------- ×100
Total density of colonization
RESULTS
Colonization frequencies of different endophytic
fungi in different parts of the plant are
presented in table 1. From the perusal of the
table it is evident that leaves are more colonized
by endophytic fungi than the stems and roots.
Out of 240 segments of leaf, 169 segments were
found to be colonized by endophytic fungi
(70.47%). Colonization frequency percentage in
stem and roots was 61.25 and 60 percent
respectively. Leaf segments were colonized by as
many as 20 endophytic fungal species, whereas
stem segments were colonized by 17 species.
However, root segments were colonized by 15
species only. The twenty fungal species belonged
to 10 genera. In these genera, Penicillium
species (5) dominated all other fungal generic
species followed by Aspergillus (4), Fusarium (3)
and Alternaria (2). All other genera are
represented by a single species.
Table 01: Colonization Frequency of endophytic fungi isolated from Litsea glutinosa
S.No Endophytic Fungi Root (120) Stem (240) Leaf (240)
NOI CF% NOI CF% NOI CF%
01 Alternaria alternata 02 1.6 05 2.0 06 2.5
02 Alternaria solani - - 03 1.2 04 1.6
03 Aspergillus flavus 09 7.5 20 8.3 16 6.6
04 A.nidulans - - 04 1.6 05 2.0
05 A.niger 09 7.5 18 7.5 20 8.3
06 A.terreus 03 2.5 05 2.0 05 2.0
07 Cladosporium sphaerospermum 02 1.6 - - 04 1.6
08 Colletotrichum acutatum - - 05 2.0 06 2.5
09 Euricoa sp - - - - 03 1.2
10 Fusarium oxysporum 05 4.1 10 4.1 12 5.0
11 Fusarium semitectum 03 2.5 07 2.9 09 3.7
12 F.solani 08 6.6 16 6.6 18 7.5
13 Neurospora crassa 04 3.3 06 2.5 07 2.9
14 Penicillium chrysogenum 04 3.3 10 4.1 12 5.0
15 P.citrinum 03 2.5 04 1.6 06 2.5
16 P.notatum 04 3.3 10 4.1 12 5.0
17 Penicillium roqueforti - - 06 2.5 05 2.0
18 P.rubram 05 4.1 08 3.3 05 2.0
19 Trichoderma viride 06 5.0 - - 02 0.8
20 Verticillium dahliae 05 4.1 10 4.1 12 5.0
72 60% 147 61.25% 169 70.47%
*NOI = Number of isolates *CF% = Colonization Frequency
Abhinesh et al 56
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
In leaves, highest colonization frequency was
shown by A.niger followed by F.solani. Least
colonization frequency was observed with
Trichoderma viride. Colonization frequency of
A.flavus (8.3%) was highest in stem followed by
A.niger (7.5%) and F.solani. Three species viz,
Cladosporium sphaerospermum, Euricoa species
and Trichoderma viride were not observed in
stem segments. In roots, like stems, highest CF
was recorded with A.flavus and A.niger followed
by F.solani. Altogether five species, Alternaria
solani, Colletotrichum acutatum, Euricoa
species, A.nidulans and Penicillium roqueforti
were not isolated from root segments.
Endophytic Infection Rate (EIR) of different
endophytic fungi in three parts of the Litsea
glutinosa is presented in table -2. For all the
fungi put together, EIR was highest (31.5%) for
leaf followed by stem (26.7%) and root (25%).
Among different fungi, highest EIR was shown
by Verticillium dahlia (58%) followed by Euricoa
species (50%) A.nidulans and Neurospora crassa
(42.8%) for leaf and least by Penicillium rubrum.
In case of stem, highest EIR was recorded by
Fusarium semitectum (42.8%) which was
followed by Verticillium dahliae and
P.chrysogenum. Cladosporium sphaerospermum
has shown highest EIR (50%) in root segments
followed by Verticillium dahliae (40%). Least
EIR was shown by F.oxysporum (20%). The
results also reveal that for all three plant parts
EIR was highest with Verticillium dahliae for
leaf with Fusarium semitectum (42.8%) for stem
and Cladosporium sphaerospermum for root.
Interestingly, Euricoa species though totally
absent in root and stem, was recorded with
maximum EIR in leaf.
Table 02: Endophytic fungi isolated from L.glutinosa and its Endophytic Infection Rate
(EIR)
*S = Screened *I = Infected *EIR= Endophytic Infection Rate
S.No. Endophytic fungi Root
(120)
Stem
(240)
Leaf
(240)
S I EIR% S I EIR% S I EIR%
01 Alternaria alternata 02 - - 05 01 20 06 02 33.3
02 Altrernaria solani - - - 03 01 33.3 03 01 33.3
03 Aspergillus flavus 09 03 30.3 20 04 20 16 03 18.7
04 Aspergillus nidulans - - - 03 01 33.3 05 02 42.8
05 Aspergillus niger 09 02 20.2 18 03 16.6 20 04 20
06 Aspergillus terreus 03 01 33.3 05 01 20 05 01 20
07 Cladosporium sphaerospermum 02 01 50 - - - 04 01 41.6
08 Colletotrichum acutatum - - - 05 02 40 06 02 33.3
09 Euricoa sps - - - - - - 02 01 50
10 Fusarium oxysporum 05 01 20 10 03 30 12 05 41.6
11 Fusarium semitectum 03 01 33.3 07 03 42.8 09 03 33.3
12 Fusarium solani 08 02 25 16 02 12.5 18 03 16.6
13 Neurospora sps 04 01 25 06 02 33.3 07 03 42.8
14 Penicilium chrysogenium 04 01 25 10 04 40 12 05 40
15 Pencillium citrinum 03 01 33.3 04 - - 06 02 33.3
16 Penicillium notatum 04 01 25 10 03 30 12 03 25
17 Penicillium roqueforti - - - 06 02 33.3 15 04 26.6
18 Penicillium rubrum 05 01 20 08 03 37.5 05 - -
19 Trichoderma viride 06 - - - - - 02 - -
20 Verticilium dahliae 05 02 40 10 04 40 12 07 58%
Total 72 18 25% 146 39 26.7% 165 52 31.5%
Abhinesh et al 57
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
Results pertaining to seasonal distribution of
endophytic fungi in different plant parts of
Litsea glutinosa are presented in table- 3. A
critical analysis of the table reveals many
interesting points. Firstly, the composition of
endophytic flora in root, stem and leaf varied
with the season. More number of fungal taxa has
been observed in rainy season followed by winter
and least in summer. Secondly, the colonization
frequency of endophytic fungi also varied with
the season and also with plant part. In case of
root, more colonization frequency was observed
in winter season. However, with regard to stem
and leaves more colonization frequencies are
observed in rainy season. Among the three
different seasons, summer season was observed
to be least favorable for colonization. Some
endophytic fungal species like Aspergillus flavus,
A.niger, Fusarium solani and a few sterile forms
were recorded in all three seasons and also with
all plant parts. In contrast, some fungal species
were recorded in a particular season and in
association with a particular plant part. For
example, Euricoa species was recorded with only
leaves that too in winter season. Similarly,
Trichoderma viride, most common soil fungus,
was found to be associated with root in winter
season only.
Table 03: Endophytic fungi isolated from Litsea glutinosa during Different seasons
S.NO Endophytic fungi Root(40) per
season
Stem(80) per
season
Leaf(80) per
season
Winter season (Oct- Jan) NOI CF% NOI CF% NOI CF%
01 Alternaria alternata 02 5 05 6.25 06 7.5
02 Aspergillus flavus 04 10 08 10 05 6.25
03 A.niger 04 10 07 8.75 08 10
04 Colletotrichum acutatum - - 03 3.75 03 3.75
05 Fusarium oxysporum 05 12.5 10 12.5 12 15
06 F.solani 03 7.5 05 6.25 07 8.75
07 Gliocladium solani - - 03 3.75 03 3.75
08 Neurospora crassa 04 10 06 7.5 07 8.75
09 Penicillium notatum 02 5 04 5 05 6.25
10 P.rubrum 02 5 03 3.75 02 2.5
11 Trichoderma viride 06 15 - - - -
12 Sterile forms 04 10 03 3.75 03 3.75
Total 36 90% 57 71.25% 61 76.25%
Summer season (Feb-May)
13 Aspergillus flavus 03 7.5 04 5 04 5
14 A.niger 03 7.5 03 3.75 04 5
15 Cladosporium
sphaerospermum
02 5 - - 04 5
16 Euricoa sp - - - - 02 2.5
17 Fusarium semitectum 03 7.5 03 3.75 03 3.75
18 F.solani 02 5 04 5 05 6.25
19 Penicillium chrysogenium 04 10 10 12.5 12 15
20 P.citrinum 03 7.5 04 5 06 7.5
21 Phomopsis sp - - 03 3.75 03 3.75
22 Sterile forms 04 10 02 2.5 03 3.75
Total 24 60% 33 41.25% 46 57.5%
Rainy season (June-Sept)
23 Aspergillus flavus 02 5 08 10 07 8.75
24 Aspergillus nidulans - - 04 5 05 6.25
25 A.niger 02 5 08 10 08 10
26 A.terreus 03 7.5 05 6.25 05 6.25
Abhinesh et al 58
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
27 Colletotrichum acutatum - - 03 3.75 03 3.75
28 Fusarium semitectum 02 5 04 5 06 7.5
29 F.solani 03 7.5 07 8.75 06 7.5
30 Gliocladium solani - - 02 2.5 03 3.75
31 Penicillium notatum 02 5 06 7.5 07 8.75
32 P.rubrum 03 7.5 05 6.25 03 3.75
33 Phomopsis sp - - 04 5 03 3.75
34 Verticilium dahliae 05 12.5 10 12.5 12 15
35 Sterile forms 05 12.5 04 5 04 5
Total 27 67.5% 70 87.5% 72 90%
*NOI = Number of isolates
*CF% = Colonization Frequency
Seasonal variation in RPO of endophytic fungi
varied with the fungal group and season (fig.02).
In general, hyphomycetes dominated in all the
plant parts and in all the seasons. However,
RPO of hyphomycetes is more in leaf tissue.
Similarly, winter season has found to be
favorable for hyphomycetes. Interestingly, RPO
of sterile forms was more in roots in all the three
seasons.
* Hyp. = Hyphomycetes * Coe. = Coelomycetes * St.f. = Sterile forms
Fig. 2: Relative Percentage Occurrence (RPO) of endophytic fungi isolated from Litsea
glutinosa with seasonal variation
DISCUSSION
The research on endophytic fungi though began
rather late, intensive investigations have been
carried out in recent years in view of their
multitude applications and ecological
significance [35]. Many of the endophytic fungi
still remain unexplored [36]. Despite the
extensive plant diversity, a few plants have been
studied for their endophytic diversity and their
potential to produce bioactive compounds.
Endohytic fungi occupy unique biological niches
as they grow in many divers environments [37].
Endophytic fungi colonize almost all plant parts
and are environment sensitive for growth and
reproduction. Thus, it makes a sense to evaluate
different plant parts and also under different
environmental conditions in order to get the
holistic view of their distribution and to exploit
their potential applications. The objective behind
the present investigation was to isolate the
endophytic fungi from Litsea glutinosa, a highly
valued medicinal plant, and screen them for
medicinal properties.
The results of the present study reveal that
different plant parts of the test plant are
associated with different endophytic fungi
belonging to diverse taxonomic groups. Several
researchers working on different plants have
also noted the association of endophytic fungi
[38]. However, a wide variation has been
Abhinesh et al 59
J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61
observed with different plant parts. Variation in
the distribution of endophytic fungi with
different plant parts can be attributed for
different reasons, such as anatomy and
histochemistry of plant part and its exposure to
environment [39]. Hence, it is suggested that
bioprospecting for endophyte natural products
should be host plant based as opposed to fungal
taxon based [40]. The present study also reveals
the variation in the incidence of endophytic
fungi with different seasons. Selim et al (2012)
suggested that environmental conditions which
effect on host plant growth influence the number
and variety of endophytic populations and affect
on metabolites produced by endophytes. Under
certain environmental conditions, for example
prevailing in certain seasons may turn a
mutualistic association into parasitic
association.
CONCLUSSION
It is evident from the present study that Litsea
glutinosa is associated with different endophytic
fungi of different taxonomic groups with its
different parts viz, root, stem and leaf. However,
the composition, infection frequency varied with
the plant part. This study also reveals the
changes in the incidence of endophytic fungal
flora, with environmental conditions, under
which the behavior of endophytic fungi vis-à-vis
host may change. Thus, these observations lead
to conclusion that the role of endophytic fungi
with respect to production of secondary
metabolites and response to environmental
conditions in the milieu of host need to be
understood in a broader perspective.
ACKNOWLEDGEMENT
The authors express their sincere thanks to
Head, Department of Botany, Kakatiya
University, for encouragement and facilities.
M.Abhinesh and A.Aruna are thankful to UGC,
New Delhi for financial assistance in the form of
RGNF. A part of the work is also sponsored by
UGC under SAP DRS- III (Lr.No.F.5-
25/2015/DRS-III (SAP-II), dt. 09.02.2015). We
are also thankful to Dr.S.Ram Reddy Emeritus
Professor for constructive suggestions at various
stages.
CONFLICTS OF INTEREST
The authors declare that there is no conflict of
interests regarding the publication of this paper.
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Cite this article as:
M.Abhinesh, A.Aruna, J.Ramesh, V.Krishna Reddy. Diversity and Distribution of Endophytic
Fungi Associated with Litsea Glutinosa (Lour.) C.B. Rob, an Ethno Medicinal Plant. J Pharm
Chem Biol Sci 2018; 6(1):52-61