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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.

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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

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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

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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%

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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

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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

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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