Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(3): 370-375 (2018)

Bio-prospecting fungal endophytes of high altitude

medicinal plants for commercially imperative enzymes

Neha Kapoor

, Pranchal Rajput

, Md Abu Mushtaque

and Lokesh Gambhir

Department of Microbiology, Uttaranchal (PG) College of Biomedical Sciences and Hospital, Dehradun

Department of Biotechnology, Agra College, Agra (U.P)

Department of Life Sciences, Shri Guru Ram Rai University, Dehradun, Uttarakhand

ABSTRACT

Endophytic fungi have been a focal point of research as repository of extreme chemical diversity. Isolation of fungal

endophytes from medicinal plants has led to detection of plethora of novel agents encompassing bioactive potential.

In the present work, Withania sominifera, Ocimum basillicum and Syzygium aromaticum from high altitude region

were selected for bio-prospecting of fungal endophytes. 14 fungal endophytes were recovered from different parts of

Withania sominifera, Ocimum basillicum and Syzygium aromaticum. Maximum fungal colonization was recovered

from Withania sominifera and Syzygium aromaticum (42.8 %). In the preliminary screening for production of com-

mercially important enzymes including protease, amylase, cellulose and asparaginase activity, all fungal endophytes

of Ocimum basillicum exhibited potent activity. However, In case of proteolytic activity, #9SASTD exhibited maxi-

mum proteolytic potential. Maximum amylase and cellulase production was observed in #2SASTD and #14WSLF

respectively. Interestingly, isolates form Withania sominifera and Syzygium aromaticum exhibited potent asparagi-

nase activity with maximum potential in #22WSLD. Thus, the data clearly indicates the potential of high altitude

medicinal plants as source of endophytic repository that can be taken as measure to prevent exploitation of endan-

gered medicinal plants for commercial use. Further studies are warranted for characterization of the fungal isolates.

KEY WORDS: ASPARAGINASE, ENDOPHYTE,

OCIMUM BASILLICUM, SYZYGIUM AROMATICUM, WITHANIA SOMINIFERA

370

ARTICLE INFORMATION:

*Corresponding Author: gambhir.lokesh@gmail.com

Received 12

July, 2018

Accepted after revision 21

Sep, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.3/4

Neha Kapoor et al.

INTRODUCTION

Endophytic fungi comprise a group of heterogeneous

fungi which live asymptomatically inside the plant

tissues without showing any sign of their existence

(Strobel and Daisy, 2003; Muller et al, 2016). Fungal

endophytes are considered to be “Gold Mine” of novel

bioactive compounds with immense therapeutic poten-

tial in the pharmaceutical sector. Various anticancer,

anti-in ammatory, antioxidant, antimalarial drugs have

been reported from fungal endophytes that are currently

being exploited as control measure for various medical

conditions such as hyperchlosteremia, Leukemia, renal

failure, cardiovascular disorders (Gouda et al. 2016;

Raviraja et al. 2006). Fungal endophytes are also proved

to mimic the bioactive properties of the host plant due to

the horizontal gene transfer (Strobel, 2002; Strobel et al.

2004; Jia et al. 2016; Venieraki et al. 2017; Huang et al.

2018). Potential of medicinal plants form high altitude

region in pharmaceutical industry is undebatable. Over

exploitation of medicinal plants has been a threat of

increase in number of endangered plants. Hence, fungal

endophytes inhabiting the plants with medicinal values

are under exploitation by various groups of researchers

in the process of  nding a novel chemical scaffold with

therapeutic potential (Kapoor and Saxena, 2016; Kapoor

and Saxena, 2018).

The present study was oriented towards the explora-

tion of fungal endophytes inhabiting Withania sominif-

era, Syzygium aromaticum and Ocimum basillicum of

Uttarakhand region, India. The main rationale behind

selection of these plants from Uttarakhand region lays

within the fact that endophytic myco ora of Uttara-

khand region is very sparsely explored for bioactive

entities.

MATERIALS AND METHODS

PLANT SAMPLE COLLECTION AND ISOLATION

OF ENDOPHYTIC FUNGI

Healthy parts (stem and leaves) of medicinal plants viz.

Withania sominifera, Syzygium aromaticum and Oci-

mum basillicum were collected from Kanhaiya Vihar

region, Dehradun during winter season. The samples

were transported to laboratory in sealed pouches and

stored at 4˚C. For the isolation of endophytes, surface

sterilization of stems and leaves were carried out by

dipping in 0.1% sodium hypochlorite solution for 2-3

min followed by 70% Ethanol for 1 min and then sub-

sequent washing in 30% Ethanol for 30-45 sec. The sur-

face sterilized samples was cross sectioned into small

pieces of 1-2 mm size aseptically and were inoculated

on to pre sterilized Potato Dextrose Agar (PDA) plates.

The plates were then incubated at 26±2ºC, 16h/8h light/

dark condition for 8-10 days. The plates were regularly

monitored for any fungal growth. The fungal hyphae

emerging out of the segment was transferred to fresh

PDA plate aseptically with the help of inoculation loop

to obtain pure culture (Mitchell et al. 2008).

SECONDARY METABOLITE PRODUCTION

All the fungal endophytes were subjected to secondary

metabolite production by following the method of Ravi-

raja et al. 2006. Brie y describing, 5mm mycelial plug

of 4-5 day old active culture was inoculated into 100

ml pre-sterilized Potato Dextrose Broth (PDB) followed

by incubation at 26±2ºC, 120 rpm for 7-10 days. After

the culmination of incubation period, the culture  ltrate

was separated from mycelial mass by  ltration through

Whatman  lter paper No. 4 followed by centrifugation

at 10,000 rpm for 10 min. The supernatant so obtained

was stored at 4˚C till further use.

SCREENING OF ENZYMATIC ACTIVITIES

For the protease activity, 1 % skim milk agar plates were

prepared and 5 mm wells were punched out by sterile

cork borer. To each well, 30 μl of each culture  ltrate

was added followed by incubation at 37˚C degrees for

24 h. Non-inoculated PDB served as control. After incu-

bation, a clear zone around the wells indicates the pro-

teolytic activity. The zone diameter was measured and

expressed as Mean ± SD.

(a) Cellulolytic activity:

Cellulase activity was assessed by preparing Modi ed

Czepak Dox (MCD) agar plates supplemented with 1

% carboxymethyl cellulose (CMC). 30 μl of each cul-

ture  ltrate was dispensed in 5 mm well prepared by

sterile cork borer in MCD-CMC agar plates. The plates

were incubated at 37˚ C for 18-24 h. Un-inoculated PDB

served as control. After the incubation is over, the plates

were  ooded with aqueous congo red solution. After

15 min, appearance of yellow zone around the fungal

colony indicated Cellulolytic activity (Lingappa et al.

1962). The zone diameter was measured and represented

as Mean ± SD.

(b) Amylolytic activity:

Amylase activity was assessed by preparing the 1%

starch agar plate followed by preparation of 5 mm well

with the help of pre-sterilized cork borer. Brie y, 30 μl

culture  ltrate of each fungus was loaded into the wells

followed by incubation at 37˚ C for 18-24 h. Un-inoc-

ulated PDB served as control. After the incubation, the

plates were  ooded with 1 % iodine solution. Appear-

ance of clear zone around the well indicated amylolytic

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIO-PROSPECTING FUNGAL ENDOPHYTES OF HIGH ALTITUDE MEDICINAL PLANTS 371

Neha Kapoor et al.

FIGURE 1. Fungal endophytes isolated from different parts of medicinal plants

collected from Uttarakhand

activity (Hankin and Anagnostakis, 1975). The zone

diameter was measured and represented as Mean ± SD.

Production of Asparaginase by the fungal endophytes

was assessed by modi ed Ditch plate assay (Mahajan

et al. 2013). Brie y, L- asparaginase-agar (2 %) plates

containing phenol red (0.009 %) were prepared and each

plate was divided into four quadrants followed by prep-

aration of 5mm wells in each quadrant using sterilized

cork borer. Further, 30 μl of culture  ltrates of each fun-

gal endophyte were loaded onto the premade wells in

L-asparaginase agar plates. The plates were incubated

at 37°C for 24h. After the culmination of incubation

period, the plates were observed for the pink halo forma-

tion around the wells. The zone diameter was recorded

and expressed as Mean ± SD.

RESULTS

A total of 14 fungal endophytes were recovered from

different parts of Withania sominifera, Ocimum basilli-

cum and Syzygium aromaticum (Table 1, Figure 1). Max-

imum fungal colonization was recovered from Withania

sominifera and Syzygium aromaticum(42.8%) followed

by Ocimum basillicum(14.2%). The host tissue of each

plant sample exhibited a variation in colonization of

the endophytic myco ora. The maximum colonization

of fungal endophytes was observed in leaf (57.1%) fol-

lowed by stem (28.5%) and internal tissue/vascular tissue

in the stem (14.2%). No endophyte was recovered from

the stem and stem internal tissue of Withania sominifera

and leaf of Ocimum basillicum (Table 2).

SCREENING OF BIO-ACTIVITIES:

In the preliminary screening of protease, amylase and

cellulase activity, both the isolates recovered from Oci-

mum basillicum were found to be positive for proteo-

lytic, amylolytic and cellulolytic activity. Further, the

isolates recovered from Syzygium aromaticum were

potent producers of amylase and cellulase. Out of 14

isolates, 7 fungal endophytes were found to exhibit pro-

teolytic activity with maximum potential in #9SASTD

Table 1. Showing culture code of fungal endophyte, plant name, plant part and place of plant

collection

S. No. Culture Code Plant part Plant Name Place of collection

1. #16OPSTD Stem Ocimum basillicum Kanhaiyavihar, Dehradun

2. #9OPSTITD Stem internal tissue Ocimum basillicum Kanhaiyavihar, Dehradun

3. #6WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

4. #9 WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

5. #10 WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

6. #14 WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

7. #22 WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

8. #24 WSLD Leaf Withania somnifera Kanhaiyavihar, Dehradun

9. #7SALD Leaf Syzygium aromaticum Kanhaiyavihar, Dehradun

10. #16SALD Leaf Syzygium aromaticum Kanhaiyavihar, Dehradun

11. #2SASTD Stem Syzygium aromaticum Kanhaiyavihar, Dehradun

12. #9SASTD Stem Syzygium aromaticum Kanhaiyavihar, Dehradun

13. #15SASTD Stem Syzygium aromaticum Kanhaiyavihar, Dehradun

14 #16SASTITD Stem Internal tissue Syzygium aromaticum Kanhaiyavihar, Dehradun

372 BIO-PROSPECTING FUNGAL ENDOPHYTES OF HIGH ALTITUDE MEDICINAL PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Neha Kapoor et al.

Table 2. Summary of endophytic fungi isolated from different tissues of host plant

S. No Host plant

Endophytic fungi

Total

Leaf Stem Stem internal Tissue

1. Withania sominifera 600 06

2. Syzygium aromaticum 231 06

3. Ocimum basillicum 011 2

Total 8 4 2 14

Table 3. showing bioactivity pro ling of culture  ltrates of fungal endophytes

expressed as zone size in mm

S. No

Culture code Mean zone size (mm)

Protease Cellulase Amylase Asparaginase

1. #16OPSTD 8 ± 0 11 ± 0 7 ± 0 -

2. #9OPSTITD 11 ± 0 10 ± 0 9 ± 0 -

3. #6WSLD - 15 ± 0 12 ± 0 14.33 ± 0.57

4. #9 WSLD - - - -

5. #10WSLD - - - -

6. #14WSLD 9 ± 0 16 ± 0 11 ± 0 18.67 ± 0.57

7. #22WSLD - - 9.5 ± 0.70 21.67 ± 0.57

8. #24 WSLD - - - -

9. #7SALD 14.5 ± 0.70 8 ± 0 14 ± 0 -

10. #16SALD - 10 ± 0 8 ± 0 16 ± 0

11. #2SASTD 15 ± 0 10 ± 0 16.5 ± 0.70 11.33 ± 0.57

12. #9SASTD 15.5 ± 0.57 11 ± 0 13 ± 0 18.33 ± 0.57

13. #15SASTD - 10 ± 0 9.5 ± 0.70 -

14. #16SASTITD 7 ± 0 9 ± 0 11 ± 0 -

FIGURE 2. Enzyme production by endophytic fungal isolates: a) Proteolytic activity, b)

cellulase activity, c) amylase activity, d)-f) L-asparaginase activity of culture  ltrates of

different fungal endophytes

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIO-PROSPECTING FUNGAL ENDOPHYTES OF HIGH ALTITUDE MEDICINAL PLANTS 373

Neha Kapoor et al.

closely followed by #2SASTD and #7SALD. Further-

more, #14WSLD and #16OBSTD was weak producer of

proteolytic enzymes.Further, all the isolates of Syzygium

aromaticum were found to positive In case of amylase

and cellulase activity. However, maximum amylase pro-

duction was observed in #2SASTD and maximum cellu-

lase production was exhibited by #14WSLF followed by

#6WSLF (Table 3, Figure 2). In case of asparaginase pro-

duction assay, #22WSLD exhibited maximum asparagi-

nase production with zone size of 21.3 mm followed by

#14WSLD and #9SASTD with zone size of 18.6 mm and

17.6 mm respectively. Moderate level of enzyme produc-

tion was observed in #16SALD and #6WSLD with zone

size of 16 mm & 14.3 mm respectively (Table 3, Figure

2). Further, the isolates of Ocimum basillicum did not

showed the activity.

DISCUSSION

Endophytes have been targeted as bioactive repository

with immense potential for industrial and pharmaceu-

tical interventions. Among the different types, fun-

gal endophytes have found a niche of being a sublime

resource for harnessing novel bioactive agents against

spectrum of disorders (Strobel, 2003; Gunatilaka, 2006).

Fungal endophytes produce diverse chemistry of mol-

ecules depending upon the host plant requirements

against different kinds of stresses. Thus, choosing a plant

for sampling to isolate a fungal endophyte is a crucial

aspect. For this reason the medicinal plants documented

in traditional indigenous preparations provide an ample

group as repository of fungal endophytes. Among the

geographically diverse medicinal plants, high altitude

inhabiting medicinal plants have found their own niche

in ethnopharmacology (Rajagopal et al. 2012). Owing to

their immense bioactive potential, high altitude medici-

nal plants have been exploited to an extent of endanger-

ment. Thus we hypothesis that, high altitude medicinal

plants may be explored for endophytic fungal diversity

and can further be screened for bioactivities (Chutulo

and Chalannavar, 2018)

The present study was undertaken to bio-prospect

the fungal endophytes from medicinal of high altitude

regions of Uttarakhand for pharmaceutical interventions.

Withania sominifera, Ocimum basillicum and Syzygium

aromaticum were selected for endophyte isolation based

on their ethno-pharmacological potential. We observed

higher colonization fungal endophytes in Withania

sominifera and Ocimum basillicum. Isolates were fur-

ther screened for industrially imperative enzymes. Inter-

estingly, endophytic fungal isolates exhibited varying

degree of enzyme production. These results corroborate

our hypothesis that high altitude medicinal plants may

be explored for isolation of fungal endophytes with

potent bioactivities. The present study shows promising

signs for further puri cation and evenness of enzymes

which may found application in pharmaceutical indus-

try.

ACKNOWLEDGEMENT

Authors thank the Managing Director, Uttaranchal (PG)

College of Biomedical Sciences and Hospital for provid-

ing necessary infrastructural facilities to carry out this

work.

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