Engineering and Scientific International Journal (ESIJ) ISSN 2394-7187(Online)

Volume 8, Issue 1, January – March 2021 ISSN 2394 - 7179 (Print)

Endophytic Fungi and their Metabolites Isolated from

Bauhinia Racemosa Lamk Plant
Venkatesan Govindan
Assistant Professor, P.G. and Research Department of Botany, Mannai Rajagopalaswami Government Arts College,
Mannargudi-614001, Thiruvarur - District, Tamil Nadu, India

Abstract— Bauhinia racemosa family, Caesalpiniaceae is one of the precious resources of the earth. It is traditionally used
in the indigenous system of medicine Ayurveda, Unani, and Sidha for the treatment of several ailments like headache, fever,
skin and blood diseases, jaundice, chronic dysentery, diarrhea, and leucorrhoea, infection of malaria, boil, glandular swelling,
tumors, and cancer. It is also used to cure scorpion bite, to relieve food poisoning in cattle and as a contraceptive by women.
Endophytic fungi live in leafage of medicinally important plants are diverse and abundant; also little is known of their
terrestrial and habitat variation. Age differences in endophyte infections on young, mature, and senescent leaves of a
medicinal plant Bauhinia racemosa were studied. Enzyme evaluation, it was observed that the endophytic fungi species
Phyllosticta sp., Colletotrichum sp., Aspergillus species were presented four enzymes amylase, pectinase, lipolytic, and
cellulose activity. Particularly have indicated only cellulose was produced in Aspergillus niger. The endophytic fungus was
tested against standard bacterial and fungal cultures. In vitro antimicrobial test was carried out by the agar well diffusion
method. The minimum inhibitory concentration test was performed by the modified agar well diffusion method. Endophytic
fungi showed a broad spectrum of antimicrobial activity as it inhibited Gram-positive bacteria in Bacillus, and fungi in
Colletotrichum.

Keywords — Bauhinia Racemosa; Bioactive Metabolites; Endophyte Fungi; Tissues Specificity.

diverse novel products with significant bioactivity has
1. Introduction obtained promoted regard [12]. Address the common
observation that foliar endophyte infections increase in
Endophytes are microbes that colonize the internal density with leafage, assessing the relative importance of
plant tissue underneath the epidermal cell layers without age-specific, leaf toughness, and duration of disclosure to
causing any obvious symptom or symptomatic infection to fungal diversity. It explicitly identifies ecological factors
their host [1]. Hence, it is more difficult to complete the underlying patterns of endophyte colonization within
global fungal enlist, as compared to other organisms such healthy leaves of this tropical tree of Bauhinia racemosa. A
as animals, plants. Fungi play key roles in ecosystems as few additional investigations have focused on the
decomposers, mutualisms, and pathogens, while in most antimicrobial activity of endophytic fungi against clinical
cases, the role of individual fungus in nature is still isolates of Gram-positive bacteria and fungi.
unknown [2]. In most of the cases, each tissue segment was
infected by more than one fungal species [3]. Several 2. Materials and Methods
ecological groups of plants such as mangroves [4],
halophytes [5], fresh water [6] and trees of tropical forest 2.1 Botanical description
[7, 8, 9] have been studied for their endophyte assemblages.
Endophytes usually inhabit above-ground plant tissues
Bauhinia racemosa (Lamk), belonging to the family
which distinguish them from better known mycorrhizal
Caesalpiniaceae is a small deciduous tree used in the
symbionts. The endophytic fungi are ubiquitous and vastly
primordial treatment of medicine (Fig.1), the tree growing
diverse in host plants. Every plant examined to date
throughout India and Srilanka. Its importance played a
harbors at least one species of endophytic fungus and many
significant role in human civilization since ancient times. It
plants, especially woody plants, may contain literally
has universally one of the major roles in the treatment of
hundreds of thousands of species [3, 10]. Currently, studies
human Injury and diseases worldwide. Currently, the
on endophytes of medicinal plants have given due attention
demand for medicinal plants is increasing for both
as they oversee to produce of natural products beneficial to
developed and developing countries due to the growing
the human being [11]. Apart from isolating probable strains
recognition of natural products. It is traditionally used for
from medicinal plants, variety studies focus on different
the treatment of various ailments like vermicides, urinary
features of ecology. Endophytic fungi are reasonably
discharges, thirst headache, quatrain fever, fistula,
unexplored producers of metabolites useful to
tuberculoses glands, skin diseases, throat troubles, tumors,
pharmaceutical and agricultural industries. A single
diseases of the blood, etc. It is having antifilarial activity,
endophyte produces diverse bioactive metabolites. While
anthelmintic, antimicrobial, antihistaminic, anti-
the result, the function of endophytes in the production of

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DOI: 10.30726/esij/v8.i1.2021.81004
Engineering and Scientific International Journal (ESIJ) ISSN 2394-7187(Online)
Volume 8, Issue 1, January – March 2021 ISSN 2394 - 7179 (Print)

inflammatory, analgesic, antimalarial, anti-oxidant, and appearance of the yellow zone around the fungal
various other pharmacological activities. The leaves of B. colonization in red medium indicates cellulase activity.
racemosa are used for bidis production like so the plant is
called as bidi leaf tree, and also used for good fodder in 3. Results and Discussion
sheep, goats, and cattle.
Fungal endophytes were isolated from the tissues of
2.2 Surface Sterilization and Culture Protocols the Bauhinia racemosa. The leafage has found in dissimilar
fungal species were isolated. The tissues of the young,
The host plant studied was collected from Mannargudi, mature, and senescence leaves reveal 8, 9, and 24 species,
Tamil Nadu, and South India. These stages of plant leaves respectively, and also the number of isolates is disguised
were collected for the investigation. Leaf samples were from age of leaf such as young (20), mature (109),
collected from healthy plants. In these plants from Young, senescence (119) of leaves (Fig. 2). The fungal species
mature, and senescent leaves were randomly collect fifty were constantly isolated. Hyphomycetes were the abundant
leaves a few plants and their one hundred and fifty tissue group followed by Coelomycetes, Ascomycetes, and
segments were cut from fifty leaves. However, sterilization unidentified forms.
techniques were followed before cutting these segments.
The plant leaves are washed thoroughly with running water
and then the leaves have sterilized as followed. After the
surface sterilization, the leaves were cut into 0.5 cm2 bits
(segments) each leaf. The samples were washed in running
water, dipped in 70% ethanol for 60 seconds, immersed in
2.5% NaoC1 for 90 seconds, and then washed in sterile
water for 60 seconds or three times. The sterilized samples
were placed on the PDA medium amended with antibiotics
contained in Petri dishes. The Petri dish was sealed with
ParafilmTM and incubated in a light chamber at 26+1ºC for
7 to 21 days [13, 14]. The light regimen given was 12
hours light followed by 12 hours of darkness. Fungi that
grew from the segments were periodically observed and the
endophytes were identified. Fig. 1: Photo is shown on the plant of Bauhinia racemosa

2.3 Statistical Analysis

Colonization frequency (CF %) is the number of

colonies/ number of total X 100. The number of colonies
and the number of totals is the number of segments
colonized by each endophyte and the total number of
segments observed respectively [15].

2.4 Biological Active Analytical Evaluation

The isolated endophytic fungi from these aquatic

plants of Bauhinia racemosa and selected dominant and
common hyphomycetes and Coleomycetes group of Fig. 2: The number of fungal endophytes and species
endophytes were screened extracellular enzyme activity. isolated from Young, Mature, and Senescence leaves
The enzyme examine was made on prepared by both a
spread- plate technique with spores and mycelium [16]. Fungal endophytes reside in healthy tissues of all
tropical and temperate terrestrial plant taxa studied to date.
2.5 Cellulase Actitivity These fungal were diverse and abundant in leaves of
tropical woody angiosperms. Although, the fungal diversity
Yeast peptone medium containing Na-Carboxy- was varied from leaves (ages) tissue specificity. Studies
methylcellulose (0.5%) was used for Cellulase activity. have illustrated that plant location and leaf age impact the
Later than 3-5 days of fungal colony growth, the Petric density of endophyte infection in leaves of Bauhinia
plates were streamed with 0.2% aqueous Congo red strain racemosa. However, ecological factors have not been
compound and destained with 1M NaCl (15 min each). The investigated in detail. In order to understand the status of

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Engineering and Scientific International Journal (ESIJ) ISSN 2394-7187(Online)
Volume 8, Issue 1, January – March 2021 ISSN 2394 - 7179 (Print)

endophytes assemblage in different stages of the leaf, the frequency, as well as fungal species, increased in senescent
young, mature and senescent leaves were screened. Bright leaves. Phomopsis sp. Colletotrichum sp. and Phyllosticta
green, thin, tender, young leaves; dark green, thick mature sp were obtained from all the ages of leaves. The fungal
leaves and yellow, old senescent leaves of B. racemosa tree endophyte colonization is high richness becoming to
were collected and screened. The yellow senescent leaves occupy in leaf tissues from young to mature leaves.
were collected by gently tapping the petioles. Only those Endophytes isolated from mature B. racemosa more
senescent leaves that could be detached by this method rapidly on rich diversity mature leaf. Also, we believe this
were included for the study. This indicated that they were information allows us to discover underlying patterns of
in the late stage of senescence and would have fallen in a endophytic colonization within healthy leaves of this
few days. The procedure for inoculation of endophytes tropical tree [5, 17].
from leaves of these all ages of leaves was standard
sterilization. The senescent leaves were more densely Endophytic fungi are rich sources of bioactive natural
colonized by endophytes when compared with young products that can be used to gratify of Pharmaceutical,
leaves in the host of B. rasemosa. The senescent leaves medicinal, agriculture and industries. These ubiquitous
were found to assume that to be more species of fungi as fungi of Aspergillus niger were screened for four enzymes
endophytes. The fungal spores can be transmitted from tested. This endophyte showed that produce amylase,
air/soil to leaf in aerial tissues/seedling of the surface the lipolytic, cellulase, laccase enzymes such as cellulase
wetting leaves. However, these conceptions cannot be enzyme are rich produce (Fig. 4) and also similar recorded
proclaimed for a single host studied. The endophytes [18, 19, 20]. Cellulose, the substrate of cellulose, is the
diversity and richness is determined the importance of most plenty of polysaccharide present on earth. It is the
several factors such as canopy, leafage, leaf toughness, leaf main substance in fungi and plant materials. In the present
chemistry and duration of exposure to air etc. In this study, pretreated lignocellulose was used for the maximum
studies have demonstrated that plant leaves environments production of cellulase from Aspergillus niger. The
and leaf ages influence the diversity of endophytes present cellulose resource of Aspergillus niger was commonly
in leaves of B. racemosa (Fig. 3). For this tree species, we screened by plate assay method. For this, Yeast peptone
recorded varied these endophytic fungi have been found to (YP) medium amended with 0.5% carboxymethylcellulose
study in different leaf ages (young, mature, senescent). (CMC) and the plates were incubated for 3 days. After
Among 20 (13.3%) colonies isolates, 8 fungal species be incubation plates were stained with 0.2% Congo-red
held in young leaves, 109 (72.6%) colonies isolates, 9 solution, followed by de-staining with 1M NaCl solution
fungal species be held in mature leaves and 119 (79. 3%) for 15 minutes. This test has gained; the cellulases are a
colonies isolates, 24 species held in senescent leaves from complex group of enzymes which are secreted by a broad
450 leaves (each age 150 bits) segments leaves were range of microorganisms including fungi, bacteria, and
isolated (Fig. 2). actinomycetes. Cellulose-degrading fungi can be directly
used in industrial production, but the optimum conditions
for enzyme production and utilization must be considered
simultaneously, because their environmental requirements
are not uniform. Filamentous fungi are outward hyphae,
that is, their major mode of obtaining nutrients is to secrete
hydrolytic enzymes and then absorb the products through
the plasma membrane (Fig. 5).

Fig. 3: Imaginary diagram show on penetrate and increase of

endophytic fungi in leaf

Nevertheless of age at genesis, all mature leaves of

plant seedling contained fungal endophytes. It is detected
in many diverse differences in morphology between leaves
that matured in the plant and leaves that were mature at
outset. The endophyte species had a higher colonization Fig. 4: Screening of cellulolytic activity of Aspergillus niger

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Volume 8, Issue 1, January – March 2021 ISSN 2394 - 7179 (Print)

[3] Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA.
Aretropical fungal endophytes hyperdiverse?. Ecology Letter. 3;
2000: 267-274.
[4] Suryanarayanan TS, Kumaresan V, Johnson JA. Foliar Fungal
endophytes from two species of the mangrove Rhizophora. Canadian
Journal of Microbiology. 44; 1998: 1003-1006.
[5] Suryanarayanan TS, Kumaresan V. Endophytic fungi of some
halophytes from an estuarine mangrove forest. Mycological
Research. 104; 2000: 1465-1467.
[6] Venkatesan G, Arun G. Endophytes Fungi Associated with a water
Hyacinth of Eichhornia Crassipes (Mart.) Solms. International
Journal of Scientific Research in Biological Sciences. 7(3); 2020:
62-66.
[7] Suryanarayanan TS, Murali TS, Venkatesan G. Occurrence and
distribution of fungal endophytes in tropical forests across a rainfall
Fig. 5: Diagram showing on the diffusion of gradient. Canadian Journal of Microbiology. 80; 2002: 818-826.
[8] Murali TS, Suryanarayanan TS, Venkatesan G. Fungal endophyte
endophytic fungi in leaf tissue communities in two tropical forests of southern India: diversity and
host affiliation. Mycological Progress, 6(3); 2007: 191-199.
The endophytic fungi of Colletotrichum species of [9] Suryanarayanan TS, Murali TS, Thirunavukkarasu N, Rajulu MBG,
Venkatesan G, Sukumar R. Endophytic fungal communities in
Bauhinia racemosa were showing good antimicrobial woody perennials of three tropical forest types of the Western Ghats,
activity against standard Bacillus subtilis bacterial cultures. southern India. Biodiversity Conservation. 20; 2011: 913-928.
In vitro antimicrobial test was produced by agar well [10] Petrini O. Taxonomy of of endophytic fungi of aerial plant tissues.
diffusion method on Mueller Hinton agar and Sabouraud In: Microbiology of the Phyllosphere (eds. N.J. Fokkema, J.Van den
Heuvel). 1986: pp 175-187. Cambridge University Press,
dextrose agar for bacterial and fungal cultures respectively. Cambridge.
The minimum inhibitory concentration test was performed [11] Gunatilaka AAL. “Natural products from plant-associated micro-
by a modified agar well diffusion method. The endophytic organisms: Distribution, structural diversity, bioactivity, and impli-
fungi showed a broad spectrum of antimicrobial activity as cations of their occurrence”. Journal of Natural Product. 69; 2006:
509-526.
it inhibited Gram-positive bacteria (Bacillus subtilis ) [12] Prabavathy D, Valli Nachiyar C. Screening for Extracellular
verses fungi ( Colletotrichum species ). Enzymes and Production of Cellulase by an Endophytic Aspergillus
sp, Using Cauliflower Stalk as substrate. International Journal on
Applied Bioengineering. 6(2); 2012: 40-48 .
4. Conclusion [13] Bills GF, Polishook JD. Recovery of endophytic fungi from
Chamaecyparis thyoides. Sydowia. 44; 1992: 1-12.
[14] Suryanarayanan TS. Light incubation: a neglected procedure in
Endophytic fungi of woody plants proceed between mycology. The Mycologist. 6; 1992: 144.
hosts as spores, germinating epiphytically, and penetrating [15] Hata K, Futai K. Endophytic fungi associated with healthy pine
leaf cuticles to grow intercellularly within healthy tissues. needles and needles infested by the pine needles gall midge,
Therefore, successful colonization of host tissues by foliar Thecodiplosis japonesis. Canadian Journal of Botany. 73; 1995:
384-390.
endophytes likely is correlated with factors influencing the [16] Hankin L, Anagnostakis SL. The use of solid media for detection of
local abundance of aerial and epiphytic propagules, and enzyme production by fungi. Mycologia. 67; 1975: 597-607.
with diverse aspects of host plant suitability, including host [17] Arnold AE, Edward Allen H. Canopy cover and leaf age affect
genotype and leaf characteristics. This study denotes refer colonization by tropical fungal endophytes: Ecological pattern and
process in Theobroma cacao (Malvaceae). Mycologia. 95(3); 2003:
that high inoculums volume, beneficial conditions for the 388–398.
survival of epiphytic propagules, and multiple applications [18] Narasimha G, Reddi Pradeep M, Sridevi A. Chemical Pretreatment
throughout the leaf and plant lifetimes should increase of Agricultural Feedstock for Enhanced Production of Cellulase by
colonization of healthy tissues by foliar endophytes under Mutant Fungus, Aspergillus Niger. Journal of Applied Biotechnol
Bioeng. 1(1); 2016: 01-05.
field conditions. Hence, in this investigation finding can be [19] Naseema B, Ismat Fatima R, Neha S, MohdHaris S, Mohd Kalim
further developed by performing the young, mature, and AK, Salman A. Production of Bioactive Secondary Metabolites from
senescent leaves of endophytic fungal diversity, Endophytic fungi. International Research Journal of Engineering and
identification, and utilizing these species to produce Technology. 3(6); 2016: 1859-1866.
[20] Venkatesan G, Ramesh Kumar J. Diversity of endophytes fungi
secondary metabolites and antibacterial biological activity assemblages in an aquatic eel weed plant of Vallisneria spiralis L,
for various applications. International Journal of Life Sciences. 8(2); 2020: 299-307.

Reference

[1] Strobel G, Daisy B. Bioprospecting for microbial endophytes and

their natural products. Microbiology and Molecular Biology
Reviews. 67(4); 2003: 491–502.
[2] Schmit JP, Mueller GM. An estimate of the lower limit ofglobal
fungal diversity. Biodiversity Conservation. 16; 2007: 99-111.

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DOI: 10.30726/esij/v8.i1.2021.81004