ISSN: 2320-5407 Int. J. Adv. Res.

9(05), 702-708

Journal Homepage: - www.journalijar.com

Article DOI: 10.21474/IJAR01/12896

DOI URL: http://dx.doi.org/10.21474/IJAR01/12896

RESEARCH ARTICLE
ISOLATE ENDOPHYTIC FUNGI FROM LOCAL CATHARANTHUS ROSEUS AND ANALYZE THEIR
EXTRACELLULAR ENZYME ACTIVITIES

Nguyen Chi Mai1, Pham Thi Hoe1, Vu Huong Giang1, Le Quynh Lien1, Nguyen Tuong Van2 and Tran My
Linh1
1. Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST).
2. Institute of Biotechnology, VAST.
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Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History Endophytic microbia are known as natural sources for producing
Received: 20 March 2021 valuable enzymes. In this study, four endophytic fungi were isolated
Final Accepted: 24 April 2021 from roots of local Catharanthus roseus (L.) G. Don var. roseus
Published: May 2021 (purple flower) and C. roseus var. ocellatus (red stamens white flower)
widely grown in Nha Trang. They were identified as Fusarium solani
Key words:-
Catharanthus Roseus, Endophytic RN1, Chaetomium funicola RN3, Penicillium rugulosum RN4 and
Fungi, Extracellular Enzymes Chaetomium homopilatum WN1 based on morphologies colonies and
spores. The activity analysis of their extracellular enzymes indicted all
isolated endophytic fungi are able to produce protease, cellulose,
xylanase as well as amylase. This is the first report on the endophytic
fungi inhabited in C. roseus plant growing in the coastal regions of
Vietnam, which could provide an attractive source for bioactive
enzyme exploitation.

Copy Right, IJAR, 2021,. All rights reserved.

……………………………………………………………………………………………………....
Introduction:-
Endophytes are microorganisms living inside plants for at least a part of their life cycle without causing any disease
symptoms to the host plant (Petrini, 1991), in which, endophytic fungi are the most common groups found in plants
and present in most their organs and tissues (Wang and Dai, 2011). One million of 1.5 million species of existing
fungi in nature are reported to be endophytic. It is well-known that they play a vital role in enhancing the growth of
plants and well known for their capability to produce bioactive compounds. Bioactive compounds synthesized by
endophytic fungi have diverse of natural properties, such as antibiotics, anticancers, antioxidants, biopesticides,
antiparasitics and agrochemicals (Pinheiro et al., 2013). In addition, several highly stable and commercially
valuable extracellular enzymes had been exploited from endophytic fungi. Among 40 endophytic fungi isolated
from Ocimum species (Tulsi), Pavithra et al. (2012) found 50% of isolates to produce amylase and protease and
more than 27% to produce tyrosinase. Uzma et al. (2016) reported 29% of 112 endophytic fungi isolated from six
wild medicinal plants growing in Bisle region (Western Ghats, Karnatakwas) were positive for amylase, 28% for
cellulase, 18% for pectinase and 40% for asparaginase activity. Alberto et al. (2016) identified 62% of endophytic
fungal isolates of four medicinal plant species exhibited amylase, approximately 93% cellulase, 50% pectinase, and
64% protease activity.

Cantharanthus roseus is a herbaceous plant of the Apocynaceae family and native to the subtropics. It can produce
various secondary compounds with diverse medicinal activities, for example treatments of cancer, diabetes, high
blood pressure, antibacterial, antifungal etc… (Singh et al., 2001; Ferreres et al., 2008; Gajalakshmi and Rajeswari,

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Corresponding Author:- Tran My Linh
Address:- Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam.
ISSN: 2320-5407 Int. J. Adv. Res. 9(05), 702-708

2013; Das and Sharangi, 2017). Recent studies have shown that a number of endophytes of C. roseus also play an
important role in the biosynthesis of host-specific secondary metabolites. From the endophytic Fusarium
oxysporum, Kumar et al. (2013) extracted an adequate amount of vinblastine and vincristine – two important
anticancer drugs. Palem et al. (2016) also reported another endophytic fungus; Talaromyces radicus produced
vinblastine and vincristine, which stimulated apoptosis in some human cancer cell lines. Similarly, Kuriakose et al.
(2016) also found vincristine synthesized by an endophytic fungus, Eutypella spp-CrP14. Beside the synthesis of
secondary compounds, endophytic fungi of C. roseus also produce a number of valuable extracellular enzymes.
From 5 endophytic fungi isolated from root and leaf samples of C. roseus collected in the north of Peninsular,
Malaysia, Ayob and Simarani (2016) observed that all isolates exhibited cellulase activity; amylase activity from
Colletotrichum sp. and F. solani and protease activity only from F. solani.

In this study, the endophytic fungi isolated from C. roseus wildly growing in the central coast of Vietnam (Nha
Trang) were identified and screened for their extracellular enzyme production. These findings might provide a
considerable source of useful bioactive compounds.

Materials and Methods:-

Isolation and identification of endophytic fungi of C. roseus:
Roots of white and purple flower C. roseus plants collected in the coast of Nha Trang, Khanh Hoa, Vietnam were
washed thoroughly with running tap water first. The samples was cut into 1 cm pieces, then surface sterilized by
dipping them in 0.1% Tween 20 for 10 min, followed by 70% ethanol for 1 min, then rinsed in sterile distilled
water thrice. After sterilization, samples were ground in the ceramic pestle and mortar and placed into a flask
containing 8 ml of sterilized water. The flask was shaken for 15 min at room temperature. The extract solution (100
µl) was spread on humic acid plates supplemented with 100 mg/l ampicillin to suppress bacterial growth. Plates
were incubated at 25°C for at least 7 days in darkness. After incubation, the colony diameters were measured. The
production of soluble pigments was noted. Colonies were photographed with a Canon EOS 400D. Morphological
characteristics such as growth pattern, hyphae, colony color, surface texture, aerial mycelium, sporulation using
standard manuals and taxonomically to the genus level on the basis of macroscopic and microscopic morphological
characters under Scanning Electron Microscope FESEM S4800.

Detection of extracellular enzyme activities:

The activities of extracellular enzymes produced by fungal endophytes was assessed by growing them on Hansen
pH 6 (20 g/l glucose, 10 g/l tryptone, 2 g/l K2HPO4, 2 g/l MgSO4, 18 g/l agar) at 30oC for 7 days. Mycelial plugs (5
mm in diameter) were placed on the solid Czapek plates (3 g/l NaNO 3, 1 g/l K2HPO4, 0.5 g/l MgSO4.7H2O, 0.5 g/l
KCl, 0.01 g/l FeSO4.7H2O, 20 g/l agar) supplemented with 1% dissolved substrates, including casein, CMC,
cellulose, starch, xylan to assess protease, cellulase, amylase and xylanase activities. After incubation for 3-7 days
at 25oC, the zone of enzyme activity surrounding the fungal colony was measured. Proteolytic activity was detected
by casein hydrolysis on agar plates via the formation of a clear zone around colonies after covering the plate with
10% acid trichloacetic for 30 min. The degradation zones of cellulase, amylase and xylanase activities were
detected by flooding the plates with Lugol dye. The diameter (D, mm) of degradation zone was used to assess
enzyme activity. D values ≥ 25, ≥ 20, ≥ 15 and ≤ 10 mm were considered as very strong, strong, medium and weak
enzyme production, respectively. All the experiments were performed in triplicates and the means were analyzed
statistically with the SPSS program version 20. The significant differences between the means were determined
through Duncan’s multiple range test.

Result and Discussion:-

Isolation, morphological identification and characterization of endophytic fungi:
A total of 04 endophytic fungi isolates were obtained from the roots of local C. roseus, in which RN1, RN3, RN4
isolated from pink flower and WN1 from white flower C. roseus plants. Fungi were identified to the species level
based on macroscopic and microscopic morphological and cultural characteristics.

Isolates RN1 culture has rapid growing and white-greyish aerial mycelia (Figure 1A). The colonies with wooly or
cottony surface reached 3.5 to 4.0 cm after 7 days at room temperature of 24-26 °C on Czapek medium.
Conidiosphores, 45-80 x 2.5-3.0 μm (length x width), are in mono or polyphyalidics (Figure 1B). Microconidia 8-
16 x 2-4 μm with 0-1 septa and thick wall formed after 2-3 day culture of a conidiosphore. Macroconidia 35-55 x
4.5-6.0 μm with 3-5-7 septa, cylinder to rhombus with slightly curved at other ends were formed at 4-7th day of

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culture (Figure 1C). Clamydospores 8-12 x 8-10 μm in globose and ovoid shape, smooth or rough surface also
appeared after 7-14th day of culture. According to the keys proposed by Domsch and Gams (1980) RN1 is identified
as Fusarium solani (Mart.) Sacc.

Figure 1:- Cultural and morphological characteristics of Fusarium solani RN1. (A) Colonies on Czapek medium at
250C, 7 days ; (B) Conidiosphores and conidia X 1000 magnification ; (C) Macroconidia and clamydospores X 1000
magnification.

For RN3 isolate, it grew as fast as 3.5-5.0 mm/day and the colony was covered by white or light gray aerial mycelia
in the front and yellow color in the backside (Figure 2A). Ascomata 120-200 µm of RN3 appeared after two weeks
of culture, which had dark brown or light gray color, ovoid to spherical (Figure 2B). The lateral hairs were straight
or slightly curved, septate, rough, up to 4–5 µm wide at base. Clavate asci contained 8 ascospores, light red at early
stage, 22-30 x 8-11 µm in size. Ascospores were in limoniform, slightly curved pointed to one or two ends, 6.0-7.5 x
4-4.5 µm with a brown germ pore and thin-walled at mature (Figure 2C). This isolate is matched with morphology
of Chaetomium funicola Cooke offered by Axr et al. (1986).

Figure 2:- Cultural and morphological characteristics of Chaetomium funicola RN3. (A) Colonies on Czapek
medium at 25°C, 14th day; (B) Ascoma X 400 magnification; (C) Ascospores X 1000 magnification.

Strain RN4 was identified as Penicillium rugulosum Thom (Raper and Thom, 1949). The isolate developed as low
as 1.5-2.5 cm in 14 days on Czapek medium, flat or partly partitioned colony surface (Figure 3A) and exhibited
mycellium hyaline, yellow-green background with alternating red-orange strands holding conidiophores ; less
secretion and soluble pigments ; light red or burgundy backside. Conidiophores 100-150 x 3.0-3.5 µm were
biverticillate, symmetry; 5, 7 or 8 metulae 10-14 x 2.5-3.0 µm. Phialide 10-12 x 2.2-2.5 µm exhibited somewhat
pointed apex. Conidia 3.0-3.5 x 2.5-3.0 µm were globose or ellipsoidal, smooth or rough-thick walled (Figure 3B).

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Figure 3:- Cultural and morphological characteristics of Penicillium rugulosum RN4: (A) Colonies on Czapek
medium at 25°C, 14th day; (B) Conidiophore and conidia X 1000 magnification.

Similarly, WN1 colonies developed as fast as 2.5-3.5 mm/day at 25°C, white or gray-brown in color with white
aerial mycelia (Figure 4A). Ascomata were formed after 2-3 weeks, olive brown or greyish, or globose, 100-170
µm; lateral hairs wider at base (Figure 4B). Clavate asci contained 8 ascospores. Ascospores 6.5-8.5 µm x 6.0-7.5
µm x 5-6 µm were discharged in pyriform with apiculate at both ends and a germ pore (Figure 4C).
Chlamydospores 7-14 µm x 6-9 µm were globose, colorless or light brown formed on the top or beside aerial
mycelia. According to von Axr et al. (1986) WN1 belongs to Chaetomium homopilatum Omvik.

Figure 4:- Cultural and morphological characteristics of Chaetomium homopilatum WN1 : (A) Colonies on Czapek
medium at 25°C, 7th day ; (B) Ascomata X 1000 magnification; (C) Asci X 1000 magnification.

The endophytic fungi have been isolated from the different medicinal plants. So far, this study is the first report
involving the endophytic fungi from C. roseus inhabiting the central coastal area of Vietnam. Three isolates are
from Sodariomycetes class, only Penicillium rugulosum belongs to Eurotiomycetes class. Among endophytic fungi
isolated from 29 medicinal plants, Huang et al. (2008) found total of 45 endophytic fungi from different tissues of C.
roseus grown in five locations of China, however no Furasium, Chaetomium and Pelicilium genera were recorded.
In India, most of the indetified fungal species from C. roseus belonged to Hyphomycetes (Kharwar et al., 2008). The
difference in region from where plants are isolated could be one possible reason for different isolated microbial
communities.

Extracellular enzyme production and activity of C. roseus endophytic fungi:

Extracellular enzyme activities of the studied fungal strains were screened based on their performances on culture
medium supplemented with specific substrates. The larger zone of enzyme activity surrounding the fungal colony
showed the stronger enzyme activities.

Table 1:- Analysis of extracellular enzyme activities from isolated endophytic fungi.
Host plants Endophytic fungal strain Diameter of degradation substrate zone (mm)
Casein Cellulose CMC Starch Xylan
While flower C. homopilatum WN1 32±0.55 25±0.38 29±0.43 0 25±0.78

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F. solani RN1 32±0.48 35±0.68 41±0.54 30±0.41 37±0.43

Pink flower C. funicola RN3 41±0.44 40±0.56 35±0.73 36±0.29 28±0.65
P. rugulosum RN4 21±0.37 20±0.47 13±0.23 23±0.18 28±0.37

RN1 RN3 RN4 WN1

Protease

Cellulase
(Cellulose
substrate)

Cellulase
(CMC
substrate)

Amylase

Xylanase
Xylanase

Figure 5:- Enzymes produced by isolated endophytic fungi based on substrate degraded zones (RN1: F. solani,
RN3: C. funicola, RN4: P. rugulosum, WN1: C. homopilatum).

As shown in Figure 5, all endophytic fungi isolated from local C. roseus in Nha Trang were able to produce
extracellular protease, cellulase, xylanase and amylase; except no amylase acitivity for C. homopilatum WN1. C.
funicola RN3 exhibited the strongest protease activity with the diameter of substrate degradation zone to be 41 mm.
Cellulase activities were strongly expressed in two isolates of F. solani RN1 and C. funicola RN3 on both culture
medium supplemented with cellulose and CMC substrates. Amylase activity was found in 3 strains of F. solani
RN1, C. funicola RN3 and P. rugulosum RN4, with substrate degradation diameters of 30 mm, 36 mm and 23 mm,
respectively. The xylanase activity was also expressed in four isolates, in which the strongest was from F. solani
RN1 (37 mm), followed by C. funicola RN3, P. rugulosum RN4 (28 mm) and C. homopilatum WN1 (25 mm).

These obtained results were in agreement with the previous reports on extracellular enzyme activity of these fungal
strains isolated from different plant species. According to Ayob and Simarani (2016) F. solani isolates from C.
roseus exhibited cellulase and amylase activities. Darwish and Abdel-Azeem (2020) recorded endophytic
Chaetomium genus was able to produce several enzymes including amylase, dextranase, laccase, protease, chitinase,
β-1, 3-glucanse, which were applied in agriculture, beer and wine fermentation, detergents and food production.

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In this study, we determined that the P. rugulosum RN4 isolated from C. roseus varieties in Nha Trang (Vietnam)
showed strong xylanase activity, relatively strong protease and amylase activities, and moderate cellulase activity. It
also exhibited β-glucosidase and xylanase activities. Penicillium genus was known as the producer of several
enzymes for degradating proteins, xylans, starches, lipids, etc. of agricultural wastes (Techapun et al., 2003). Yoon
et al. (2007) reported β-glucosidase was strongly expressed in 106 Penicillium strains of and pectinase and xylanase
activities were detected in 24 and 84 species, respectively. Two endophytic Penicillium strains isolated from
medicinal plants, Camellia caduca and Schima khasiana, exhibited strong protease activity; and moderate activities
of lipase, cellulose and xylanse (Bhagobaty and Joshi, 2012).

Conclusion:-
In the present study, four endophytic fungi were isolated from root samples of white and purple flower C. roseus
varieties in Nha Trang coastal area (Khanh Hoa, Vietnam) and identified as F. solani RN1, C. funicola RN3, P.
rugulosum RN4 and C. homopilatum WN1. This is the first report on the endophytic fungi inhabited in C. roseus
plant growing in a coastal region of Vietnam. The analysis of extracellular enzyme activities indicated the sufficient
production of protease, cellulase, and xylanase from all of them and amylase from F. solani RN1, C. funicola RN3,
P. rugulosum RN4. In the coming time, further investigation will be carried out to confirm the practical application
of obtained endophytic fungi isolates.

Acknowledgements:-
This research was funded by Vietnam Academy of Science and Technology (VAST) via the Project Code No.
QTBY01.05/18-19. The authors also would like to thank Dr. Phan Thi Hong Thao, Department of Soil
Microbiology, Instistute of Biotechnology for the supportive discussion in isolation of endophytic microorganisms.

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