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Isolation and Identification of Endophytic Fungi From Justicia Adhatoda of Samba District (J&K)
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| Paper Id :
18143 Submission Date :
12/08/2023 Acceptance Date :
22/08/2023 Publication Date :
25/08/2023
This is an open-access research paper/article distributed under the terms of the Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. DOI:10.5281/zenodo.8412839 For verification of this paper, please visit on
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| Abstract |
Endophytic fungi live inside the plants, without causing
any harm to the hosts and its produce the secondary metabolites which are known
to possess antimicrobial activity. Justicia adhatodahas been used in Ayurveda,
Unani, and Homeopathy medicine. In this study, 40 samples of plants were
collectedfrom area of Samba district (J&K) and screened for the endophytic
fungi.The plant first rinsed in running tap water to remove the dust and the
other debris present in it. Segments of approximately 0.5 cm were cut with
sterile lancet blades and surface sterilized by agitating in 70% ethanol (5 s),
followed by treatment with 4% NaOCl (90 s) and then rinsed in sterile distilled
water (10 s). 40 (leaf and stem) segments from different plants are processed
for the isolation of endophytic fungi. A total of 27 endophytic fungi was
isolated and identified. The leaf segments showed a maximum repository for
endophytic fungi than the stem segments. Among the 27 endophytic fungi, the
predominant endophytic fungi isolated belonged to the genera of Alternaria alternata,Cladosporium
oxysporum, Aspergillus fumigatus, and some sterile fungi ,followed by Cercosporaoxysporum.
In this study, it is also observed that the majority of the fungi belonged to dematiaceous
hyphomycetes. From this study it is concluded that the isolation of endophytic
fungi from Justaciaadhatoda may be a good sourcefor some novel bioactive compound. However,
further studies are required to screen these endophytic fungi for production of
novel bioactive compounds. |
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| Keywords | Medicinal Plant ,Endophytic Fungi, and Justicia Adhatoda. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Introduction | Endophytes are microorganisms that are present inside the living tissue of various plants (root, fruit, stem, seed, leaf, etc.) which are establishing mutual relationship without any symptom of diseases(Arnold et al., 2003). These endophytes protect their hosts from infectious agents and adverse conditions by secreting bioactive secondary metabolites. The endophytic fungi play important physiological and ecological roles in their host life. Many traditional medicinal plants are known as to play a crucial role in providing primary health care since the dawn of civilization. The knowledge of medicinal plants has been accumulated from different medicinal systems such as Ayurveda, Unani, and Siddha. In India, it is reported that traditional healers use 2500 plant species and 100 species of plants serve as regular source of medicine. During the last few decades, there has been an increasing interest in the study of these medicinal plants has been witnessed in different parts of the world mainly due to many problems associated with synthetic drugs and with the emergence of multi-drug resistant pathogens (Gritto MJ, et.al., 2015). Out of these, 10,000 species are known for producing medicine .Indian System of Medicine uses around 3000 plant species belonging to more than 1000 genera (Vaidyanathan D, et.al. 2015). |
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| Aim of study |
Endophytic fungi live and grow inside the plants, without causing any harm to the hosts and known to produce the secondary metabolites are potential antimicrobial activity. Justacia adhatoda has been used in Ayurveda, Unani, and Homeopathy medicine. Therefore, present investigation is conducted with traditional known medicinal plant, collected from area of samba district (J & K) . In this study endophytic fungi to be isolate with an confined technique and identify them. |
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| Review of Literature | Recent investigations have been intensified by the
potentialities of endophytic fungus strains in production of bioactive
metabolites such as taxol, pestaloside, torreyanic acid and enzymes, xylanase,
isoflavonoids, and asparagines(Dar RA, et.al.2015). Justicia adhatodabelongs to (Acanthaceae), known commonly as Malabar
nut tree, is a shrub growing throughout the Indian peninsula. The plant is used
in the indigenous system of medicine in India and is a well-known expectorant
in both Ayurvedic and Unani Systems of Medicine (Chopra R. N.etal
1956;Kapoor LD, 2001). The leaves of this plants are used to
treat malarial fever, chronic fever, intrinsic haemorrhage, cough, asthma,
leprosy, skin diseases, and piles [Sharma P. V. 1996]. The plant is reported to show abortifacient (Wakhloo R.
L., et.,al 1979) antimicrobial (Doshi J. J.
and Patel V. K.1983, Mathew A.
S., et., al. 1998), and antitussive activities (Dhuley J. N, 1999). The crude extract of A.
vasica leaves was found to have conspicuous antifeedant and
toxic effects on the larvae of S.
littoralis (Sadek M. M, 2003). The plant contains alkaloids such as Vasicine, vasicinone,
deoxyvasicine, vasicol, adhatodinine, and vasicinol (Claeson U.
P., et.,al.2000). Other constituents include vitamin C, saponins, flavonoids
as well as steroids, and fatty acids (Wealth of
India A Dictionary of Indian Raw Materials: Volume II,1998). Vasicine is reported to have bronchodilatory, respiratory stimulant,
and uterine stimulant effects (Gupta O. P., et.,al.1977). Vasicine acetate showed antimycobacterial activity (Ignacimuthu
S., and Shanmugam N.2010). Essential oils of the
leaves of A. vasica are
also known to contain ketone, terpene, and phenolic ether which have antitumor,
antioxidant, antiaging, antimutation, and sedative effects; the high phenolic
content of essential oils contributes to their antimicrobial properties (Wealth of
India,1998). Various researcher have been able to isolate number of
endophytic fungi from some medicinal plant and also reported that they may role
growth promoter in plant as well as health care in human beings( Ibrahimet.,al
2021;Ahmed, MAK. Et.,al.2021, Eram,D.,et., al.2018). Selim K.,et., al. (2018) worked on Biodiversity and
antimicrobial activity of endophytic associated with Egyptian medicinal Plants. Therefore, the present investigation was conducted to explore
the medicinal plant for endophytic fungi. |
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| Methodology | Collection of plant material
Healthy parts of plant like leaves and stem of Justicia adhatoda were collected from area of Samba district (Jammu-J&K). The plant materials were brought to the laboratory in sterile polythene bags and keep it in cold temperature (10oC) in the refrigerator.
Isolation of endophytic fungi
The plant materials were taken out from the refrigerator and place under room temperature .They were washed with running tap water; leaf segments were equally cut by sterilized scalpel from the mid portions of healthy leaves to include the midrib. The cut segments were surface sterilized by immersing into the following series of solutions: sterile dis. H2O for 60 s, 70% ethanol for 60 s, 2.5% sodium hypochlorite for 4 min, 70% ethanol for 30 s, and a final rinsing in sterile dis. H2O three times. About 100 µL of the final rinse water was inoculated on Malt Extract Agar (MEA) medium to success the surface sterilization. The sterilized plant leaves were cut into five segments (5 mm), and 20 leaf segments per individual plant were placed on the surface of MEA plate (five segment for each plate), supplemented with 0.05 g of streptomycin sulfate per 100 mL of medium to inhibit bacterial growth and incubated at 28 ◦C ± 2 ◦C. The plates were checked daily for any fungal growth; single isolates grown out from the tissues were re-inoculated on fresh MEA plates and maintained at 4 ◦C in MEA slants (ALKahtani, et.,al 2020).
Identification of endophytic isolates
The endophytic fungal isolates were identified upto genus level based on the basis of micro and macro morphological characters such as colony morphology, pigmentation, growth pattern, spore structures, and other hyphal characteristics with the help of relevant literature and keys(Ellis, 1971;Gilman, 1957; Raper and Fennel, 1965; Booth, 1971; Barron, 1972; Sutton 1980). The microscopic examination was also done to study their reproductive spores. Cultures which failed to produce spores were grown on different minimal media and incubated for several weeks to months |
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| Analysis | Statistical analysis To know the endophyte richness, the frequency of fungal
endophytes harbored in plant species were calculated by the number of segments
colonized by endophyte species divided by a total number of segments examined
×100 (Divya CR et., al 2016). Colonization frequency (CF%) |
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| Result and Discussion | About 40 segments (10 segments of
each part respectively) of plant were screened for the isolation of the
endophytic fungi. A total of 30 endophytic fungi was isolated and identified
viz.. The leaf segments showed a maximum repository for endophytic fungi than
the stem segments. Among the 30 number of endophytic fungi , 15 genera belongs
to 22 species viz of Alternaria, sp. were isolated (table
2). Tables 1 and 2 showed the CF value and taxonomic position of
endophytic fungi. The predominant endophytic fungi isolated belonged to the
genera of Alternaria alternata, Aspergillus fumigatus, Aspergillus
niger, A. terrious and Cladosporium oxysporium followed by
Cercospora oxysporum, , Bipolaris , Colletotrichum sp , Curvularia
sp , Dreschelra sp, Emericilla nidulans Fusarium oxysporum, Penicillium
brevicompactum, Phoma sp, Phomopsis sp, Mucor sp, Verticillium sp. Some
fungi which did not produce any reproductive structure, as they produced
sterile mycelia (no sporulation). These fungi did not sporulate in spite of
repeated subculturing on to sporulating media (PDA, Potato dextrose
agar, and tap water agar) and hence are grouped on mycelia sterilia. In this
study, the majority of the fungi (Table 3) belonged to dematiaceous
hyphomycetes or group of fungi imperfecti or deuteromycetes (44%) , followed by
hyaline hyphomycetes (25.9%) ,sterile mycelium fungi (11.1%), Coelomycetes (7.4%), zygomycetes (3.7%) and Ascomycetes (7.4%). The colonization frequency
was found to be 67.5 % (table.1). Discussion This study was carried out to isolate and identify of endophytic fungi from Medicinal Plants Justacia adhatoda from area of Samba District (J&K). In the study, a total of 27 fungal colonies were isolated from 40 segments ( 20 from eachstem and leaf). Among the 27 endophytic fungi, the predominant endophytic fungi isolated belonged to the genera of Alternaria alternata, Aspergillus fumigatus, Aspergillus niger, A. terrious and Cladosporium oxysporium followed by Cercospora oxysporum, Bipolaris , Colletotrichum sp , Curvularia sp , Dreschelra sp, Emericilla nidulans, Fusarium oxysporum, Penicillium brevicompactum, Phoma sp, Phomopsis sp, Mucor sp, Verticillium sp. These results were similar to the studies of Rajeshwari et al (2017), they isolated and identified numbers of endophytic fungi from the medicinal plant Terminalia chebul . Dhanalakshmi et al.(2013), who had isolated Alternaria spp., Aspergillus spp., Bipolaris spp., Exophiala spp., Nigrospora spp., and Penicillium spp. from Moringa oleifera as dominant endophytic fungi. Barnabas et al.(2013) also reported Aspergillus spp., as the predominant isolate in the leaves, stem, and roots of M. oleifera .They have also reported that most of fungal species belonged to hyaline hyphomycetes (40%), coelomycetes (8.33%), dematiaceous hyphomycetes (29%), zygomycetes (12.5%), and mycelia sterilia (12.5%). The same results are showed the presence of endophytic fungi in Avicennia officinalis, that isolated the endophytic fungi namely reported Aspergillus, Penicillium, Curvularia, Cladosporium, Phoma, and Fusarium species (Job N, et.,al. 2015). According to some reports of workers, Alternaria sp. as endophytes have an potential to produce many antimicrobial metabolites.The endophytic fungi are able to produce many antimicrobial metabolites, such as colletotric acid (Zou et al., 2000) and griseofulvin (Park et al., 2005) reported from Alternaria species ubiquitous fungal genus including saprobic, endophytic and pathogenic species. Table : 1 Endophytic fungi
isolated from plant Adhtodavasica
Table 2: Taxonomic position of endophytic
fungi
Table 3: Percentage of isolated fungal class
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| Conclusion |
Medicinal plants are good source for isolation of
endophytic fungi that colonize the tissue without causing apparent symptoms.
Endophytic organisms have received considerable attention as they are found to
protect their hosts against pests, pathogens and even domestic herbivores. In
this study, a total of 27 endophytic fungi were isolated from the Justaciaadhatoda. |
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| Acknowledgement | The authors are thankful to, Institution , Pt. Prem Nath Dogra ,Govt. Degree College , Samba for providing necessary facilities required for this investigation. The assistance of my senior during collection of plant samples; Dr. Geeta Sumbali ( Retd Head of department, deptt. of botany, university of Jammu help in identification is acknowledged. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| References | 1. Ahmed Mohamed Aly
Khalil , Saad El-Din Hassan , Sultan M.
Alsharif , Ahmed M. Eid , Emad El-Din
Ewais , Ehab Azab , Adil A. Gobouri , Amr Elkelish and Amr Fouda (2021).
Isolation and Characterization of Fungal Endophytes Isolated from Medicinal
Plant Ephedra pachyclada as Plant Growth-Promoting. Biomolecules, 11, 140 2. ALKahtani, M.D.;
Fouda, A.; Attia, K.A.; Al-Otaibi, F.; Eid, A.M.; Ewais, E.E.-D.; Hijri, M.;
St-Arnaud, M.; Hassan, S.E.-D.; Khan, N. Isolation and characterization of
plant growth promoting endophytic bacteria from desert plants and their
application as bioinoculants for sustainable agriculture. Agronomy 2020, 10,
1325. 3. Arnold A.E., Mejia
L.C., Kyllo D., Rojas E.I., Maynard Z., Robbins N., Herre E.A. (2003). Fungal
endophyte limit pathogen damage in a tropical tree. Proceedings of National
Academy of Sciences USA, 100:15649–15654. 4. Barnabas J, Murthy
S, Jagdeesh S. Antimicrobial properties of endophytic fungi isolated from
Cynodondactylon and Moringa oleifera. Int J Biol Pharm Res 2013;4(2):98-104. 5. Barron ,G.L.(1972).The
genera of hyphomycetes from soil. Robert . E. Kriegar Publishing Co. Inc New
York, pp364 6. Booth, C. (1971). The genius Fusarium . CMI,
Kew, Survey, England pp237. 7. Chopra R.
N., Nayar S. L., Chopra I. C. Glossary of Indian Medicinal Plants. New
Delhi, India: Council of Scientific and Industrial Research; 1956. 8. Claeson
U. P., Malmfors T., Wikman G., Bruhn J. G. Adhatodavasica: a critical review of
ethnopharmacological and toxicological data. Journal of
Ethnopharmacology. 2000;72(1-2):1–20. 9. Daniya Eram,
Manojkumar Arthikala, GovindappaMelappa and Gustavo Santoyo (2018). Alternaria
species: endophytic fungi as alternative sources of bioactive compounds.
Italian Journal of Mycology vol. 47 ;40-54. 10. Dar RA, Rather SA,
Mushtaq S, Qazi PH. Purification and characterization of endophytic fungal
strains from four different high value medicinal plants of Kashmir valley. Int
J Phytopharm 2015;5(1):8-11. 11. Dhanalakshmi R,
Umamaheswari S, Sugandhi P, Prasanth DA. Biodiversity of the endophytic fungi
isolated from Moringa oleifera of Yercaud hills. Int J Pharm Sci Res
2013;4(3):1064-8. 12. Dhuley
J. N. Antitussive effect of Adhatodavasica extract on mechanical or chemical
stimulation-induced coughing in animals. Journal of
Ethnopharmacology. 1999;67(3):361–365. 13. Divya CR, Sharada
MS, Ashwini V. Isolation and identification of endophytic fungi in Trigonella
foenum-graceum L. and their antibacterial activity. Imp J Interdiscip Res
2016;2(9):516-21. 14. Doshi J.
J., Patel V. K., Venkatakrishna-Batt H. Effect of Adhatodavasica massage
in pyorrhoea. International Journal of Crude Drug Research. 1983;21(4):173–176. 15. Ellis, M.B.(1971).
Demataceous hyphomycetes. CMI, Kew.Surrey, England pp.608. 16. Eram,D.,Arthikala.,M,Melappa,G.,andsantoya,G.(2018).
Alternaria species: endophytic fungi as alternative source of bioactive
compounds. Italian J.Mycol. 47;40-54. 17. Gilman ,
J.C.(1957). A manual of soil fungi.(2nd ed)IOWA State university
press USA, PP 450 18. Gritto MJ,
Nandagopalan V, Doss A. Ethno-botanical study on the traditional healers in
Pachamalai hills of Eastern Ghats, Tamilnadu, South India. J Med Plants Stud
2015;3(2):80-5. 19. Gupta O.
P., Sharma M. L., Ghatak B. J. R., Atal C. K. Potent uterine activity of
alkaloid vasicine. Indian Journal of Medical Research. 1977;66(5):865–871. 20. Ignacimuthu
S., Shanmugam N. Antimycobacterial activity of two natural alkaloids, vasicine
acetate and 2-acetyl benzylamine, isolated from Indian shrub Adhatodavasica Ness.
leaves. Journal of Biosciences. 2010;35(4):565–570. 21. Job N, Manomi S,
Philip R. Isolation and characterization of endophytic fungi from Avicennia
officinalis. Int J Res Biomed Biotechnol 2015;5(1):4-8. 22. Kapoor
L. D. Handbook of Ayurvedic Medicinal Plants. Boca Raton, Fla,
USA: CRC Press; 2001. (pp. 416–417). 23. Mathew
A. S., Patel K. N., Shah B. K. Investigation on antifeedant and anthelmentic
potential of Adhatodavasica nees. Indian Journal of
Natural Products and Resources. 1998;14:11–16. 24. Mutiat Ibrahim1* ,
Elizabeth Oyebanji2 , Muinah Fowora3 , Ayobami Aiyeolemi1 , Chiamaka Orabuchi1
, Babajide Akinnawo1 and Adedotun A. Adekunle4 Extracts of endophytic fungi
from leaves of selected Nigerian ethnomedicinal plants exhibited antioxidant
activity. BMC Complementary Medicine and Therapies. 2021) 21:98 ,1-13 25. Rajeshwari,S.,
Revaths,R., and ArvindPrasanth, D.(2017) .Isolation and identification of
endophytic fungi from Terminalia chebula of eastern Ghat, Tamil Nadu. Assian J.
Pharm.Clinical Res. 10(4)21-23. 26. Sadek M.
M. Antifeedant and toxic activity of Adhatodavasica leaf
extract against Spodoptera littoralis (Lep., Noctuidae) Journal
of Applied Entomology. 2003;127(7):396–404. 27. Selim K.,ElBech
A.,AbdelEl-Rahman,T.,El-diwany-A(2018). Biodiversity and antimicrobial activity
of endophytic associated with Egyptian medicinal Plants. Fermentation 4,49. 28. Sharma
P. V. Classical Uses of Medicinal Plants. 1st. Varanasi,
India: ChaukhambhaVisvabharti; 1996. 29. Sutton, B.S
(1980). The Coelomycetes. CMI, kew, Survey, England. Pp696. 30. Vaidyanathan D,
Senthilkumar MS, Sisubalan N, Basha MG. Studies on ethnomedicinal plants used
by Malayali Gounder Tribes in Pachamalai of Eastern Ghats, Tamil Nadu, India.
Adv Appl Sci Res 2014;5(1):244-53. 3. 31. Wakhloo
R. L., Wakhloo D., Gupta O. P., Atal C. K. Vasicine hydrochloride, a new drug
for interruption of pregnancy. Journal of Obstetrics and Gynaecology of
India. 1979;29:939–940. 32. Wealth
of India A Dictionary of Indian Raw Materials: Volume II: B (revised) New
Delhi, India: Publications and Information Directorate; 1998. |
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