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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

The work is licensed under Research Article

Diversity and Distribution of Endophytic Fungi Associated with glutinosa (Lour.) C.B. Rob, an Ethno Medicinal

M.Abhinesh, A.Aruna, J.Ramesh, V.Krishna Reddy*

Toxicology laboratory, Department of Botany, Kakatiya University, Warangal-506009,

*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

ABSTRACT Endophytic fungi exhibit a great variation with respect to host, , 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; coloniza tion frequency; Endophytic infection rate; seasonal variation ; relative percentage occurrence

INTRODUCTION The Indian laurel (Litsea glutinosa (Lour.) identified as a high valued medicinal plant with C.B.Rob.) is a rainforest tree in the laurel many therapeutic properties. family, . Vernacular names of this The Indian people use the bark and leaves of the plant include soft bollygum, bollygum, bolly tree in the form of a demulcent as well as mild beech, brown bollygum, brown astringent for treating conditions like dysentery bollywood, sycamore and brown beech. Litsea and diarrhea [4]. Different plant parts are also glutinosa is native to India, used in relieving pain (gouty joints), bruises, south to , and the sprains, cut wounds, hemorrhages and helps in western Pacific islands. It has been observed arousing sexual power [5]. Roots of Litsea throughout Asia, including China, India, glutinosa used as emollient and also useful in Bhutan, Myanmar, Nepal, , Thailand treating trauma and fractural limbs. Oil from and Vietnam [1, 2]. It has been introduced to La the berries is used for rheumatism [6]. On the Reunion, and [2] and in the other hand, people in China use the oil extracted KwaZulu-Natal province in South Africa [3]. from the seeds for making soaps. Recent This plant, since ancient days, has been pharmacognocological studies conducted revealed that the plant is a source of

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arabinoxylans, essential oils as well as other present work aimed at to understand the compounds possessing antiseptic attributes. diversity of endophytic fungi associated with Leaves and bark are rich in mucilage and Litsea glutinosa. In order to get a complete contain an alkaloid, laurotetanine, which causes picture of associated endophytic fungi, plant titanic spasm in animals. Leaves also contain growing in different edaphic regions under amino acids like cystine, glycine, L-alanine, ß- different seasons and different plant parts were alanine, valine, tyrosine, proline, phenylalanine selected for isolation of endophytic fungi. and leucine. A new flavonoid - naringerin along with naringin, kaempferol-3- and 7-glucosides, MATERIAL AND METHODS quercetin and its 3-rhamnoside, pelargonidin-3- Sample collection and 5-glucosides have also been isolated from In the present study endophytic fungal species leaves. Seeds yield fatty oil which is a rich were isolated from different parts of the Litsea source of lauric acid. The essential oil of the glutinosa, (fig 01.a) growing in different forest fruits contains more than 40 compounds of regions of Warangal district, Telangana (India). which ß-ocimene occurs in high proportion. Healthy and mature plant parts (root, stem and Other predominant biochemicals include leaf) were carefully chosen for sampling. The caryophyllene oxide and ß-caryophyllene. Two plant parts were brought to the laboratory in new alkaloids - sebiferine and litseferine have sterile polythene bags and processed for isolation been isolated from trunk bark. Actinodaphnine of endophytic fungi within 24 – 48 hours after and its N-methyl derivatives, boldine and sampling. norboldine have also been isolated from this plant [7, 8]. Isolation of Endophytic Fungi The term endophytic fungi refers to an organism Endophytic fungi were isolated by following which lives within photosynthetic plant tissue by methods employed by Petrini (1986) and forming a symbiotic relationship with host and Hallman et al. (2007) [12, 28]. First, the plant without any harmful effect to the host plant [9, material was rinsed in tap water to remove the 10]. About a million endophytic fungal species dust and debris, then cut into small pieces by a are reported to be present in all [11]. sterilized blade under aseptic conditions [29]. Mostly Ascomycetes, Deuteromycetes and Each sample was surface sterilized by 70% Basidiomycetes class fungi are reported as ethanol for 1 minute and after that immersed in endophytic fungi [12, 13]. Endophytic fungi sodium hypochloride (NaOCl) / mercuric chloride exhibit a wide diversity of microbial adaptations (HgCl2) for 30 seconds to 1 minute. The samples that have evolved in special and unusual were rinsed in sterile distilled water for 1 environments, making them a great source of minute and then allowed to surface dry on filter study and research for new drugs for medical, paper. After proper drying, 4 segments were industrial and agriculture uses [14, 15, 16 and inoculated on PDA plate supplemented with 17]. These fungi also represent an important and antibiotic (streptomycin) and incubated at 28 ± quantifiable component of fungal biodiversity in 2ºC for 5 to 7 days with 12 hours light and dark plants that impinge on plant community cycle [30]. Pure colonies (like fig.01. b) were diversity and structure [18, 19,20 and 21]. transferred on to PDA slants. Pure cultures of Endophytic fungi are well known to produce a isolated fungal strains were preserved on potato variety of bioactive secondary metabolites such dextrose agar (PDA) slants at 4 to 5ºC with as alkaloids, terpenoids, steroids, quinones, proper labeling and were sub cultured from time isocoumarins, lignans, phenylpropanoids, to time. phenols, and lactones [22, 23]. Medicinal plants are reported to harbor endophytes which in turn Identification of Endophytic Fungi protect the host from infectious agents and Lactophenol-cotton blue microscopic slides were enable the plants to survive against adverse made from the isolated fungal cultures and conditions [24]. Most of the medicinal plants examined under light microscope and were surveyed for the occurrence of endophytic fluorescent microscope then photomicrographs fungi from various parts and were exploited for were taken (fig 01.c & d) for the identification. bioprospecting purpose [25, 26 and 27]. The

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(a) Litsea glutinosa (b) Trichoderma viride colony

(C) Aspergillus flavus (d) Alternaria alternate

Fig. 01: Endophytic fungi isolated from Litsea glutinosa

Colony morphology, surface texture, fungal isolates from host plant tissue segments pigmentation and spore morphology were used were analyzed in terms of Colonization to identify the endophytic fungi at species level Frequency percentage (CF %), Relative using standard manuals [31]. Endophytic fungi Percentage Occurrence (RPO) and Endophytic that did not produce the conidia even after Infection Rate (EIR) which were calculated by repeated subculturing on different media were using the following formulae [34]. treated as "sterile form"[32, 33]. The endophytic

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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 respectively. Leaf segments were colonized by as Colonization frequencies of different endophytic many as 20 endophytic fungal species, whereas fungi in different parts of the plant are stem segments were colonized by 17 species. presented in table 1. From the perusal of the However, root segments were colonized by 15 table it is evident that leaves are more colonized species only. The twenty fungal species belonged by endophytic fungi than the stems and roots. to 10 genera. In these genera, Out of 240 segments of leaf, 169 segments were species (5) dominated all other fungal generic found to be colonized by endophytic fungi species followed by Aspergillus (4), Fusarium (3) (70.47%). Colonization frequency percentage in and Alternaria (2). All other genera are stem and roots was 61.25 and 60 percent 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 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 Among different fungi, highest EIR was shown shown by A.niger followed by F.solani. Least by Verticillium dahlia (58%) followed by Euricoa colonization frequency was observed with species (50%) A.nidulans and Neurospora crassa Trichoderma viride. Colonization frequency of (42.8%) for leaf and least by Penicillium rubrum. A.flavus (8.3%) was highest in stem followed by In case of stem, highest EIR was recorded by A.niger (7.5%) and F.solani. Three species viz, Fusarium semitectum (42.8%) which was Cladosporium sphaerospermum, Euricoa species followed by Verticillium dahliae and and Trichoderma viride were not observed in P.chrysogenum. Cladosporium sphaerospermum stem segments. In roots, like stems, highest CF has shown highest EIR (50%) in root segments was recorded with A.flavus and A.niger followed followed by Verticillium dahliae (40%). Least by F.solani. Altogether five species, Alternaria EIR was shown by F.oxysporum (20%). The solani, Colletotrichum acutatum, Euricoa results also reveal that for all three plant parts species, A.nidulans and Penicillium roqueforti EIR was highest with Verticillium dahliae for were not isolated from root segments. leaf with Fusarium semitectum (42.8%) for stem Endophytic Infection Rate (EIR) of different and Cladosporium sphaerospermum for root. endophytic fungi in three parts of the Litsea Interestingly, Euricoa species though totally glutinosa is presented in table -2. For all the absent in root and stem, was recorded with fungi put together, EIR was highest (31.5%) for maximum EIR in leaf. leaf followed by stem (26.7%) and root (25%).

Table 02: Endophytic fungi isolated from L.glutinosa and its Endophytic Infection Rate (EIR) S.No. Endophytic fungi Root Stem Leaf (120) (240) (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%

*S = Screened *I = Infected *EIR= Endophytic Infection Rate

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Results pertaining to seasonal distribution of different seasons, summer season was observed endophytic fungi in different plant parts of to be least favorable for colonization. Some Litsea glutinosa are presented in table- 3. A endophytic fungal species like Aspergillus flavus, critical analysis of the table reveals many A.niger, Fusarium solani and a few sterile forms interesting points. Firstly, the composition of were recorded in all three seasons and also with endophytic flora in root, stem and leaf varied all plant parts. In contrast, some fungal species with the season. More number of fungal taxa has were recorded in a particular season and in been observed in rainy season followed by winter association with a particular plant part. For and least in summer. Secondly, the colonization example, Euricoa species was recorded with only frequency of endophytic fungi also varied with leaves that too in winter season. Similarly, the season and also with plant part. In case of Trichoderma viride, most common soil , root, more colonization frequency was observed was found to be associated with root in winter in winter season. However, with regard to stem season only. and leaves more colonization frequencies are observed in rainy season. Among the three

Table 03: Endophytic fungi isolated from Litsea glutinosa during Different seasons S.NO Endophytic fungi Root(40) per Stem(80) per Leaf(80) per season season 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 02 5 - - 04 5 sphaerospermum 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 Similarly, winter season has found to be varied with the fungal group and season (fig.02). favorable for hyphomycetes. Interestingly, RPO In general, hyphomycetes dominated in all the of sterile forms was more in roots in all the three plant parts and in all the seasons. However, seasons. RPO of hyphomycetes is more in leaf tissue.

* 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 different plant parts and also under different The research on endophytic fungi though began environmental conditions in order to get the rather late, intensive investigations have been holistic view of their distribution and to exploit carried out in recent years in view of their their potential applications. The objective behind multitude applications and ecological the present investigation was to isolate the significance [35]. Many of the endophytic fungi endophytic fungi from Litsea glutinosa, a highly still remain unexplored [36]. Despite the valued medicinal plant, and screen them for extensive plant diversity, a few plants have been medicinal properties. studied for their endophytic diversity and their The results of the present study reveal that potential to produce bioactive compounds. different plant parts of the test plant are Endohytic fungi occupy unique biological niches associated with different endophytic fungi as they grow in many divers environments [37]. belonging to diverse taxonomic groups. Several Endophytic fungi colonize almost all plant parts researchers working on different plants have and are environment sensitive for growth and also noted the association of endophytic fungi reproduction. Thus, it makes a sense to evaluate [38]. However, a wide variation has been

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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

J Pharm Chem Biol Sci, March - May 2018; 6(1):52-61