STUDY ON ENDOPHYTIC FUNGI PRESENT IN Catharanthus roseus AND Gloriosa superba

PROJECT REPORT submitted in partial fulfillment of the requirements for the award of the degree of BACHELOR OF TECHNOLOGY In BIOTECHNOLOGY By Anila Kulandai .K (10904021) Under the guidance of Mrs.S.Rupachandra Msc.,Mphil., (Lecturer, Department of Biotechnology)

DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF BIOENGINEERING FACULTY OF ENGINEERING AND TECHNOLOGY SRM UNIVERSITY KATTANKULATHUR 603 203 April 2008

CERTIFICATE
Certified that the project report entitled STUDY ON ENDOPHYTIC FUNGI PRESENT IN Catharanthus roseus AND Gloriosa superba submitted byANILA KULANDAI .K(10904021) is a record of project work done by her under my supervision. This project has not formed the basis for the award of any degree,diploma,associateship or fellowship.

INTERNAL GUIDE

HEAD OF THE DEPARTMENT

For the purpose of viva voce 1. 2.

DECLARATION
I do hereby declare that the project report entitled STUDY ON ENDOPHYTIC FUNGI PRESENT IN Catharanthus roseus AND Gloriosa superba is a record of original work carried out by me under the supervision of Mrs.S.Rupachandra

Msc.,Mphil.,, Lecturer, Department of Biotechnology, SRM University, Kattankulathur. This project has not been submitted earlier in part or full for the award of any degree, diploma,associateship or fellowship.

Kattankulathur Date

---------------------------

ACKNOWLEDGEMENT
I am extremely obliged to the almighty for his blessings in completing my project. I would like to thank all people who have helped and inspired me during my project work. Firstly,I would like to thank Dr. K. Ramasamy, M.Sc., M.S., Ph.D., Dean ,SRM UNIVERSITY, for his support and encouragement in my project work. I would also like to thank Dr Kantha Arunachalam Ph.D.,MASc., HOD Dept of Biotechnology, SRM UNIVERSITY,for her support in my project work. I would like to express my gratitude to Dr.R. S. David Paul Raj

Msc.,Ph.D.Assistant professor, SRM UNIVERSITY for his help, support and valuable hints for my project work. My heart felt thanks to my internal guide Mrs.S.Rupachandra Msc., Mphil., SRM UNIVERSITY who made herself available even through her very heavy travel work and teaching schedule. Firstly, I thank Dr.P.Augustine, HOD, Plant biotech department, Loyola College for allowing me to do the project in the Loyola College. His indispensable guidance and help played the major role in completing my project work in the most successful way.

Special thanks to Mr.Preetam, Loyola College for his support and valuable ideas for progress with our work.

I would also gratefully thank my friends Anil Kumar P, Praveen Kumar A, Elizabeth Amalarasi for their timely help and encouragement.

My sincere thanks to lab assistant Mr.Victor , Loyola College for his enduring support.

Lastly and most importantly, I wish to thank my parents and my brother for their continuous encouragement that was vital in completing the project work to them I dedicate this work

CONTENTS

S.NO CONTENTS 1 2 3 4 5 6 7 8 INTRODUCTION OBJECTIVES REVIEW OF LITERATURE MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY REFERENCES

PAGE NO 1 10 11 35 48 53 55 IV

LIST OF TABLES

TABLE NO 1 2

LIST OF TABLES
Extraction of endophytic fungus from Catharanthus roseus and Gloriosa superba Details of thin layer chromatography of crude extract of endophytic fungus from Catharanthus roseus

Details of thin layer chromatogarphy of crude extract of endophytic fungus from Gloriosa superba Rf values of crude extract of endophytic fungus from Catharanthus roseus And Gloriosa superba

5 6

Antimicrobial testing Enzyme activity of Catharanthus roseus and Gloriosa superba

7a,7b.

Analysis of pigmented compounds

LIST OF FIGURES

FIG NO FIG 1 FIG 2 FIG 3 FIG 4 FIG 5 FIG 6

LIST OF FIGURES Catharanthus roseus Gloriosa superba Isolation of endophytic fungi from Catharanthus roseus Isolation of endophytic fungi from Gloriosa superba Identification of endophytic fungi, hyphae structures Identification of endophytic fungi .presence of sickle shaped microconidia, macroconidia

Fig 7a, 7b, 7c. Subculture results of Catharanthus roseus Fig 8a, 8b,8c,8d Subculture results of Gloriosa superb Fig 9a ,9b Mass production of endophytic fungus from Catharanthus roseus ,Gloriosa superba Fig 11a.11b.11c. 11d Fig 12a.12b.12c. 12d.12e Fig 13a, 13b,13c Antimicrobial activity

Enzyme activity

Graph showing analysis of pigmented compounds, Catharanthus roseus

Fig 14a,14b.15c Graph showing analysis of pigmented compounds Gloriosa superb

LIST OF ABBREVIATIONS
1. PDA-Potato Dextrose Agar 2. SDA-Sabouraud Dextrose Agar 3. NA-Nutrient Agar 4. CMC-Carboxy Methyl Cellulose 5. CCA-Collidal Chitin Agar 6. PTA-Peptone Tween Agar 7. DMSO-Dimethyl Sulfoxide 8.MHA-Mueller Hinton Agar 9. KI- Potassium Iodide

ABSTRACT

Fungal endophytes (microfungi that live within healthy plant tissues) represent an unknown pro-portion of fungal diversity even though they are associated with all plants. While there is a growing appreciation of their ecological importance and human uses, little is known about their host specificity, geographic structure, or phylogenetic relationships. Thus we did a study on the endophytic fungi present in Catharanthus roseus and Gloriosa superba.The endophytic fungi, Fusarium sp were isolated from surface sterilized root of Catharanthus roseus and corm of Gloriosa superba.The fungal crude extract, obtained using ethyl acetate showed antimicrobial activity against the tested pathogens. The production of extra cellular enzyme such as amylase, cellulase, chitase, gelatinase and lipase by the isolated endophytic fungi on solid medium was also determined. The fungi showed amylase and gelatinase activity.The fungal crude extract was also subjected to thin layer chromatography and Rf value was calculated. Analysis of pigmented compounds was also done using three solvents such as methanol, water and ethyl acetate. The highest peaks obtained using visible spectrum shows the maximum absorption of pigmented compounds ranging from 400-700nm.

1.0.INTRODUCTION
Plants have been described as chemical factories that are capable of synthesizing unlimited numbers of highly complex and unusual chemical substances whose structures could escape the imagination of synthetic chemists forever. Fossil records date human use of plants as medicines at least to the Middle Paleolithic age some 60,000 years ago. Plants are capable of producing of an overwhelming variety of secondary metabolites, both in terms of complexity and quantity. The more we use medicinal herbs on a commercial scale the more important it is to ensure that they come from sustainable sources, so that these plants will continue to exist in wild places. Paclitaxel, the world's most widely used cancer drug, is derived from the bark of several species of yew trees and has defied all attempts of commercial synthesis. An average of six trees is needed for just a single dose. Its use has decimated wild yew populations across the world with 80 per cent of the trees in China's Yunnan province, once famous for its yew forests, destroyed within a three-year period, according to the report
A global survey carried out by more than 100 botanists and other scientists has found that more than 10,000 species of medicinal plants face extinction, including many used in prescription drugs that cannot be commercially synthesise .Because native plant habitats are destroyed almost daily, many medicinally valuable plants will be gone before scientists can even investigate them. Of the estimated 250,000 plant species on earth, only 2% have been thoroughly screened for chemicals with potential medicinal use. Approximately 119 pure chemical substances extracted from higher plants are used in medicine throughout the world (Farnsworth et al., 1985); these 119 plant-derived drugs are obtained from less than 90 species of plants (Farnsworth et al., 1985).

1.1.ENDOPHYTIC MICROORGANISM Endophytic microorganisms are those that inhabit the interior of plants, especially leaves, branches and stems showing no apparently harm to the host (azevedo, 1998).In the 70s endophytes were initially considered neutral, not causing benefits nor showing detriment to plants, but later on they studied that, it was possible to conclude that in many cases they had an important role in host protection against predators and pathogens.

Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships ranging from symbiotic to pathogenic. Bacon et al. give an inclusive and widely accepted definition of endophytes: microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects.

Endophytic microorganisms received considerable attention in last 20 years, when their capacity to protect their hosts against insectspest, pathogens and even domestic herbivores as sheep and cattle was recognized. In addition, it is worthy of note that some plants generating bioactive natural products have associated endophytes that produce the same natural products.

Mycorrhyzae and nitrogen fixing bacteria also live in an intimate relationship with their hosts and could be considered as endophytic microorganisms. However, Mycorrhyzae are distinguished from other root endophytes by the fact that they posses external structures as hyphae .Likewise, nitrogen fixing endophytic bacteria such as rhizobium which form external structures called modules are distinguished from other root endophytic bacteria .

Webber (1981) was probably the first researcher to report an example of plant protection giving by an endophytic fungi, in which the endophyte phomopsis oblonga protected elm trees against the beetle physocnenum brebilineum.

1.1.1.ENDOPHYTIC FUNGI

The most frequently isolated endophytes are the fungi. Dreyfuss and Chapela estimate there may be at least 1 million species of endophytic fungi alone. They grow within their plant hosts without causing apparent disease symptoms and growth in this habitat involves continual metabolic interaction between fungus and host and consequently should produce more secondary metabolites.

Currently, endophytic fungi are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. Of the approximately 300 000 higher plant species that exist on the earth, each individual plant, of the millions that exist here, is host to one or more endophytes. It seems obvious that they are a rich and reliable source of genetic diversity and may represent previously undescribed species. Finally, novel microbes (as defined at the morphological and/or molecular levels) often have associated with them novel natural products. This fact alone helps eliminate the problems of dereplication in compound discovery.

Tan and Zou recently reviewed the diversity of metabolites that have been isolated from endophytic fungi emphasizing their potential ecological role. These secondary metabolites of endophytes are synthesized via various metabolic pathways, e.g. polyketide, isoprenoid, or amino acid derivation, and belong to diverse structural groups, i.e. steroids, xanthones, phenols, isocoumarins, perylene derivatives, quinines, furandiones, terpenoids, depsipeptides, and cytochalasines. Some of them represent novel structural groups, for example the palmarumycins and benzopyranone

1.2. Catharanthus roseus

1.2.1.CLASSIFICATION: Catharanthus roseus (L.) G. Don Kingdom Division Class Family Genus Species Plantae Magnoliophyta Magnoliopsida Apocynaceae Catharanthus G. Don Catharanthus roseus (L.) G. Don

1.2.2.MORPHOLOGY It is an evergreen subshrub or herbaceous plant growing to 1 m tall. The leaves are oval to oblong, 2.59 cm long and 13.5 cm broad, glossy green, hairless, with a pale midrib and a short petiole 11.8 cm long; they are arranged in opposite pairs. The flowers are white to dark pink with a darker red centre, with a basal tube 2.5-3 cm long and a corolla 25 cm diameter with five petal-like lobes. The fruit is a pair of follicles 24 cm long and 3 mm broad. 1.2.3.COMMON NAMES English names include Cape Periwinkle, Rose Periwinkle, Rosy Periwinkle, and "Old-maid.and in India it is known as, Billaganneru, Ainskati, Nayantra, Rattanjot

Sada,Bahar,Sadaphul,Ushamanjairi. And in Tamil it is called as Nityakalyani.

1.2.4. HABITAT Madagascar periwinkle does best in poor, well-drained soils. Flowering will suffer if soils are too fertile. In frost free climates it develops a woody stem near the base and can get 2-3 ft (0.6-0.9 m) tall and spread out just as wide. Madagascar periwinkle should be watered moderately during the growing season, but it is relatively drought resistant once established. In fact, it is unusually drought resistant for an annual. Madagascar periwinkle is not at all tolerant of overwatering.

1.2.5.DISTRIBUTION Catharanthus roseus is native to Madagascar. abundantly neutralized in many regions particularly in arid coastal locations. It has escaped cultivation and naturalized in most of the tropical world where it often becomes a rampant weed. It is established in several areas in the southern U.S. Madagascar periwinkle is grown commercially for its medicinal uses in Australia, Africa, India and southern Europe.

1.2.6.PLANT CHEMISTRY Over 70 different indole alkaloids: including vincaleukoblastine (vinblastine) vincamine, alstonine, leurocristine, vinomine, 22- Oxovincaleukoblastine (vineristine), vintsine, leurosine ajmalicine, vinine, vinoxine, reserpine. There are two classes of active compound in vinca, alkaloids and tannins. The major alkaloid is known as vincamine.A closely related semi-synthetic derivativative of vincamine widely used as medicine is known as ethyl-apovincaminate or orvinpocetine .The other major alkaloids present are Vinristine, Vinblastine, Reserpine, Ibogaine and Yohimbine.

1.2.7. MEDICINAL PROPERTIES The anti cancer drug namely Vincristine and Vinblastine are produced from Periwinkle.Most commonly used in treatment of the following type of cancers:Lymphomas,Hodgkin's Disease,Breast cancer,Acute Lymphocytic Leukemia Soft tissue sarcomas,Multiple

Myeloma,Neuroblastoma.

Fig 1.Catharanthus roseus

The rootbark contains the alkaloid Alstonine which has been used traditionally for its calming effect and its ability to reduce blood pressure. The alkaloid vincamine has vasodilating, blood thinning, and memory-enhancing actions. It has been shown in double-blind studies to help alleviate a type of dementia known as vascular dementia, in which the arteries supplying blood to the brain develop atherosclerotic plaques.catharanthine, leurosine sulphate, lochnerine, tetrahydroalstonine, vindoline and vindolinine lower blood sugar levels (thus easing the symptoms of diabetes)Reserpine and serpentine are powerful tranquilizers. More recently, extracts from Madagascar Madagascar periwinkle have been shown to be effective in the treatment of various kinds of leukemia, skin cancer and lymph cancer. In congo the stem,leaves,and roots of catharanthus roseus are used against diabetes and diarrhea.On the coast of Tanzania, a decoction of the leaves is drink for stomach problems Extracts of Vinca have significant anticancer activity against numerouscell types. The greatest activity is seen against multi-drug resistant tumor types which suggest that there are compounds in Vinca rosea that are synergistic or additive with anti-neoplastic elements by inhibiting resistance to them.Taken as a daily supplement, it improves the blood supply to the brain, increases oxygen and glucose for the brain to use, helps prevent abnormal coagulation of blood, and it raises brain levels of the neurotransmitter serotonin. Recently, a laboratory study showed that chemicals in Catharanthus roseus may also prevent the growth of new blood vessels that support tumor growth. These drugs should only be used by healthcare professionals; however, because they may cause severe side effects.

1.3 Gloriosa superba

Gloriosa superba derives its name Gloriosa from the word gloriosus, which means handsome and superba from the word superb means splendid or majestic kind. Gloriosa superba is the national flower of Zimbabwe. It is also the state flower of Tamil Nadu state 1.3.1.CLASSIFICATION Kingdom Division Class Family Genus Species Plantae Magnoliophyta Liliopsida Liliacea Gloriosa superba

1.3.2.MORPHOLOGY Gloriosa superba is a striking tuberous climbing plant. Herbaceous tendril climber; rootstock tuberous, naked; stem 3 to 6 m long, sparingly branched; leaves sessile or nearly so, opposite or 3-nately whorled, tip tendrillar; flowers axillary, solitary, nearly 10 cm, at first greenish, becoming yellow and finally scarlet or red; capsules nearly 5 cm long. 1.3.3.COMMON NAMES In English it is commonly known as flame lily, fire lily, gloriosa lily, glory lily, superb lily, climbing lily, creeping lilya; in India as Harihari, Kalihari and in Tamil it is called as Sen-kandhal, Karthigai Poo.

1.3.4.HABITAT It is found in whole of the India up to the height of 6000 feet. he plant grows in sandyloam soil in the mixed deciduous forests in sunny positions. It is very tolerant of nutrient-

poor soils. It occurs in thickets, forest edges and boundaries of cultivated areas in warm countries up to a height of 2530 m. It is also widely grown as an ornamental plant in cool temperate countries under glass or in conservatories (Neuwinger, 1994; Inchem, 2004). 1.3.5.DISTRIBUTION G. superba is a native of tropical Africa and is now found growing naturally in many countries of tropical Asia including Bangladesh, India, Sri Lanka, Malaysia and Myanmar. n India, it is mainly found in Nasik, Ratnagiri, Savanthwadi (Maharastra); Uttara Kannada, Hassan, Chikmangalur, Coorg, Mysore (Karnataka); Cannanore, Palakkad, Trivandrum (Kerala); Tamil Nadu and Goa (CES, 2004). It is also planted outdoors in the southern United States. In cool temperate countries, it is treated as a greenhouse or conservatory plant. 1.3.6.PLANT CHEMISTRY Colchicine is the major compound isolated from the rhizomes0.2 to 0.3 %.. Seed contain high level of colchicines. Cornigerine, 3-demethyl-N-formyl-N-deacetyl-b-

lumicolchicine, 3-demethyl-g-lumicolchicine, 3-demethyl colchicines have been isolated from plant. b-sitosterol, its glucoside, a long chain fatty acid, b and g-lumiccolchicines from fresh tubers and luteolin, colchicines, N-formyldeacetylcolchicines and glucosides of 3-demethylcolchicine have been isolated from flowers. It also contains gloriosine 1.3.7.MEDICINAL PROPERTIES Colchicine is the major compound isolated from the rhizomes. They have abortifacient, antipyretic, anti-inflammatory and antileprotic properties. In Ayurveda, the tuber is anthelmintic and abortifacient. In Ayurveda and Yunani systems of medicine it is a reputed medicine. According to Ayurveda, tuber is pungent, bitter, acrid, heating, anthemirtic, laxative, alexiteric, abortifacient, and useful in ulcers, leprosy, piles, inflammations, abdominal pains and itching .

Fig 2.Gloriosa superba

Ayurveda uses its roots as abortifacient, acrid, alexiteric, anthelmintic, antipyretic, bitter, depurative, digestive, emetic, expectorant, gastrointestinal irritant, purgative,

rejuvenative, stomachic, thermogenic, tonic, in dealing debility, dyspepsia, flatulence, haemorrhoids, helminthiasis, inflammations, in promoting labour pain and expulsion of the placenta; stimulates uterine contractions (fresh plant juice and rhizome); antimalarial and laxative (tuber). Glory lily is among some of the modern medicine's most important plants actually facing local extinction. This plant has been a source of medicine right from the ancient time. Gloriosa superba is believed as most important herb that is exported, and collection of seeds and roots for the foreign market is causing a shortage of raw material for local drug industries in India. If endangered plants like G. superba are allowed to become damaged through excessive collection, a whole series of traditional medicines and plants which have been in use for thousands of years will be threatened. It is therefore need of the hour to come forward and rescue this important glorious herb. Hence the present study aims to study the endophytic fungus from the important medicinal plants Catharanthus roseus and Gloriosa superba well known for its anticancer activities.

2.0.OBJECTIVES

This project work was carried out with the following objectives

Isolation and identification of endophytic fungi

To determine the production of extracellular enzyme such as amylase,cellulase, chitase,gelatinase and lipase by the isolated endophytic fungi on solid medium

To test the antimicrobial activity of the fungal crude extract

Separation of compounds present in fungal crude extract by thin layer chromatography Analysis of pigmented compounds

3.0. REVIEW OF LITERATURE

Nature has been a source of several medicines for treating various types of diseases in humans and animals for many years. Natural products are naturally derived metabolites and/or by products from microorganisms, plants, or animals. These products still play a major role in drug treatment either as the drug, or as a forebear in the synthesis or design of the agent. Even with untold centuries of human experience behind us, and a movement into a modern era of chemistry and automation, it is still evident that natural product-based compounds have had an immense impact on modern medicine. Contributing to this world-wide attention towards formulations based on natural products, are their low or absent toxicity, their complete biodegradability, their availability from renewable sources, and, in most cases, their low cost when compared with those of compounds obtained by total chemical synthesis.

According to Barbara Schulz et al., (Sep 2002), In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. Since natural products are adapted to a specific function in nature, the search for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit such a biotope.

3.1.ENDOPHYTIC FUNGI

The endophytes of medicinal plants provide a good source for compounds of biological activity and endophytes are an untapped reservoir of potentially novel, effective drugs (Stierle et al., 1995).

A widely accepted definition of endophytes is that they are microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects (Bacon & White, 2000). 3As defined by Webster's dictionary, an endophyte is a "plant living within another plant." Endophytes occupy unique ecological niches and can influence the distribution, ecology, physiology and biochemistry of plants (Sridhar and Raviraja 1995) Endophytic fungi have been reported from diverse plants including grasses, mosses, ferns, conifers and angiosperms (Clay 1989) Microbial endophytes are associated with a large number of plants. (Kobayashi DY et al., 2000) Endophytes are ubiquitous microbial associates found in most species of plants (Clay 2004) and show an astonishing diversity (A.E Arnold et al 2000).,Given this diversity, there must be a role for these hidden microbes in structuring natural communities and affecting ecosystem functions (K.Clay et al., 2001) The most frequently isolated endophytes are fungi (Bacon & White, 2000), but bacteria also form endophytic relationships. The vast majority of plants have not been studied for the endophytes that may inhabit them. Endophytes are likely to be a rich and reliable source of genetic diversity and biological novelty (Bacon & White, 2000), and experience has shown that novel endophytic microbes usually produce novel natural products (Strobel, 2002, 2003). According to Barbara Schulz et al., (Sep 2002),In the course of the last 12 years, [similar] 6500 endophytic fungi from herbaceous plants and trees, screened them for biological activities, and have isolated and determined the structures of the biologically active compounds. Correlations were found between biological activity and biotope, e.g. a higher proportion of the fungal endophytes, in contrast to the soil isolates, inhibited at least one of the test organisms for antialgal and herbicidal activities. The substances isolated originated from different biosynthetic pathways: isoprenoid, polyketide, amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols, isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines.

According to Barbara Schulz et al., (Sep 2002) the fungal endophytes possess the exoenzymes necessary to colonize their hosts and they grow well in the apoplastic washing fluid of the host. The fungal endophyteplant host interaction is characterized by a finely tuned equilibrium between fungal virulence and plant defence. If this balance is disturbed by either a decrease in plant defence or an increase in fungal virulence, disease develops. Not only must the endophyte synthesize metabolites to compete first with epiphytes and then with pathogens in order to colonize the host, but presumably also to regulate metabolism of the host in their delicately balanced association. The utilization of a biotope such as that of the fungal endophyte is one aspect of intelligent screening; another very important one is the taxonomy of the fungus in order to avoid redundant structural isolations. It is not a random walk through a random forest. Many groups of fungi in different biotopes are waiting to be exploited. According to Orlando Petrini et al., (2006) Endophytic fungi are a taxonomically and ecologically heterogenous group of organisms, mainly belonging to the Ascomycotina and Deuteromycotina. The isolation methods affect the species composition of the endophyte assemblage in a given host. The number of endophyte taxa isolated from a host species is usually large; however, only few, normally host specific species or strains are dominant. Endophyte assemblages are specific at the host species level, but species composition and frequencies are significantly affected by site-specific conditions. Moreover, the relative importance and number of endophytic species vary among individuals within sites.

According to A.Demain (1981), microorganisms have long served mankind by virtue of the myriad of the enzymes and secondary compounds that they produce According to A.T.Bull (2004) only a relatively small number of microbes are used directly in industrial applications (e.g. cheese, wine and beer making), in environmental clean-up operations and in the biological control of pests and pathogens. It seems that we have by no means exhausted the world of its hidden microbes, and a much more comprehensive search of the Earth's various niches might yet reveal novel microbes which have direct usefulness to human societies. These uses could be either of the microbes itself, or one or more of its natural products. The diversity of microbial life is enormous and the niches in which microbes live are truly amazing, ranging from deep ocean sediments to the Earth's thermal pools

One specialized and unique biological niche that supports the growth of microbes is the intracellular space between cells of higher plants. Furthermore, because so little work on these endophytes has been done, it is suspected that untold numbers of novel fungal and bacterial genera exist as plant-associated microbes and their diversity might parallel that of the higher plants It turns out that each plant supports a suite of microorganisms known as endophytes G.Strobel et al., (2003). According to C.W.Bacon et al., (2000) these microorganisms do not cause overt symptoms on the plants in which they live Such a specific search is governed by the fact that some endophytes could have coevolved with their respective higher plant and, as a result, already exist compatibly with a higher life-form. Thus, a concerted search for novel endophytic microbes have begun, with the prospect that they could produce novel bioactive products as well as possess processes that might prove useful. It is obvious that any discovery of novel microbes will have implications in virtually all of the standard processes of industrial microbiology because scale-up fermentation of the microbe will be necessary. Several reviews have discussed the products of endophytic microbes and their promise for use in medicine, agriculture and industry. (R.X Tan et al., 2001)

3.2.IMPORTANCE OF ENDOPHYTIC FUNGI

The nature and biological role of endophytic fungi with their plant host is variable. Endophytic fungi are known to have mutualistic relations to their hosts, often protecting plants against herbivory, insect attack or tissue invading pathogens (1-3); Defining the number of fungi on earth has always been a point of discussion (Fries 1825, Bisby & Ainsworth 1943), but has gained prominence in scientific literature towards the latter part of the twentieth century. While this exercise might on the surface appear to be of peripheral importance, it is fundamental to understanding and protecting the worlds biodiversity. Thus, a number of studies have in recent years focused on enumerating the worlds fungal biodiversity (Pirozynski 1972, Pascoe 1990,

Hawksworth 1991, Dreyfuss & Chapela 1994, Rossman 1994, Hyde 1996, Hyde et al. 1997, Hawksworth 1998, Frhlich & Hyde 1999, Hawksworth 2001, 2004). They have provided the foundation for studies aimed at a better understanding of fungal biodiversity worldwide, and results have been used to motivate for bioconservation and fungal biodiversity studies.

It is widely recognised that fungi have been relatively poorly collected and studied from most countries, regions and habitats. This is at least in comparison to plants and larger animals that are considerably easier to collect and identify than fungi. One might then have expected that the predicted numbers of fungi on earth would have been considerably greater than the 1.5 M suggested by Hawksworth (1991). Clearly different authors that have considered the likely total number of fungi have had differing views of an appropriate answer, but the discrepancy between the results of most of these studies is not particularly great. It is currently accepted that the 1.5 M estimate is highly conservative. The 100 000 that have thus far been described, therefore represents no more than 7 % of the estimated total.

3.2.1. PRODUCTION OF ANTICANCER DRUG BY ENDOPHYTIC FUNGI

3.2.2.TAXOL
Taxol is a potent antimitotic agent with excellent activity against a range of cancers. It was first isolated from the bark of yew trees and approved by FDA (Food and Drug Administration) in 1992 (Wani et al., 1971; Kohler and Goldspiel, 1994). The supply of Taxol has been limited since the discovery of this natural product, and, with increasing applications in chemotherapy, the availability and cost of the drug will remain important issues. Since the first Taxol-producing fungus Taxomyces andreanae was isolated in 1993 (Stierle et al., 1993), there have been a few reports on the isolation of Taxol-producing endophytic fungi (Strobel et al., 1996; Li et al., 1996; Wang et al., 2000), demonstrating that organisms other than Taxus sp. could produce Taxol. Thus, fermentation processes using Taxol-producing microorganisms may be an alternative

promising way to produce Taxol. BT2, a newly isolated endophytic fungus from Taxus chinensis var. mairei, was observed to produce Taxol. Besides Taxol, a potent anticancer drug, BT2 could also yield taxane baccatin III, which was an important intermediate for Taxol and semi-synthesis of Taxol in industry. (B.H. Guo, Y.C. Wang et al.) Although the amount of Taxol produced by most endophytic fungi associated with taxus trees is relatively small when compared with that of the trees, the short generation time and high growth rate of fungi make it worth while to continue our investigation of these species.

3.2.3.VINBLASTINE AND VINCRISTINE

Vinblastine and vincristine, tubulin binding indole alkaloids, originally isolated from the Catharanthus roseus commonly known as sadabahar in India are regarded as important anticancer agents. Ten endophytic fungi were isolated from C. roseus, out of which one fungus AA-CRL-6 was found to produce vinblastine and vincristine. Fungal vinblastine was isolated and purified by thin layer chromatography and high performance liquid chromatography and characterized by ultra violet absorption and mass spectroscopy. Cytotoxicity towards cancer cell line HL-60 (leukemia), A431 (epidermal carcinoma) and MCF-7 (breast cancer) was determined by MTT assay. Further characterizations of vinblastine and vincristine are in progress.

According to Su Valley, et al (2000) Catharanthus roseus reported for the first time from Catharanthus roseus (L.) G. Don stem phloem isolated from Fusarium oxysporum Fusarium oxysporu m, a plant for the endophytic fungi, and with TLC and HPLC bacteria strains cultured 97 CG3 of an analysis of preliminary results show that the fungus can produce anticancer drug vincristine components.

An endophytic fungus, Fusarium oxysparum [WTBZ (97CG3) was isolated from the phloem (inner bark) of Catharanthus roseus (L.) G. Don for the first time. Metabolic products of cultured 97CG3 hav e been analysed by TLC and HPLC. It was shown that the fungus can produce vincr istine which has an anticancer activity.

3.2.4.PRODUCTION OF ANTIBIOTICS

One of the most promising endophytic micro-organisms to be isolated would be an actinomycete, or specifically a streptomycete, since these organisms often produce antibiotics. Recently, one of the first antibiotic-producing endophytic streptomyctes was isolated from a medicinal plant (snakevine, Kennedia nigriscans) used by Aboriginal Australians to treat and dress bleeding wounds (Castillo et al., 2002). The snakevine consistently yielded a yellowish-orange culture of Streptomyces sp. which was subsequently demonstrated to produce a unique family of functionalized peptide antibiotics, the munumbicins (Castillo et al., 2002). Other streptomycetes may be found as endophytes in other higher plants, and ethnobotanical or other approaches could be used to find them.

3.2.5.ENDOPHYTES OFFER NATURAL PROTECTION TO HOST According to Walter Maccheroni Jr et al.,(2000) ,the control of pests and diseases affecting cultivable plants is made mainly by the use of chemical compounds. The use of agrochemicals to control insects and plant pathogens represents, however, a high risk to field workers, consumers and environment. They are responsible, for eliminating important species of insects that control other pests and microorganisms that are performing a critical role in the environment, inhibiting growth and multiplication of useful microbes. The natural and biological control is an alternative way that may contribute to reduce or even eliminate the use of chemical products in agriculture. The natural and biological control of agricultural pests gained more attention during the past decades. Latin America, having vast agricultural areas, most of them located in the tropics, is severely affected by pests and diseases. Ways to control them are important to reduce costs and protect the environment. One group of useful microbes that are affected by man modifications is the endophytes. Endophytic microorganisms are those that inhabit at least for one part of their life cycle, the interior of plants. Although already described during the XIX Century, they did not received considerable

attention. Only in the last twenty years they were recognized with capacity to protect the host-plants against insect-pests and diseases. Also they could confer to plants, other important characteristics like greater resistance to some stress conditions as, for instance, hydric stress. On the other hand, they can produce phytohormones and other substances of biotechnological interest as enzymes and pharmaceutical drugs including antibiotics and antitumour drugs.In the early 80s researchers started to show that endophytic fungi and bacteria could give to their host-plants, protection against insects and phytopathogenic microorganisms.

Fungal endophytes may induce plant resistance responses by means of structural/morphological and physiological or biochemical changes in the plant. Biochemical responses include the synthesis of defence-related chemicals, such as phenolic compounds, against pest and pathogens (Ramamoorthy et al., 2001). Phenolic compounds may occur as constitutive molecules present in healthy plants or as substances synthesized by plants in response to bacterial or fungal infection (Mansfield, 1983), and are well recognized as plant resistance factors against nematodes (Giebel, 1974; 1982; Bajaj et al., 1983; Peng and Moens, 2004; Zinov'eva et al., 2004; Pegard et al., 2005). Banana cultivars resistant to R. similis were reported to contain higher amounts of constitutive phenolics compared to susceptible cultivars (Fogain and Gowen, 1996; Valette et al., 1998; Collingborn et al., 2000; Dochez, 2004). Schulz et al. (1999) also demonstrated that higher amounts of phenolic metabolites were produced in barley (Hordeum vulgare L.) inoculated with an endophytic Fusarium sp. Non-clavicipitaceous endophytes cause discrete and symptomless infections in the aerial tissues of plants and survive within these tissues at least for part of their life cycle. These endophytes are taxonomically restricted to the ascomycetes and their anamorphs and span several ecological functional groups such as mutualists (Redman et al., 2002; Arnold et al., 2003), commensals (Deckert et al., 2001) and latent pathogens (Sinclair and Cerkauskas, 1996; Photita et al., 2004, 2005; Gonthier et al., 2006; Suryanarayanan and Murali, 2006).Recent studies have shown that the leaves of tropical plants are densely colonized by endophytes although their species richness varies with the type of the tropical plant communities (Arnold et al., 2003; Suryanarayanan et al.,There are a few cases of non-clavicipitaceous endophytes functioning as

insect antagonists and offering natural protection to their hosts (Webber, 1981; Carroll, 1991; Wilson, 2000).

Several reports correlated decrease in attack frequency of insect-pests and presence of endophytic fungi. Mainly in grasses, certain genera of endophytic fungi were shown to be able to control insect-pests. On the other hand they can be also responsible for intoxication, weight loss, decrease of milk production and other effects in cattle and other herbivores fed on these plants.nowadays, many examples of reduced host-damage provoked by insect attack, due to the presence of endophytes, are known. The examples are mainly derived from temperate hosts as grasses and woody plants.The mechanisms of insect control displayed by endophytic fungi are quite well studied. Some were based in the capacity to render the plant unpalatable to several types of pests as grasshoppers, aphids, beetles and other insects. Endophytes may also produce several toxins as ergot alkaloids and neurotoxines, which cause death, decreased of development rate and other effects against insects.The expression of insect resistance by the host-plants may be affected by several factors as soil fertility, hydric stress, temperature, soil pH, plant and endophyte genotypes among others. So, there is a complex interaction among plant-endophyte-pests related to the environment. Walter Maccheroni Jr et al.,(2000)

Some endophytes are entomopathogenic fungi, that is, are able to infect insects and kill them.Other indirect effect of endophytes is the control of ectoparasites in domestic animals. Insects, as for instance the horn fly, a cattle ectoparasite, feed on manure derived from animals fed with grasses infected with endophytes are killed or have reduced development.

According to Walter Maccheroni Jr et al.,(2000) ,more recently, Recombinant DNA techniques, commonly known as Genetic Engineering, have been applied as a tool to improve endophytic microorganisms, aiming to the introduction of new characteristics of agronomic interests as biological control of pests. Genetic modified endophytic bacteria able to secrete toxin against insect-pests, inoculated inside plants protect them against attacks of target insects. There are several advantages of using endophytic inoculated plants with genetic engineered microorganisms when compared to the use of transgenic plants or chemical products to control pests. Plant inoculation of valuable strains of endophytic microbes used for biological

control is starting to be used with success in agriculture. These techniques are expected to become popular as it is nowadays the inoculation of nitrogen fixing bacteria in leguminosae plants, already largely used in agriculture.Most of the research with endophytes has been carried out using plants from temperate regions. The data from tropical regions are scarce but preliminary results are shown that tropical plant hosts contain a great diversity of endophytic microorganisms, many of them not yet classified and possibly belonging to new genera and species. Potentially they are of biotechnological importance. New pharmaceutical compounds, secondary metabolites, agents for biological control of insect-pests and plant diseases besides other useful characteristics could be found, by further exploration of tropical endophytic microorganisms. This is a broad field of investigation that is almost entirely open to new findings. It is expected that new ways of interactions between endophytes and their hosts will be found. It is expected also that new drugs of biotechnological importance, produced by endophytes will be described, with the increasing of studies focusing these tropical microorganisms.

While the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens (Bacon & White, 2000) Plants growing in areas of great biodiversity have the prospect of harbouring endophytes with great biodiversity (Strobel, 2003)
The effects of some microbes on plant fitness are well understood, but endophytic fungi in grasses have received less attention than mycorrhizae, nitrogen-fixing bacteria and microbial pathogens, presumably because their function is not always clear. The first records of endophytic fungi in grasses date back to the late 19th century, but the symbiosis attained more recognition from the 1970s onwards, following the discovery of the toxicity to livestock of endophyte-produced alkaloids. The grass endophytes that cause such effects belong to the family Clavicipitaceae (Ascomycota) and have a sexual and an asexual life cycle (C.L. Schardl. 2004)

Endophytes are potential producers of novel antimicrobial secondary metabolites. Endophytes might therefore prove interesting subjects for the study of coordinated control of plant growth regulators, anti-fungal and anti-herbivory metabolites. Endophytic fungi are of biotechnological interest due to their potential use as genetic vectors as a source of secondary metabolites and as biological control agents. . A critical review of the literature suggests that the beneficial action of these endophytes is based on direct antimicrobial and insecticidal activity due to alkaloid production.

3.2.6. ANTI-MICROBIAL STUDY

Anti-microbial agents are undeniably one of the most important therapeutic discoveries of the 20th century. However, with the antibiotic era barely five decades old, mankind is now faced with the global problem of emerging resistance in virtually all pathogens (Peterson and Dalhoff, 2004). Surveys have revealed that almost no group of antibiotics has been introduced to which resistance had not been observed (Eloff, 2000). This is indeed quite alarming when considering that in 1990, out of the 39.5 million of death in the developing world, 9.2 million were estimated to have been caused by infectious and parasitic diseases, and that 98% of death in children in developing countries resulted mostly from infectious diseases (Murray and Lopez, 1997). Bacterial resistance is beyond doubt the consequence of years of widespread indiscriminate use, incessant misuse and abuse of antibiotics (Peterson and Dalhoff, 2004). In human medicine alone, the US Centre for Disease Control and Prevention estimates that approximately one-third of the 150 million prescriptions for antibiotics written each year were not needed. Because of the limited life span of antibiotics, it is of utmost importance to find appropriate solutions to impede, or perhaps even reduce, the development of drug resistance associated with many microbial species (Martini and Eloff, 1998).Since time immemorial, medicinal plants had been a dependable source of therapeutics for the treatment of various ailments (Hoareau and Da Silve, 1999) but since the advent of the use of fermentation-based antibiotics work on anti-microbial agents from plants sources has been greatly overshadowed (Mitscher et al., 1987). The rapid propagation in antibiotic resistance and the increasing interest in natural products, however, have placed medicinal plants back in the front lights as a reliable source for the discovery of active anti-microbial agents and possibly even novel classes of antibiotics (Shultes, 1992). Plants are complex chemical storehouses of undiscovered biodynamic compounds with unrealized

potential for use in modern medicine (Plotkin, 1988). It has long been established that naturally occurring substances in plants have anti-bacterial and anti-fungal activities

Nearly 75 % of the world's antibiotics are produced by the Streptomyces spp. (Arai, 1976; Goodfellow et al., 1988; Demain, 1981). These bacteria have, for the most part, been obtained from soil samples in various parts of the world (Waksman, 1967). The advent of new strains of drug-resistant infectious bacteria, new agents of disease, and old agents of disease whose menace has never been truly contained, e.g. malaria, all emphasize the need for a continued search for novel antibiotics (NIH, 2001). In the meantime, many large pharmaceutical companies have given up or reduced discovery work for control of infectious diseases. It is apparent that plants can serve as a reservoir of endophytic streptomycetes, and evidence thus far indicates that the antibiotics from these sources are novel, interesting and hold pharmaceutical and agricultural promise (Castillo et al., 2002, 2003; Kunoh, 2002). This microbiological reserve residing in the rainforests of the world has never been tapped for its antibiotic potential (Strobel, 2002). The coronamycins provide another exam example of the potential that endophytes hold for antibiotic discovery.
Plants have long provided mankind with a source of medicinal agents, with natural products once serving as source of all drugs . Though synthetic chemical also have long been used as active agents in reducing the incidence of plants, animals and humans diseases, they are costly, have potentially harmful effect on the environment and may induce pathogen resistance. Thus, biological controls or the use of microorganisms or their secretions to prevent diseases offer an attractive alternative or supplement to disease management without the negative impact of chemical control. In natural product discovery programs, typical procedures included isolating microorganisms from samples, growing at various temperatures in a variety of selective or nonselective media and testing the extracts in a spectrum of targeted screens for activity for potential industrial or pharmaceutical applications. For a successful fungal screening, a varied and novel repertoire of either well-known or unexplored fungi is desirable. The most promising trend in isolating new fungi is the move towards investigating novel endophytes, with the idea that unusual endophytes may produce untapped natural products.

3.3. Endophytic fungi Fusarium

Fusarium is common in the soil and is an important plant pathogen. Fusarium solani is the most common species but Fusarium oxysporum,Fusarium moniforme and Fusarium proliferatum are also pathogens,in keratomycoses , Fusarium oxysporum and Fusarium solani are the most common species. Fusarium like Aspergillus ,has hyaline septate,branching hyphae,with diameters of 3 to 8 m. Fusarium grows rapidly on many types of media,and the mycelial colonies are various colours.Fusarium is identified microscopically by its sickle shaped or banana shaped macroconidia and clusters of fusiform micro conidia.

Valette et al., 1998; Collingborn et al., 2000; Dochez, 2004). Schulz et al. (1999) also demonstrated that higher amounts of phenolic metabolites were produced in barley (Hordeum vulgare L.) inoculated with an endophytic Fusarium sp.

According to J.Hertage, Emlyn and Glyn Vaughn in the book INTRODUCTORY MICROBIOLOGY, The most common method used to visualize fungi is to use lacto phenol cotton blue. Light green is an alternative stain to cotton blue that can be used to stain fungal structure. However it does not stain fungi so intensely and it tends to fade with time. It is therefore not suitable for preparing mounts that are to be kept for long periods. Cotton blue stains hyphae blue, particularly young hyphae, and the intensity of staining increases with time. Old mounts can become over stained .Lacto phenol is prepared by dissolving phenol crystals, lactic acid and glycerol in distilled water. When it is used on its own as a mountant, lacto phenol can be employed to observe the natural colours of fungal structures. To visualize fungi microscopically, a portion of the fungal growth is removed from the culture using a sterile needle or wire hook. The sample is then placed in a drop of alcohol on the surface of a clean microscope slide. The alcohol is used to drive out air that becomes trapped between the fungal structures. A drop of lacto phenol cotton blue is then added to the preparation. Care should be taken no to add to

much mountant to the preparation and if this accidentally happens, then the excess should be gently removed using blotting paper. The edge of a cover slip is then placed onto the drop of mountant carefully and the cover slip is lowered over the preparations, avoiding the introduction of air bubbles. Some authorities recommend that the preparation is then gently heated to enhance staining, but this is generally unnecessary and may cause over staining unless extreme cautions is applied. Mounts prepared in lacto phenol can be kept for several weeks or months. According to Dexter H.Howard in the book PATHOGENIC FUNGI IN HUMANS AND ANIMALS, many Fusarium species have characteristic colours at least in uncontaminated isolates growing on PDA.many begin as pale colonies, and F.oxysporum, F.solani, F.verticillioides and F.proliferatum may remain quite pale in some isolates. As a multimembered species complex F.solani is especially variable .the chestnut red brown colours that form in some of its subtypes need to be distinguished fro the vinaceous (red-wine coloured) and violaceous (purplish) colours that are characteristics of F.oxysporum and members of F.subgenus Liseola, as well as the carmine (vivid Lipstick red) pigments that forming F.chlamydosporum and a variety of Fusarium species not known from human infection. it cannot be overstressed that these characters are highly variable when different growth media are used and is frequently true in the

identification of non-Onygenalean molds.

According to John F.Leslie ,Brett A.Summerll in the book THE FUSARIUM LABORATORY MANUAL ,conidia formed on PDA are not as consistent in either size or shape as those formed on CLA and SNA medium, and thus are much less reliable for use for identification purposes.however,colony morphology pigmentation and growth rate of cultures of most Fusarium species on PDA(Potato Dextrose Agar)are reasonable consistent if the medium is prepared in a consistent manner, if the cultures are initiated from standard inocula and incubated under standard condition. These colony characteristics often are useful secondary criteria for identification.PDA is used by some researchers for the isolation of Fusarium species. And water agar medium is recommended for germinating conidia used to initiate Fusarium cultures. As hyphal growth is sparse on this medium, it is suitable for growing cultures from which individual hyphal tips are taken to initiate new colonies. In some instance, the sparse growth on water agar medium facilitiates the isolation of Fusarium species from plant materials, particularly roots.

3.3.1.Enzyme activity of Fusarium sp.

Endophytes usually produce the enzymes necessary for the colonization of plant tissues. Substrate utilization studies and isozyme analysis have demonstrated that most endophytes are able to utilize most plant cell components. The production of growth promoting factors and of metabolites useful in the pharmaceutical and agricultural industry is widespread among endophytic fungi.Fungal enzymes are gaining importance in agriculture, industry and human health, as they are often more stable (at high temperature and extreme pH) than the enzymes derived from plants and animals. Fungal enzymes are used in manufacturing food, beverages, confectioneries, textile and leather and help simplifying the processing of raw materials.

Wood-inhabiting marine fungi serve as a potential source of exoenzymes (e.g. Rohrmann and Molitoris, 1992; Raghukumar et al., 1994, 1999; Pointing et al., 1998, 1999).

Metabolites exploited in pharmaceutical and agricultural industries are widespread among the endophytic fungi (Petrini et al., 1992 ). For example, the anticancer drug, taxol, which was previously thought to be produced only by the plant genus Taxus (yew) has been found in many genera of endophytic fungi (Alternaria, Fusarium, Monochaetia, Pestalotia, Pestalotiopsis, Pithomyces and Taxomyces) ( Strobel et al., 1996 ). Marine mangrove fungi have proven to be an important source of new bioactive compounds ( Lin et al., 2001 ) and enzymes (e.g . Grant et al., 1996; Pointing et al., 1998; Pointing and Hyde, 2000). Endophytes might involve in decomposition when the tissue becomes senescent or die. Hence, they elaborate the enzymes necessary for degradation of lignocellulosic materials. Kumaresan and Suryanarayanan (2002) studied the extracellular enzyme production by the foliar endophytic fungi of Rhizophora apiculata and demonstrated their involvement in litter degradation after senescence.

According to (Vitale. S et al., 2005) Pectolytic enzymes were produced by Fusarium sambucinum in vitro and during colonization of potato tubers.

Fusarium oxysporum F3 produced N-acetyl---glucosaminidase when grown on wheat bran and chitin as carbon sources in solid-state fermentation. Two isozymes of N-acetyl--glucosaminidase, called N-acetyl---glucosaminidases I and II, were isolated from the culture filtrate of F. oxysporum F3. (Konstantinos Gkargkas et al., 2003)

According to G.L.Maria et al., (2005) Fusarium species lack chitinase activity when colloidal chitin agar (Cinagappa and Lockwood, 1962) medium was used.

According to Guickshank et al., (1975) NA medium amended with 4% gelatin was used to test the gelatinase activity.

According to Collins and Lyne, 1980 NA (Nutrient Agar) medium supplemented with1%soluble starch was used to test amylase activity. And 1%iodine in 2% potassium iodide was used as staining solution. According to G.L.Maria et al., (2005) Fusarium species showed amylase activity on GYP (Glucose Yeast Extract Peptone) medium.

According to Yeoh et al., (1973) medium containing 2g- NaNO3, 1g -K2HPO4, 0.05g MgSO4.7H2O, 0.05g Kcl, 0.01g FeSO4.7H20, 16g Agar, amended with 1%CMC-Na salt per Litre is used to test cellulase activity. 0.1ml Hcl +5ml1%iodine in 2%KI was used as staining solutions.

According to Villan and Blasins (1974) PIA medium was used to test the lipase activity. The opalescent zone formed around the colony confirmed the lipase activity.

According to Larran .s et al., (2002) the surface sterilization technique most efficient for eliminating epiphytes.

3.4.Catharanthus roseus

In the Handbook of medicinal plants By-zohara yaniv,uriel bachrach ,Antitumor medicines are quite common in folk medicine.alreadyin the papyrus ebers (1550 bc).garlic is praised as effective against cancer.the antitumor activity of colchicines from Colchicum autumnale L. one of the oldest documented medicinal plants was discovered when the effective mechanism of the substance on the acute gout attack was detected.the arabs used the plant long before Linnaeus named it after Colchis at the black sea.in the 17th century European medicine widely embraced it.meadow saffron was used both to treat gout as well as rheumatic complaints.these nuclear indications and the high toxicity induced nothnagel and rossback to advise against the application of colchicines even upto around 1900.neverthless ,until today it has remained the only specific therapy against acute gout attack. The most significant discovery in the field of antitumor substances deriving from folk medicine is owed to a coincidence.at the end of 1950s,two research groups independent of each other the collip laboratories(university of wester Ontario)and the lily research laboratories made a major finding in their serch for antidiabetes.while studying the hypoglycemic properties of catharanthus roseus ,a small shrub indigenous to Madagascar and now widespread in all tropical regions of the world,the research workers lostmany rats which had been given as extract of the leaves due to Psudomonas infectin.further investigation revealed that their death was due to a sever decline in the number of their lymphocytes.this discovery finally directed the reseachers attention toward the antitumor activities of catharanthus alkaloids.in the beginning four indole alkaloids,which were found to inhbit cell growth ,were discovered :vinblastine

(vincaleucoblastine),

vincristine(leurocristine)vinleurosine(leurosine)and

vinrosidin(leurosidine).vincristine is now used in Single drug therapy for acute leukemia,in combination coination chemotherapy it is indicated for the treatment of hodgkins disease,non-

hodgkins

lymphoma,breast

cancer,uterine

and

cervical

cancer,smll

cell

bronchial

cancer,rhabdomy osarcoma and various sarcomas .vinblastine is indicated in the treatment of Hodgkin.s disease, non-hodgkins lymphoma,advanced testicucalar cancer,breast and ovary epithelioma,Kaposis sarcoma,choriocarcinomas and in some cases of histiocytosis.a semisynthetic derivative ,vindesine,which can be prepared from vinblastine ,is indicated in the treatment of acute lymphoblastic leukemia and refactory lymphomoas,certain solid tumors are also indications :breast ,esophagus,upper respiratory and digestive tract and broncho pulmonary cancer.another semisynthetic derivative ,vinorelline ,is currently used in cases of mitastatic breast cancer and bronchial cancer.the1st report on cantharanthus roseus dates more than 300 yrs . etienne de fla court-bizet in his book historie de la graunde isle Madagascar,published in 1661,describes a plant that could well have been the famous Madagascar periwinkle. Madagascar periwinkle is included in Madagascar,s pharmacpoie of crude drugs.the plant up until today is very common in folk medicine of many tropical countries.in Guyana ,a decoction is drunk against heart disease.peekolt reports from brazil that an nfusion oof the leaves is used against internal bleeding and scurvy,as a mouthwash against toothache and for cleaning and healing chronic wounds.in togo a watey solution is used to trea dysmenorrhoea and indeed ,antibiotic and antviral activities were found in catharanthus alkaloids.In congo the stem,leaves,and roots of catharanthus roseus are used against diabetes and diarrhea.On the coast of Tanzania, a decoction of the leaves is drink for stomach problems.in brazil,the plant is till used under the name Boa noite as an antidiabetics.also in the Philippines,Jamaica, South Africa,India and Australia,an infusion of leaves is drunk against diabetes.other indications in African traditional medicine are against diarrhoe,abnominal painhypertension and as an antifussive ,diuretic,purgative and antihelmintic. It is assumed that plants used in folk medicine are a rich source of possible substance with antitumor effect .J..Hartwell enumerate more than300 species that could be promising in this regard.many scientists have shown in their work that plants can actually be a good source of antineoplastic substances.

catharanthus roseus is yet to be fully studied .new substances suh as the flavonol glycoside syringetin are still being discovered.in a dual culture of Tricoderma harzianum and catharanthus roseus,a noval antimicrobial compound ,trichosetic,with a remarkable activity against grampositive bacteria was produced.the antidiabetic property of the Madagascar periwinkle are also again the focus of research.with new rapid metabolite screening method,the production of indole alkaloids by plant cells and tissue becomes more proble for future.

The Rf value of indole alkaloids present in Catharanthus roseus using solvent ethyl acetate: ethanol: ammonium solution (3:1:1) was found to be (ajmaline-0.8, catharanthine-0.7, serpentine-0.02-0.05, vindoline 0.72 and yohombine-.61) (methods in molecular biology; john M.Walker).

3.5.Gloriosa superba

According to Y.P.S BAJAJ in the book Medicinal And Aromatic Plants,Gloriosa superba L also known as the flame lily has a wide distribution in tropical and subtropical areas. The plant has numerous uses as remedies and potions to the local populations of both Africa and Asia. Clewer et al .,(1915) found that Gloriosa superba contained the alkaloid colchicine.preparations of colchicine have been used to cure acute gout.colchine is known to inhibit mitosis, interfere with the orientation of fibrils ,induce polyploidy ,and has been used in the treatment of cancer. Since the discovery of colchicine on gloriosa, a number of researchers have proposed that gloriosa could serve as a commercial source of colchine. Bellet and Gaignault (1985) compared the relative colchicine content of the genera colchicum (the traditional source of colchicine) and Gloriosa. On a dry mass basis, Colchium yielded 0.62%colchicine and 0.39%colchicoside, while Gloriosa yielded 0.9%and 0.82%respectively.this supports the argument that Gloriosa can be commercially viable source of cochicine, provided that it can be propagated at a fast rate.

Gloriosa is a member of the order Liliales and the families Colchicaceae.members of the family Colchicaceae are genotypes, having either corms or small tubers as their underground organ (Dahlgren et al 1985). Two species of Gloriosa are recorded for Southern Africa, Gloriosa superba L, and Gloriosa virescens Lindl (Pole Evans 1931) Gloriosa virescens was considered by Dyer (1976) to be a subspecies of Gloriosa superba. The major uses of Gloriosa extracts revolve around the use of the plant as a fertility drug, as well as a cure for bites and bruises.Bryant (1966).speculated that the roots of Gloriosa is effective as a cure for lice-killer,it may also act as a germicide,which could kill microbes responsible for uterine diseases. The extracts of Colchium species were first referred to in 1550 B.C by the Egyptians. (Eigsti and Dustin 1955). In 1819 Pelletier and Caventou extracted a substance with basic properties from Colchium autumnale (the meadow saffron) which they regarded as Vera trine. Geiger and Hesse in 1833 recognized this compound as a new alkaloid which they named colchicine (Pictet 1904).the chemical formula of colchicine is C22H25O6N. Clewer et al .,(1915) reported that Warden in 1880 made a study of the drug present in Gloriosa superba and found that the tuber contained a neutral, bitter principle (superbine), salicylic acid, a fluorescent principle, and three resins. Clewer et al .,(1915), using the dried tubers Gloriosa superba, collected in Sri Lanka isolated an enzyme which readily hydrolyzed amygdalin, and a considerable amount of an alkaloid. The mixture of alkaloids contained mainly colchicine (0.3%), subbaratnam (1952, 1954), using Gloriosa tubers, divided the alkaloid fractions into colchicine (mp 151-152) and a new alkaloid gloriosine (C22H25O6N) Melting point 249C-250 C.

Sarin et al .,(1974) and Thakur et al .,(1975) reported the presence of colchicines from Gloriosa, the neutral fractions giving a total of 24 alkaloid and a small amount of basic alkaloids of a non tropolone nature.

Colchicines is an extremely toxic substance which has killed a human adult in a single dose of 3mg (Watt and Breyer- Brandwijk 1962) Colchicine is less effective on cold-blooded than on warm blooded animals. In the past, the main uses of colchine were for chromosome manipulation and treatment of gout. However, at present there is renewed interest in the use of colchicines as a possible cure for cancer-related diseases (Evans et al., 1981). Colchicine and related compounds generally exert antimitotic properties, interfere with microtubule-dependent cell function, and irreversibly bind to tubulin.because colchicines itself is too toxic for human use as an antitumor drug, use has been made of its derivatives,which are less toxic.demecolcine ,trimethycolchicine acid methyl ester ,2-demethyl ,and 3-

demethylthiocolchicine have been evaluated asnti-leukeia agents.data collected on 3demethylthiocolchicine shows this compound to be a broad spectrum antitumor agent of some promise(Wagner et al .,1988). Carbonates of colchicines and thiocolchicine are suitable agents for the treatment of gout and murine malignancies (Brossi and Kerekes 1984) Due to renewed interest in the pharmacology of colchicines, a number of researchers have published work on colchicines synthesis and the production of structural analogs, so as to elimitate using plant material as a source of colchicines.(Blade font 1977;Evans et al .,1978;Boger and Brotherton 1985.1986) Colchicine is normally the alkaloid present in the highest concentrations in the corm of Colchicum .the meadow saffron or autumn crocus (Colchicum autumnale L) is the main commercial source of colchicines (Wildman and Pursey 1968). Since the discovery of colchicines in Gloriosa by clewer et al., (1915) a number of researchers have isolated and qualified colchicines from Gloriosa, and a number proposed that Gloriosa, could serve as a commercial source of colchicines. (Narain and Khoshoo 1967; Narain and Raina 1975; Srivastava and Chandra 1977; Bellet and Gaignault 1985; Finnie and Van Staden 1991) Numerous researchers have tried to isolate chemicals with the ability to induce polyploidy, Colchicines is still the only alkaloid that fulfills the different requirement of an effective polyploidizing agent (Narain and Raina 1975)

Heinz and Mee (1970) and Baylis (1976) found that polyploidy can be induced in suspension culture by the addition of colchicines. Chavadej and Beeker (1984) reasoned that in certain cases polyploidy may cause an increase in the production of medicinal compounds. Bricout et al., (1978) reported an increase in essential oil production in invitro grown plants treated with colchicines. Currah and Ockendon (1987), Novak (1983) and Guri et al (1984) have all utilized colchicines and tissue culture to study polyploidy in plant tisues. Chen and Goeden Kllenmeyen (1979) found that over 50%of the plant initiated from Hemercallis callus treated with colchicines was tetraploid. Colchicines and its biosynthesis in plant tissue have been successfully used in chemotaxonomy for classification purposes (Santavy 1980, Hegnauer 1986) According to Finne and Staden (1991), the Rf value of colchicines was found to be 0.65 using solvent chloroform: acetone: diethylamine (5:4:1). In summary, endophytes have provided many exciting scientific advancements and practical benefits. However, there is much more to look forward to in the future.

4.0.MATERIALS AND METHODS

4.1.MATERIALS

4.1.1.SAMPLES
Catharanthus roseus Gloriosa superba : whole plant (Loyola campus) : whole plant (Loyola campus)

4.1.2.MEDIA
Water agar PDA SDA PDA broth Muller Hitten broth MHA NA CMC medium CCA medium PIA medium

4.1.3.CHEMICAL AND REAGENTS

Distilled water Ethanol Sodium hypo chloride

Lacto phenol cotton blue stain 1%iodine 2% KI Hcl Chloroform Acetone Diethyl amine Ethyl acetate Absolute ethanol NH4OH Methonol 1% starch NaNO3 K2HPO4 MgSO4.7H2O Kcl FeSO4.7H20 Agar CMC-Na salt Colloidal chitin 4%gelatin Peptone MgSO.7H2O Cacl2.2H20 Tween 30

4.1.4.INSTRUMENTS
pH meter Electronic balance Vortex mixer

Laminar airflow Hot water bath Hot plate Shaker Microwave oven UV Transilluminator Inoculation loop Autoclave Micropipettes Forceps Spatula Scissors Blade Cover slip Cotton swab Rotary vacuum evaporator

4.1.5.GLASS WARES
Separating funnel Beaker Conical flask Test tube Petri plates

4.2.0.METHODS
4.2.1.COLLECTIONS OF PLANTS:
Catharanthus roseus and Gloriosa superba were collected from the botanical garden of Loyola college, Chennai. Since there is only little information available on endophytic fungi from the root of medicinal plants, endophytic fungi were isolated from roots of Catharanthus roseus and Gloriosa superba.

4.2.2.CONFIRMATION:
Plant sample were confirmed by expertise taxonomist of Loyola college.

4.2.3.SURFACE STERILIZATION OF ROOT AND CORM:

The root of Catharanthus roseus and the corm of Gloriosa superba were surface sterilized using ethanol and sodium hypochloride. The root and corm was washed in running tap water to remove the soil particles. then under laminar air flow chamber the roots were again washed with sterile distilled water, followed with ethanol 70% for 30 seconds and then treated with sodium hypochloride (3%-5% available chlorine) for three minutes.samples were exhaustively rinsed with sterile water .Sterility checks and reconstruction experiments showed that most or all epiphytic microorganisms were eliminated during this treatment.

4.2.4.INOCULATION
The root of Catharanthus roseus and the corm of Gloriosa superba was divided into pieces of 1 cm and transferred to Petri dishes containing 2.5% water agar medium under aseptic conditions .streptomycin (0.5g per ml of each) were added to the media to suppress bacterial growth. Plates were incubated at room temperature for one week.endophytic fungi growing on the media were isolated, purified and identified.

4.2.5.ISOLATION OF ENDOPHYTIC FUNGI:

The surface sterilized plant materials were inoculated on autoclave water agar media on sterile petridishes .after seven days appearance of hyphae from root and corm were observed.

4.2.6.IDENTIFICATION 4.2.6.1Microscopic studies

Small amount of hyphae from each culture was taken in a microscopic slide and mounted with lactophenol cotton blue stain and viewed under light microscope for the nature of mycelium and reproductive structures for identification. After fungal emergence in the plates, some individual hyphal tips of the various fungi were transferred to new PDA medium and SDA medium and this was repeated three times for fungus purity.

4.2.7.SUBCULTURE:
The identified endophytic fungi were subcultured on PDA and SDA media to obtain pure culture.

4.2.7.1MEDIA PREPARATION 4.2.7.2.PDA-POTATO DEXTROSE AGAR

Potato Dextrose Agar 20gm 2gm 1.5gm

Final pH-6.7 All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min.

4.2.7.3.SDA-SABORAUDS DEXTROSE AGAR

Mycological peptone Dextrose Agar 1 gm 4gm 1.5gm

Final pH (at 25c) 5.60.2 All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min.

4.2.8.MASS PRODUCTION:
Potato dextrose broth was used as mass production medium for fungal growth. Grated potato 200g in 500ml of water were boiled for 30 minutes. The mixture was cooled, mashed, and the extract was collected by squeezing through a pre-cleaned cloth filter. After adding 20gms of potato the volume of the extract was adjusted up to 1l by adding water. The broth was then inoculated with tiny agar blocks from subculture plates and kept in dark condition at room temperature .The broth was regularly observed for fungal growth.

4.2.9.EXTRACTION
After 10-12 days of incubation at room temperature the growth of endophytic fungus were observed .it was then soaked with equal volume of ethyl acetate for overnight. The solvent was separated from mycelia using separating funnel. The collected solvents was condensed by rotary vacuum evaporator.Futher to completely evaporate the solvent, the condensed samples were dispensed in petridishes and left at room temperature for obtaining the residual extract and stored at room temperature.

4.2.9.1.WORKING OF ROTARY VACUUM EVAPORATOR

Rotary Evaporator The rotary evaporator is a device for gently and efficiently evaporating solvents from a mixture. It consists of a heated rotating vessel (usually a large flask) which is maintained under a vacuum though a tube connecting it to a condenser. The rotating flask is heated by partial emersion in a hot water bath. The flask's rotation provides improved heat transfer to the contained liquid; the rotation also strongly reduces the occupance of 'bumps' caused by superheating of the liquid. The solvent vapors leave the flask by the connecting tube and are condensed in the condenser section. The condenser section is arranged so that the condensed vapors drain into another flask where they are collected. It is a very efficient way of rapidly removing large quantities of solvent.

4.2.10.TLC Thin layer chromatography

Thin layer chromatography is widely employed laboratory technique and is similar to paper chromatography. However instead of using a stationary phase of paper, it involves a stationary phase of thin layer of adsorbent like silica gel, alumina or cellulose on flat, inert substrate. Compared to paper it has the advantage of faster runs, better separations and the choice between different adsorbents. Different compounds in the sample mixture travel different distance according to how strongly they interact with the adsorbent. This allows the calculation of an Rf and can be compared to standard compounds to aid in the identification of an unknown substance. In order to determine the separation of compounds the TLC was carried out on precoated silica gel plate 60F254 plates.

4.2.10.1.Catharanthus roseus
The condensed extract of endophytic fungus from Catharanthus roseus was diluted with the solvent (ethyl acetate: ethanol: ammonium solution (3:1:1)) Exactly 5l of the sample was used for all the trials. The solvent was allowed to develop in the plate till it reached th of the length of the plate. Mobile phase used: ethyl acetate: ethanol: ammonium solution (3:1:1).

4.2.10.2.Gloriosa superba
The condensed extract of endophytic fungus from Gloriosa superba was subjected to thin layer chromatography using chloroform: acetone: diethylamine (5:4:1) Exactly 5l of the sample was used for all the trials. The solvent was allowed to develop in the plate till it reached Th of the length of the plate Mobile phase used: chloroform: acetone: diethylamine (5:4:1)

4.2.10.3.DETECTION The solvent was allowed to develop in the plate till it reached Th of the length of the plate ant then the plates were observed for the presence of bands. Detection of compounds was also done by UV visualization.

4.2.11.Anti microbial activity

In order to test the antimicrobial activity of crude extract of endophytic fungus from Catharanthus roseus and Gloriosa superba, the crude sample was tested against five different human pathogens. Staphylococcus aureus Bacillus subtilis Yersinia enterocolitica Enterobacter aerogens Candida albicanes

4.2.11.1.MEDIA PREPARATION
MHA medium was used for microbial growth. The composition is as follows

4.2.11.2.MHA MEDIUM
Beef infusion Caesin acid hydrolysate Starch Agar 7.30.2 1.775g 0.150g 1.7g 30g

Ph (28c)

All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min.

4.2.11.3.SAMPLE PREPARATION
3% sample were prepared from the crude extract of endophytic fungus from Catharanthus roseus and Gloriosa superba respectively .3 %( 0.03 in 1ml of DMSO).

4.2.11.4.INNOCULATION
Disc diffusion method was carried out to check the anti-microbial activity of the crude extract of endophytic fungus from Catharanthus roseus and Gloriosa superba. The microorganisms were inoculated for each sample in separate petriplaes under aseptic condition. The sterile disc were loaded with exactly 20l of the sample .The dried discs were placed in the plates and incubated for 48-78 hrs.

4.2.12.ENZYME ACTIVITY
The isolated endophytic fungi, present in both Catharanthus roseus and Gloriosa superba were assayed for the production of extra cellular enzymes on solid medium amended with enzymespecific substrates. All petri dishes were incubated for 1 week under laboratory conditions. Cultures were examined on a daily basis for the presence of a clear zone (halo) around the fungal colony.

4.2.12.1.AMYLASE ACTIVITY
Amylase activity was tested using starch added nutrient agar medium (Collins and Lyne, 1980). Using cork borer tiny agar blocks from One-week-old fungal isolates growing on PDA subculture plates were placed were point inoculated in the middle of Petri dishes. A 3-mm-diameter cork borer was used to remove a disc of agar from the middle of the chitin agar plates and the hole replaced with a similar sized mycelial agar disc of the fungal cultures. The plates were kept at room temperature and observed for fungal growth.

4.2.12.2.MEDIA PREPARATION pHPeptoneYeast extractBeef extractNaclAgarStarch7.2 0.5g 0.15g 0.155g 0.5g 1.6g 1%

All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min. 4.2.12.3.DETECTION The production of amylase enzyme was confirmed using staining solutions. 4.2.12.4.STAINING SOLUTION 1% Iodine in 2%KI was used as staining solutions. After the appearance of fungal growth in the medium, it was stained staining solution .Appearance of clear zone observed around the colony confirms the production of enzyme amylase.

4.2.12.5CELLULASE ACTIVITY
Cellulase activity was tested using CMC medium (carboxy methyl cellulase) (yeoh et al., 1973). Using cork borer tiny agar blocks from the subculture plates were placed on the medium. The plates were kept at room temperature and observed for fungal growth. 4.2.12.6.MEDIA PREPARATION

NaNO3 K2HPO4 MgSO4.7H2O Kcl FeSO4.7H20 0.1g

0.2g

0.005g 0.005g 0.001g

 pH

Agar CMC-NA salt 6.8

16g 1%

All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min 4.2.12.7.DETECTION The production of cellulase enzyme was confirmed using staining solutions. 4.2.12.8.STAINING SOLUTIONS 0.1ml Hcl +5ml1%iodine in 2%KI was used as staining solutions. After the appearance of fungal growth in the medium, it was stained staining solution .Appearance of clear zone observed around the colony confirms the production of enzyme cellulase.

4.2.12.9CHITINASE ACTIVITY
Chitase activity was tested using CCA medium (colloidal chitin agar) (Cinagappa and Lockwood, 1962). Using cork borer tiny agar blocks from the subculture plates were placed on the medium. The plates were kept at room temperature and observed for fungal growth. 4.2.12.10.MEDIA PREPARATION Ph 7 All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min 4.2.12.11.DETECTION The opalescent zone formed around the colony confirms the chitinase activity. Colloidal chitin 0.2g Agar 1.6g

4.2.12.12.GELATINASE ACTIVITY
Gelatinase activity was tested using gelatin (4%) added nutrient agar medium. (Guickshank et al., 1975). Using cork borer tiny agar blocks from the subculture plates were placed on the medium. The plates were kept at room temperature and observed for fungal growth.All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min. 4.2.12.13.DETECTION The opalescent zone formed around the colony confirms the gelatinase activity.

4.2.12.14.LIPASE ACTIVITY
Lipase activity was tested using PTA medium (peptone tween agar) (Villman and Blasins, 1974). Using cork borer tiny agar blocks from the subculture plates were placed on the medium. The plates were kept at room temperature and observed for fungal growth.

4.2.12.15.MEDIA PREPARATION
pH Peptone MgSO.7H2O Cacl2.2H20 Tween 30 Agar 6.5 1g 0.2g 0.01g 1ml 1.6g

All the above ingredients were dissolved in 100ml sterile distilled water and autoclaved at 15lbs for15min.

4.2.12.15.DETECTION
The opalescent zone formed around the colony confirms the lipase activity.

4.2.13.PIGMENTATION
PDA subculture plates showed more pigmentation than SDA.this confirms the present of pigmented compounds.analysis of pigmented compounds was done using water, ethyl acetate and methanol.

4.2.13.1.Catharanthus roseus
5ml of water, ethyl acetate and methanol was added to the PDA subculture plates of Catharanthus roseus and kept for overnight.The solvent obtained was scanned using UV spectrometer and the scanner was set in a range of 400 -700nm.The readings were tabulated. Respective solvent was used as reference.peaks appeared for the samples were observed.

4.2.13.2.Gloriosa superba
5ml of water, ethyl acetate and methanol was added to the PDA subculture plates of Gloriosa superba and kept for overnight.The solvent obtained was scanned using UV spectrometer and the scanner was set in a range of 400 -700nm.The readings were tabulated. Respective solvent was used as reference. Peaks appeared for the samples were observed.

5.0.RESULTS

5.1.ISOLATION OF ENDOPHYTIC FUNGI

The root of Catharanthus roseus and the corm of Gloriosa superba was divided into pieces of 1 cm and transferred to Petri dishes containing 2.5% water agar medium under aseptic conditions .streptomycin (0.5g per ml of each) were added to the media to suppress bacterial growth. Plates were observed at regular intervals . After 3-4 days the hyphal structures emerged out from the cut surface of root of Catharanthus roseus and the corm of Gloriosa superba. (Fig 3, fig 4)

5.2.IDENTIFICATION OF ENDOPHYTIC FUNGI

Small amount of hyphae from seven days old culture was taken in a microscopic slide and mounted with lactophenol cotton blue stain and viewed under light microscope. Morphology of sickle shaped microconidia, macroconidia and clamydospores resembled Fusarium species. (Fig 5, fig 6). Fusarium sp was identified based on the presence of short phialides, the shape of macroconidia and the presence of chlamydospores (Nelson et al., 1983).

5.3.SUBCULTURE
The identified endophytic fungi were subcultured on PDA and SDA media. The fungal growth was observed after 5-6 days. (Fig 7a.7b)(Fig 8a.fig 8b).When transferred and grown on PDA produced a whitish fluffy growth that after a few days gradually developed into a mycelium with a powdery felt-like surface and a pinkish-tan coloration that became more brownish with time (up to 2 weeks). The pigmentation was observed in subculture plates after 10-12days.The pigmentation of Fusarium sp was found to be more in PDA plates than in SDA plates. (Fig 7c, fig 8c, fig 8d)

ISOLATION OF ENDOPHYTIC FUNGI

Fig 3.Isolation of endophytic fungi from Catharanthus roseus

Fig 4.Isolation of endophytic fungi from Gloriosa superba

IDENTIFICATION OF ENDOPHYTIC FUNGI Fusarium sp. Fig 5 .hyphae structures

Fig 6.Presence of sickle shaped microconidia, macroconidia

SUBCULTURE RESULTS OF Catharanthus roseus Fig 7.a.PDA plates

Fig 7.b.SDA plates

SUBCULTURE RESULTS OF Catharanthus roseus

Fig 7.c.PDA (left) and SDA (right)plates.

SUBCULTURE RESULTS OF Gloriosa superba Fig 8.a.PDA plates

Fig 8.b.SDA plates

Fig 8.c.PDA plates

Fig 8.d.SDA plates

5.4. MASS PRODUCTION

PDA broth was used as mass production medium. Mass of fungal growth was observed, after 12 -14 days in Catharanthus roseus, and after 16-18 days in Gloriosa superba. (Fig 9a, fig 9b)

5.5. EXTRACTION
Non polar solvent ethyl acetate was used as extraction solvent.the extracts were dried by using rotary vacuum evaporator.the amount of total extract was 0.35g for Catharanthus roseus and 0.21g for Gloriosa superba. (TABLE 1)

5.6.TLC-THIN LAYER CHROMATOGRAPHY

5.6.1.Catharanthus roseus
Ethyl acetate extract of endophytic fungus from Catharanthus roseus was subjected to thin layer chromatography using ethyl acetate: ethanol: ammonium solution (3:1:1). Four clear bands were observed in naked eye (fig 10a).and one band was observed with UV detector at 254 nm. (TABLE 2)(Fig 10b).

5.6.2.Gloriosa superba
Ethyl acetate extract of endophytic fungus from Gloriosa superba was subjected to thin layer chromatography using chloroform: acetone: diethylamine (5:4:1) one clear band was observed in naked eye and with UV detector at 254 nm. (TABLE 3)(Fig 10 c)

TABLE 1

EXTRACTION OF endophytic fungus from Catharanthus roseus and

Gloriosa superba

CONTENTS

Catharanthus roseus

Gloriosa superba

QUANTITY USED FOR MASS PRODUCTION

1700ml

1700ml

NO.OF DAYS FOR FUNGAL GROWTH

12-14

16-18

CRUDE OBTAINED

0.35g

0.21g

TABLE 2

DETAILS OF THIN LAYER CHROMATOGRAPHY OF CRUDE EXTRACT OF ENDOPHYTIC FUNGUS FROM Catharanthus roseus

SOLVENT EXTRACTED WITH

SOLVENT USED

RATIO

DETECTION

OBSERVATION

Ethyl acetete

ethyl acetate :ethanol:ammoniu m solution

3:1:1

Naked eye

Three bands

uv

One band

TABLE 3

DETAILS OF THIN LAYER CHROMATOGARPHY OF CRUDE EXTRACT OF ENDOPHYTIC FUNGUS FROM Gloriosa superba

SOLVENT EXTRACTED WITH

SOLVENT USED

RATIO

DETECTION

OBSERVATION

Ethyl acetete

chloroform:a 5:4:1 cetone:dieth ylamine

Naked eye One band

uv

One band

Fig 9a Mass production of endophytic fungus from Catharanthus roseus

Fig 9b Mass production of endophytic fungus from Gloriosa superba

Fig 10a.TLC results of crude extract of endophytic fungus from Catharanthus roseus

Fig 10b. TLC results of crude extract of endophytic fungus from Catharanthus roseus in UV detection

Fig 10c.TLC results of crude extract of endophytic fungus from Gloriosa superba in UV detection

5.6.3.Rf values of Catharanthus roseus

From the previous literature(methods in molecular biology; john M.Walker)the Rf value of indole alkolids present in catharanthus roseus using solvent ethyl acetate: ethanol: ammonium solution (3:1:1 ) was found to be (ajmaline-0.8,catharanthine-0.7,serpentine-0.02-0.05,vindoline 0.72 and yohombine-.61 )respectively. From the present study of Catharanthus roseus the Rf value was found to be 0.09,0.30,0.43,0.96.(TABLE 4)

5.6.4.Rf values of Gloriosa superba

According to Finne and Staden (1991), the Rf value of colchicines was found to be 0.65 using solvent chloroform: acetone: diethylamine (5:4:1). From the present study of Catharanthus roseus the Rf value was found to be 0.6 and 0.9 based on this we predict that the crude extract of Gloriosa superba may constitute the compound colchicine. (TABLE 4)

5.7.ANTIMICROBIAL TESTING (fig 11a, 11b, 11c, 11d)

5.7.1.Catharanthus roseus
3% sample prepared from the crude extract of endophytic fungus from Catharanthus roseus showed both positive and negative results when tested against 5 pathogens (TABLE 5)

5.7.2.Gloriosa superba
3% sample prepared from the crude extract of endophytic fungus from Gloriosa superba showed both positive and negative results when tested against 5 pathogens. (TABLE 5)

TABLE 4

Rf VALUES OF CRUDE EXTRACT OF ENDOPHYTIC FUNGUS FROM Catharanthus roseus and Gloriosa superba

S.NO

PLANT NAME

SOLVENT USED

RESOLUTION FRONT VALUE

UV SCAN

Catharanthus ethyl acetate: ethanol: ammonium roseus

solution (3:1:1).

1st,2nd,3rd,bands gave Rf value 0.09,0.30,0.43 respectively

Single band gave Rf value 0.96

Gloriosa superba

chloroform:acetone: diethylamine(5:4:1)

Single band gave Rf value 0.6

Single band gave Rf value 0.9

TABLE 5..ANTIMICROBIAL TESTING

S.NO

PLANT NAME

ORGANISM USED

CONC

ZONE

OF

ACTIVITY (mm) 3% 3%
18

Catharanthus roseus

Staphylococcus aureus

Bacillus subtilis

22

Yersinia enterocolitica

Enterobacter aerogens Candida albicanes 2 Gloriosa superba Staphylococcus aureus

23

+ +

21 14

Bacillus subtilis

18

Yersinia enterocolitica

Enterobacter aerogens Candida albicanes

18

18

Fig 11a.Antimicrobial activity showing positive result against Candida albicans

Fig 11b.Antimicrobial activity showing positive result against Enterobacter aerogens

Fig 11c.Antimicrobial activity showing positive result against Bacillus subtilis

Fig 11dAntimicrobial activity showing positive result against Staphylococcus aureus

5.8.0.ENZYME ACTIVITY OF Catharanthus roseus AND Gloriosa superba. (TABLE 6)

The productions of extracellular enzymes by Fusarium sp, present in both Catharanthus roseus AND Gloriosa superba shows the following activity respectively.

5.8.1.AMYLASE ACTIVITY
After two days fungal growth was observed in starch added NA medium, it was then stained with staining solution of 1% iodine in 2% potassium iodide. A clear zone was observed around the colony within few minutes. This confirms the production of extracellular enzyme amylase by Fusarium sp.(fig 12a)

5.8.2.CELLULASE ACTIVITY
After two days fungal growth was observed in CMC agar medium, it was then stained with staining solution of 0.1ml Hcl +5ML1% iodine in 2% potassium iodide. Clear zone was not observed around the colony. This confirms that there is no production of extra cellular enzyme cellulase by Fusarium sp. (fig 12b)

5.8.3.CHITINASE ACTIVITY
After two days fungal growth was observed in CCA medium. The opalescent zone was not formed around the colony. This shows that their is no chitinase activity by Fusarium sp. (fig 12c)

TABLE 6. ENZYME ACTIVITY OF Catharanthus roseus AND Gloriosa superba

S.NO

ENZYME ACIVITY

PLANT USED

Catharanthus roseus 1 AMYLASE +

Gloriosa superba.

CELLULASE

CHITINASE

GELATINASE

LIPASE

Fig 12a. Enzyme activity-Amylase activity

Fig 12b. Enzyme activity-Cellulase activity

Fig 12c .Enzyme activity-Chitin activity

Fig 12d .Enzyme activity-gelatin activity

Fig 12e .Enzyme activity-lipase activity

5.8.4.GELATINASE ACTIVITY
After two days fungal growth was observed in gelatin added NA medium. The opalescent zone was observed around the colony. The opalescent zone formed around the colony confirms the gelatinase activity. (Fig 12d)

5.8.5.LIPASE ACTIVITY
After two days fungal growth was observed in PTA medium. The opalescent zone was not formed around the colony. This shows that their is no lipase activity by Fusarium sp. (fig 12e)

5.9.ANALYSIS OF PIGMENTED COMPOUNDS 5.9.1Catharanthus roseus

Water, ethyl acetate and methanol are the solvents used for the analysis of pigmented compounds. The highest peak obtained in water, ethyl acetate and methanol was found to be 1.021 in 680nm, 1.744 in 690nm, 1.35 in 680nm respectively. (FIG13a, 13b, 13c)(Table 7)

5.9.2.Gloriosa superba
Water, ethyl acetate and methanol are the solvents used for the analysis of pigmented compounds. The highest peak obtained in water, ethyl acetate and methanol was found to be 0.919 in 690nm, 1.803 in 680nm, 1.192 in690nm respectively. (FIG 14a, 14b, 14c)(Table 8)

The highest peaks obtained above using visible spectrum shows the maximum concentration of pigmented compounds ranging from 400-700nm.Further studies can be done using NMR spectroscopy to isolate pure pigmented compounds.

ANALYSIS OF PIGMENTATION COMPOUNDS Catharanthus roseusFIG 13a-using water as a solvent

FIG 13b-using ethly acetate as a solvent

FIG 13c-using methanol as a solvent

ANALYSIS OF PIGMENTATION COMPOUNDS Gloriosa superba FIG 14a-using water as a solvent

FIG 14b-using ethyl acetate as a solvent

FIG 14c using methanol as a solvent

6.0.DISCUSSIONS

Catharanthus roseus and Gloriosa superba are the plants selected for the present study. The endophytes of medicinal plants provide a good source for compounds of biological activity and endophytes are an untapped reservoir of potentially novel, effective drugs (Stierle et al.,
1995).

The endophytic fungi were isolated from the root of Catharanthus roseus and the corm of Gloriosa superba.2.5% water agar medium was used as isolation medium. (P.Sardyl et al., 1992). Water agar (containing 50 g chloramphenicol ml1 and 50 g streptomycin ml1) seemed to be a better medium for isolation of endophytic fungi than potato-dextrose agar (L.X. Cao et al., 2004). The identification of endophytic fungi was done using lacto phenol cotton blue stain. The endophytic fungus identified was found to be Fusarium species based on morphological characteristics. Fusarium is identified microscopically by its sickle shaped or banana shaped macro conidia and clusters of fusiform micro conidia. (Book Name-Smolin and Thofts: The cornea.). Antimicrobial studies clearly indicated that the fungal crude extract tested showed both anti bacterial and anti fungal properties. The production of extracellular enzymes (amylase, cellulase, chitinase, gelatinase, and lipase) by Fusarium sp was determined by culture plate method. The fungi showed only amylase and gelatinase activity. The fungal crude extract was subjected to Thin Layer Chromatography. From the previous literature(methods in molecular biology; john M.Walker)the Rf value of indole alkaloids present in Catharanthus roseus using solvent ethyl acetate: ethanol: ammonium solution (3:1:1 ) was found to be (ajmaline-0.8,catharanthine-0.7,serpentine-0.02-0.05,vindoline 0.72 and

yohombine-.61 )respectively. From the present study of Catharanthus roseus the Rf value was found to be 0.09, 0.30, 0.43, and 0.96.Thus the fungal crude extract was suspected to contain one of the major breakdown products of these indole alkaloids. According to Finne and Staden

(1991), the Rf value of colchicines was found to be 0.65 using solvent chloroform: acetone: diethylamine (5:4:1). From the present study of Gloriosa superba the Rf value was found to be 0.6 and 0.9 based on this we predict that the crude extract of Gloriosa superba may constitute the compound colchicine.

PDA subculture plates showed more pigmentation than SDA.this confirms the present of pigmented compounds.analysis of pigmented compounds was done using water, ethyl acetate and methanol. The highest peaks obtained above using visible spectrum shows the maximum concentration of pigmented compounds ranging from 400-700nm. Further studies can be done on isolation, purification and structure studies on pigmented compounds.

7.0 SUMMARY

Fungal endophytes (microfungi that live within healthy plant tissues) represent an unknown pro-portion of fungal diversity even though they are associated with all plants. While there is a growing appreciation of their ecological importance and human uses, little is known about their host specificity, geographic structure, or phylogenetic relationships. Discovery of endophytic fungi in plant tissues opened up new possibilities in the search for metabolically active compounds. According to Dreyfuss and Chapela (1994), about 4000 secondary metabolites of fungal origin have been described as biologically active.

Thus we did a study on the endophytic fungi present in Catharanthus roseus and Gloriosa superba.

The endophytic fungi, Fusarium sp were isolated from surface sterilized root of Catharanthus roseus and corm of Gloriosa superba.The fungal crude extract, obtained using ethyl acetate showed antimicrobial activity against the tested pathogens. The production of extra cellular enzyme such as amylase, cellulase, chitase, gelatinase and lipase by the isolated endophytic fungi on solid medium was also determined. The fungi showed amylase and gelatinase activity.The fungal crude extract was also subjected to thin layer chromatography and Rf value was calculated. Analysis of pigmented compounds was also done using three solvents such as methanol, water and ethyl acetate. The highest peaks obtained using visible spectrum shows the maximum absorption of pigmented compounds ranging from 400-700nm. Further studies can be done on isolation, purification and structure studies on pigmented compounds.

Fungi have proven themselves invaluable sources of natural products for agriculture as well as biomedical development for over a half century. As fungi thrive in competitive environments, it is hypothesized that their metabolic compatibility has been strongly influenced by natural selection Bioactive product discovery depends on the knowledge of habitats where fungi are abundant and the strength of culture collection .Screening this fungal resource for novel metabolites and enzymes and their application are major goals of current research to accomplish environment-friendly technological development.

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