26

Endophytic fungi and their Importance

Redman et al., (1999) studied the biochemical analysis of plant protection afforded by a

nonpathogenic endophytic mutant of Colletotrichum magna. Studies had shown previously to

protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from

anthracnose disease elicited by wild-type C. magna. Plant biochemical indicators of a localized

and systemic (peroxidase, phenylalanine ammonia-lyase, lignin and salicylic acid) “plant-defense”

response were investigated in anthracnose-resistant and susceptible cultivars of cucurbit seedlings.

Results indicated disease protection in path-1- colonized plants were correlated with the ability of

inducing plant defense mechanism in the host. A model based on the kinetics of plant-defense

activation is also presented to explain the mechanism of path-1-conferred disease protection.

Strobel and Daisy (2003) reviewed on the phenomena of bioprospecting endophytic

microbes for the natural products. Accordingly endophytes are a poorly investigated group of

microorganisms that represent an abundant and dependable source of bioactive and chemically

novel compounds with potential for exploitation in a wide variety of medical, agricultural and

industrial arenas. The mechanisms through which endophytes exist and respond to their

surroundings must be better understood in order to be more predictive about which higher plants to

seek, study and spend time isolating microfloral components. This may facilitate the product

discovery processes.

Kumar and Hyde (2004) studied the biodiversity and tissue recurrence of endophytic fungi

in Tripterygium wilfordii. Nearly 343 endophytic fungal isolates representing 60 taxa with 30

morphotypes were obtained. The endophytic assemblage comprised a number of cosmopolitan

species such as Colletotrichum sp. Glomerella sp. Pestalotiopsis sp. Phomopsis sp. and

Phyllosticta sp. Further a few endophytes isolated from twig xylem and bark was not isolated from

the roots, leaves and flowers. Hence the study concludes that species composition and frequency of

endophyte species were dependent on the tissue type. The dominant fungi isolated from different

host tissue parts however expressed a fair degree of recurrence.

Fungal biodiversity from various parts of India have been studied, interpreting their

distribution patterns and conservation strategies. Accordingly, only a fraction of total fungal

wealth has been subjected to scientific scrutiny and mycologists have to unravel the unexplored

27

and hidden wealth. One third of fungal diversity of the globe exists in India and in 1.5 million of

fungi, only 50% are characterized till date. Unfortunately only around 5–10% of fungi can be

cultured artificially. The world of fungi provides an endless source of biological diversity and a

rich source for exploitation. Hence they can play a significant role in the daily life of human beings

besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation,

natural cycling, biofertilizers and many other ways. And thus fungal biotechnology has become an

integral part of the human welfare, Manoharachary et al., (2005).

Spiering et al., (2006) studied the effects of the fungal endophyte, N. lolii on net

Photosynthesis and growth rates of perennial ryegrass (Lolium perenne). N. lolii is a fungal

endophyte of perennial ryegrass (L. perenne) that produces bioactive alkaloids which helps in

fitness of their host. The study examined the effect of N. lolii on net photosynthesis (Pn) and

growth rates in ryegrass genotypes differing in endophyte concentration in all leaf tissues. The

study revealed that N. lolii affects CO2 fixation but not light interception and photochemistry of

photosynthesis. The impact of N. lolii on plant growth and photosynthesis is independent of

endophyte concentration however the endophyte effects on Pn and plant growth are strongly

dependent on the plant growth phase.

Strobel et al., (2007) discovered a novel endophytic fungus Muscodor vitigenus from the

liana Paullinia paullinioides that produced naphthalene, an insect repellant. The extracted

naphthalene has chromatographic and mass spectral properties that are identical to authentic

naphthalene. In a preferred embodiment the naphthalene in the gas phase of M. vitigenus is useful

in the repellency of unwanted insect pests. This unique biological activity of this novel endophyte

suggests a wide range of potential practical applications particularly in the area of insect repellents,

insecticides, antimicrobials, anti helminthics and vermicides.

Vega et al., (2008) studied fungal endophyte - mediated plant defense as a novel biological

control mechanism against the coffee berry borer the most devastating pest of coffee throughout

the world. A survey of fungal endophytes in coffee plants from Hawaii, Colombia, Mexico and

Puerto Rico has revealed the presence of various genera of fungal entomopathogens including

Acremonium, Beauveria, Cladosporium, Clonostachys, and Paecilomyces. Two of these B.

bassiana and Clonostachys rosea were tested against the coffee berry borer and were shown to be

pathogenic.

28

Pimentel et al., (2011) reviewed on the use of endophytes for the production of bioactive

compounds and their use in biotransformation process. The role of endophytes on the production

of anticancer, antimicrobial and antioxidant compounds illustrating their potential for human use

was inferred. It also describes biotransformation as an auspicious method to obtain novel bioactive

compounds from microbes. Biotransformation allows the production of regio and stereo selective

compounds under mild conditions and that using endophytic fungi have been reviewed for e.g.

biotransformation of grandisin by the endophytic fungus Phomopsis sp. to a tetrahydrofuran which

showed trypanocidal activity.

Marine fungi that often occur associated with macroorganisms like algae, sponges or

tunicates and produce secondary metabolites with novel structures and potential pharmaceutical

significance. They have done the evaluation of new natural products of marine-derived fungi in

order to find novel lead structures for drug development. Nearly seven fungal strains like

Stachylidium sp., Cadophora sp., etc. living associated with marine algae and sponges were

cultivated during 40 to 60 days, and the extracts tested for bioactivity. Subsequently the natural

products were isolated, their structures determined and their bioactivity was established. Some of

the compounds being identified in the study include stachylines, marilines, marilones, etc. Almeida

(2011).

Tropical endophytes

Arnold et al., (2000) studied on fungal endophytes among tropical forests in Central

Panama. Colonization patterns, richness, host preference and spatial variation in leaves of

understorey tree species – Heisteria concinna and Ouratea lucens was studied. From 83 leaves,

418 endophyte morphospecies were isolated most of which were represented by a single isolate

(59%). The studies suggest the evidence of host preference and spatial heterogeneity. Further the

tropical endophytes are hyperdiverse and the extrapolating data estimates excluding them will

underestimate fungal species diversity.

Arnold (2001) reviewed extensively on fungal endophytes in neotropical trees their

abundance, diversity and their ecological implications. Accordingly tropical fungi are traditionally

understudied and studies of endophytic fungi in tropical forests are yet in their infancy. Fungal

endophytes may represent a ubiquitous, cryptic and ecologically interesting component of tropical

29

forests. Endophytes appear to be both ubiquitous and highly diverse in tropical forests. Further

exploration of tropical endophytes will help to clarify the fungal diversity debate and will likely

lend support to higher estimates of global fungal diversity. The study of tropical endophytes seems

promising to enrich our understanding of plant-fungus interactions in tropical forests, tropical

biodiversity and tropical ecology.

Fungal endophytes have a varied relationship between pathogen and host plant and also

infer how they limit pathogen damage in tropical trees. Endophytes represent a ubiquitous yet

cryptic component of terrestrial plant communities. The fundamental aspects of endophytic

interactions with hosts are unknown. In contrast to vertically transmitted endophytes, horizontally

transmitted endophytes of woody angiosperm are thought to contribute little to host defense. The

studies document high diversity, spatial structure and host affinity among foliar endophytes

associated with a tropical tree (Theobroma cacao, Malvaceae) across lowland Panama. The fungal

endophytes help to decrease both leaf necrosis and leaf mortality when T. cacao seedlings are

challenged with a major pathogen (Phytophthora sp.). Further endophyte-mediated protection was

greater in mature leaves, which bear less intrinsic defense against fungal pathogens than do young

leaves. Altogether the studies indicate diverse horizontally transmitted endophytes of woody

angiosperms to play an important role in host defense Arnold et al., (2003).

Suryanarayan et al., (2003) studied the endophytic fungal communities in leaves of tropical

forest trees, their diversity and distribution patterns. The cryptic guild of endophytic fungi is

regarded as a benchmark for estimating fungal biodiversity. They studied endophyte distribution,

diversity and host recurrence in 24 tree hosts (belonging to 17 plant families) of two dry tropical

forests of the Nilgiri Biosphere Reserve. Out of 81 endophyte taxa isolated from 3600 tissue

segments 56 species were isolated from more than one host. It was seen that the host density

influenced the composition and distribution of endophytes in one of the forests. The existence of

ubiquitous forms reduced the diversity of the endophytes in the plant communities. The results

concluded that dry tropical forests are not hyperdiverse with reference to endophytes compared to

wet tropical forests and that the generalists among endophytes be identified before extrapolating

data to calculate global fungal diversity.

Arnold and Lutzoni (2007) reviewed on diversity and host range of foliar fungal

endophytes with reference to tropical arenas discussing them to be hotspots to explore the putative

30

hyperdiversity of tropical leaf endophytes, endophyte communities along the latitudinal gradient of

Canadian arctic was compared to the lowland tropical forest of central Panama. Molecular

sequence data from 1403 endophyte strains showed that endophytes increase in incidence,

diversity and host breadth from arctic to tropical sites. Endophyte communities from higher

latitudes constituted relatively few species whereas tropical endophyte assemblages are dominated

by a small number of classes with a very large number of endophytic species. Leaves of tropical

trees hence represent hotspots of fungal species diversity containing numerous species not yet

recovered from other biomes. The challenge remains to recover and identify those elusive and

rarely cultured taxa with narrower host ranges and to elucidate the ecological roles of these little

known symbionts in tropical forests.

Endophytic fungi from medicinal plants

Wiyakrutta et al., (2004) isolated endophytic fungi with antimicrobial, anticancer and

antimalarial activities isolated from Thai medicinal plants. A total of 81 Thai medicinal plant

species collected from forests were examined for the presence of endophytic fungi with biological

activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected from

which extracts were prepared and tested for biological activity. Extracts of 92 isolates could inhibit

Mycobacterium tuberculosis by the microplate Alamar blue assay while extracts of 6 isolates

inhibited Plasmodium falciparum as determined by the [3H] hypoxanthine incorporation method.

Antiviral activity against Herpes simplex virus type 1 was observed in 40 isolates. Anticancer

activity against human oral epidermoid carcinoma cells and breast cancer cells was also observed

by sulphorhodamine B assay. Hence it was concluded that Thai medicinal plants inherits diverse

endophytes possessing a potential source of novel bioactive compounds.

Raviraja (2005) reported on the fungal endophytes in five medicinal plant species from

Kudremukh Range Western Ghats of India. Nearly he isolated 18 species of endophytic fungi from

bark, stem and leaf segments of five medicinal plant species growing within the Kudremukh range

in the Western Ghats of India. The dominant species were Curvularia clavata, C. lunata, C.

pallescens and F. oxysporum. The highest species richness as well as frequency of colonization of

endophytic fungi was found in the leaf segments rather than the stem and bark segments of the host

plant species. The greatest number of endophytic fungal species were found within Callicarpa

tomentosa (11 species), whereas Lobelia nicotinifolia harbored the lowest number of fungal

31

endophytes (5 species). The study provides evidence that fungal endophytes are host and tissue

specific.

Gangadevi and Muthumary (2007) studied on endophytic fungal diversity in young, old

and senescent leaves of Ocimum basilicum L. a medicinal plant. Not much is known on the

temporal and spatial variation of fungal endophytes inhabiting the foliage of medicinally important

plants. This study provides the first report on diversity of endophytic fungi of medicinal plants

from Chennai city Southern India. Added to it one of the isolates Phyllosticta sp. was found to

produce taxol in artificial culture media. The endophytic fungus is thus expected to be a potential

source of natural bioactive agent.

Mohanta et al., (2008) studied the antimicrobial potentials of endophytic fungi inhabiting

three Ethno-medicinal plants of Similipal Biosphere Reserve India. Nearly 60 fungal endophytes

belonging to 14 genera were isolated out of which 31 endophytes (51.66%) were obtained as

filamentous forms and 29 of them (48.33%) as yeast colonies. Species of Curvularia, Fusarium,

Alternaria and Penicillium were isolated as dominant and host specific endophytes. Among the

potent strains of about 13 isolates, 19.3% displayed both antibacterial and antifungal activity and

6.4% strain showed antimicrobial activity against all the test pathogens. The study reinforced the

assumption that endophytes of ethno medicinal plants could be a promising source of antimicrobial

substances.

An overview of A. racemosus and H. indicus

Alam et al., (1994) isolated, purified and partially characterized a viper venom inhibiting

factor from the root extract of the Indian medicinal plant sarsaparilla (Hemidesmus indicus R.Br.).

An organic acid was isolated and purified from the root extract of H. indicus R.Br possessing viper

venom inhibitory activity. The compound (designated HI-RVIF) was isolated by solvent extraction

Silica Gel Column Chromatography and Thin Layer Chromatography and was homogeneous in

nature. The white needle-shaped crystals were soluble in water, methanol and chloroform and had

a melting point of 155–158ºC and λmax 260 nm. Spectral analysis confirmed the presence of a

benzene ring, methoxy group and hydroxyl group the molecular weight of the compound was 168.

HI-RVIF significantly antagonized viper venom-induced lethal, haemorrhagic, coagulant and

anticoagulant activity in experimental rodents.

32

Detailed exploration of ulcer protective and healing effects of Asparagus racemosus on

various models of experimental gastroduodenal ulceration and patients with peptic ulcer was

analyzed. Their effects on mucin secretion, mucosal cell shedding, cell proliferation, antioxidant

activity, glycoproteins and PG synthesis have been reported. Clinical trials in peptic ulcer patients

were also done. Their potential ulcer protective effects both, experimental and clinical seemed to

be due to their predominant effects on various mucosal defensive factors and hence they have

potential usefulness for treatment of peptic ulcer diseases Goel and Sairam (2002).

Khanna and Kannabiran (2006) investigated the larvicidal effect of aqueous extracts of

Hemidesmus indicus roots, Gymnema sylvestre and Eclipta prostrata leaves against Culex

quinquefasciatus mosquito larvae at the concentrations of 1, 2, 3, 4 and 5% up to three days. All

extracts showed larval mortality. Larval mortality was 100% with the use of 5% concentration of

root extract of H. indicus leave extracts of G. sylvestre and E. prostrata after 2 days. Qualitative

analysis of the phytochemicals of aqueous extracts revealed the presence of carbohydrates,

saponins, phytosterols, phenols, flavonoids and tannins in all the plants. Quantitative analysis

showed that the crude saponin was the major phytochemical constituent present in highest

percentage followed by crude tannin in all three plants. It is suggested that all the three plants

possess larvicidal properties that could be developed and used as natural insecticides for mosquito

control.

Aqil et al., (2006) tested the methanolic crude extracts of 12 traditionally used Indian

medicinal plants including Hemidesmus inidcus were screened for their antioxidant and free

radical scavenging properties using a tocopherol and Butylated Hydroxy Toluene (BHT) as

standard antioxidants. Antioxidant activity was measured by Ferric Thio Cyanate (FTC) assay and

compared with the Thio Barbituric Acid (TBA) method. Free radical scavenging activity was

evaluated using Diphenyl Picryl Hydrazyl (DPPH) assay. Phytochemical analysis of plant extracts

indicated the presence of major phytocompounds, including phenolics, alkaloids, glycosides,

flavonoids and tannins. The tested plant extracts showed promising antioxidant and free radical

scavenging activity thus justifying their traditional use.

Krishnamurthy et al., (2008) researched on fungal communities in herbaceous medicinal

plants from the Malnad region of Southern India, a part of Western Ghats – hotspot of global

diversity. Nine important medicinal herbs were chosen includes H. indicus. Nearly 55 different

33

fungal species were isolated from 3600 leaf segments during wet and dry seasons the most

frequently occurring includes Chaetomium globosum, Aureobasidium pullulans, Cladosporium

cladosporioides, Nigrospora oryzae, Alternaria alternata and Curvularia lunata. Colonization

rates however varied in different seasons. Host specificity was also observed in some host plants.

Chitme et al., (2009) studied the antiosteoporotic activity of A. racemosus using

ovariectomized rat models for evaluation. Effects were evaluated by using biomechanical, bone

mineral, serum and urine biochemical parameters. Methanolic and aqueous extract obtained from

A. racemosus root has shown significant (p<0.05) effect on mineralization, ossification and

osteoclastic activity suppression were observed in histopathological examination. Obtained

treatments of ovariectomized rats by extract significantly reduced serum alkaline phosphatase

activity, serum calcium and also inhibited the ovariectomized induced excessive loss of calcium in

urine. The study concludes that phytosterols and other active constituents present in the root of A.

racemosus may effect on estrogen receptor similar to estrogen and produce antiosteoporotics

effect.

Garg and Gupta (2010) analyzed the adaptogenic activity of milk and aqueous decoction of

Asparagus racemosus Wild. in acute and chronic stress paradigms in mice. A. racemosus is

classified in Ayurveda the ancient Hindu system of medicine as a rasayana. According to this

conviction Ksheerpaka (milk decoction) of Shatavari (A. racemosus) is therapeutically more potent

than any other form of dosage. The present study was to investigate the adaptogenic activity being

tested to validate cited traditional claim regarding potentiality of milk decoction of A. racemosus

being determined via antistress effect using forced swimming stress model and chronic fatigue

stress model in rodents. The behavioral parameters (anxiety level, memory, muscle relaxation and

locomotor activity) were assessed in mice 24 h after the last chronic forced swim test. The results

indicate that milk decoction of A. racemosus has significant antistress adaptogenic activity

confirming the validity of traditional claim.

Gaur and Kaushik (2011) studied the influence of edaphic factors on distribution of

mycorrhiza associated with medicinal plants in central Himalayas. Effects of factors like pH,

organic carbon, potassium, phosphorous and soil type on the spore number of mycorrhiza

associated with three medicinal plants were studied, A. racemosus, Ocimum sp, and Catharanthus

roseus. Maximum numbers of spores were isolated from the soil of pH 6-7, with organic carbon

34

content of 0.82 to 0.85%. Phosphorous showed a negative correlation but potassium showed a

positive correlation on spore number. Also higher the moisture content it decreased the spore count

showing a negative impact.

Acremonium as an endophyte

Guo et al., (1992) studied the role of Acremonium sp. endophyte of tall fescue on inhibition

of colonization and reproduction of mycorrhizal fungi. Roots of Acremonium-free fescue were not

colonized as extensively as those of pearl millet by either fungus. The presence of Acremonium in

fescue caused a considerable reduction in extent of mycorrhizal colonization. Effects of the host on

sporulation were closely correlated with extent of colonization. Each of the three hosts supported a

similar total spore volume of the two mycorrhizal fungi. Acremonium apparently does not affect

infection perhaps because the seedlings were too young for extensive transport of toxic alkaloids

from Acremonium-infected shoot tissues to roots free of Acremonium.

Antagonistic effect of endophytes against pathogens of some plants is also prominent. This

effect has been studied in root rot pathogens of wheat. Four different endophyte species were

isolated from Rye grasses, Triticum sp. and Tall fescue. All Neotyphodium and Acremonium sp.

significantly affected the growth rate of five root-rot pathogens of wheat in PDA plates. Culture

filtrates of endophytes have had some effect against these test fungi. On conducting the

germination tests the pathogen has shown abnormal elongation of the hypha, lysis of the conidia

and abnormal germ tubes Tunali and Marshall (1995).

Hol et al., (2006) examined the interactions between the fungal endophytes and the root

herbivores of Ammophila sp and A. strictum (endophyte)-inoculated plants showed increased plant

development in terms of root biomass. The effect of A. strictum on interspecific competition was

also analyzed by plant inoculation with both nematode species. Plants inoculated with

Pratylenchus dunensis, Pratylenchus penetrans and A. strictum showed decreased total biomass

compared to A. strictum-free plants inoculated with the same nematodes

Effect of Endophytic fungi - Beaveria bassiana, Trichoderma koningii, Alternaria

alternata, Phoma sp., and A. strictum on the causal agents of maize seedling blight, stalk and root

rot was elucidated. These endophytes were isolated from maize roots while the pathogens,

Fusarium oxysporum, Fusarium pallidoroseum, Fusarium verticillioides and Cladosporium

35

herbarum were isolated from blighted maize plants. The in vitro assay of the endophytes against

the pathogens showed reducing radial growth by 25 -75% and 53 - 80%, respectively. The in vivo

studies revealed T. koningii as the best endophyte by reducing wilting rate to 25%. T. koningii and

A. Alternate could be successfully formulated and applied as alternative fungicides in the

management of maize wilt and seedling blight Orole and Adejumo (2009).

Secondary metabolites of fungal endophytes

Stierle et al., (1993) researched on taxol and taxane production by Taxomyces andreanae,

an endophytic fungus of Pacific yew, Taxus brevifolia. The hyphomyceteous fungus when grown

in a semi-synthetic liquid medium produced taxol and related compounds. Taxol was identified by

mass spectrometry, chromatography and reactivity with monoclonal antibodies specific for taxol.

Both [1-14C] acetic acid and L-[U-14

C] phenylalanine served as precursors of [14

C] taxol in fungal

cultures.

Schulz et al., (2002) reviewed on how endophytic fungi serve as a source of novel

biologically active secondary metabolites. Accordingly in course of the last 12 years, 6500

endophytic fungi were isolated from herbaceous plants and trees screened them for biological

activities and have isolated and determined the structures of the biologically active compounds.

The substances isolated were originated from different biosynthetic pathways belonging to diverse

structural groups. The potential role of the endophyte and its biologically active metabolites in its

association with its host has been investigated. 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. It was seen that

the fungal endophytes possess the exoenzymes necessary to colonize their hosts. Certain

endophytic interactions associated with roots of the host may be mutualistic improving growth of

the host and supplying the mycobiont with enough nourishment to extensively colonize the host's

roots. Further plant defense metabolites are higher in plants infected with endophytes. Hence the

interaction fungal endophyte–plant host is characterized by a finely tuned equilibrium between

fungal virulence and plant defence. Not only must the endophyte has to compete with epiphytes

and pathogens but presumably also has 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 and that fungi in different biotopes are still need to be exploited.

36

Antifungal products are vastly produced by majority of the endophytes. Griseofulvin-

producing endophyte was first reported in fungus from Abies holophylla and was evaluated in vivo

antifungal activity against plant pathogenic fungi. Based on nuclear ribosomal ITS1-5.8SITS2

sequence analysis, the fungus was identified and labeled as Xylaria sp. F0010. Two antifungal

substances, griseofulvin and dechlorogriseofulvin were purified from liquid cultures of Xylaria sp.

and identified through mass and NMR spectral analyses. Compared to dechlorogriseofulvin,

griseofulvin showed high in vivo and in vitro antifungal activity and effectively controlled the

development of rice blast (Magnaporthe grisea), rice sheath blight (Corticium sasaki), wheat leaf

rust (Puccinia recondita) and barley powdery mildew (Blumeria graminis f. sp. hordei) Park et al.,

(2004).

Hundley (2005) did structure elucidation of bioactive compounds isolated from endophytes

of Alstonia Scholaris and Acmena graveolens. In the present study, an endophyte of the genus

Xylaria was isolated from a stem of A. scholaris its mycelia and exudate extracted and the extract

assayed for growth inhibition of HeLa cancer cells in vitro. Several known compounds were

isolated and identified based on NMR, infrared and mass spectral data. The compounds identified

are 19, 20-epoxycytochalasin C; 19, 20-epoxycytochalasin D and xylobovide. Two other

compounds, fusaric acid and dehydrofusaric acid were discovered in an endophyte of the

Hypocreales family inhabiting the plant A. graveolens.

Kumala et al., (2007) were experimental in producing cytotoxic secondary metabolites

from the fermentation broth of the endophytic fungus isolated from the fruits of Brucea javanica.

In vitro cytotoxic assays were performed using leukemia cell line L1210. LC-MS analysis of the

F4 fraction of n-butanol extracts of secondary metabolites revealed bruceocin and canthin–6 one

compounds as cytotoxic constituents. These compounds were previously reported in the same host

plant. Hence the present study could demonstrate the possibility of the endophytic fungi living

symbiotically within the host plant producing cytotoxic secondary metabolites.

Wijeratne et al., (2008) isolated sesquiterpene, quinones and related metabolites from

Phyllosticta spinarum a fungal strain endophytic in Platycladus orientalis of the Sonoran Desert.

Five new metabolites (+)-(5S,10S)-4′-hydroxymethylcyclozonarone (1), 3-ketotauranin (3), 3R-

hydroxytauranin (4),12-hydroxytauranin (5), and phyllospinarone (6), together with tauranin (2)

were isolated and the structures of these new compounds were determined on the basis of their 1D

37

and 2D NMR spectroscopic data and chemical interconversions. Tauranin showed activity when

evaluated for inhibition of cell proliferation assay in a panel of five cancer cell lines that also

induced apoptosis in PC-3M and NIH 3T3 cell lines during flow cytometry.

Lactones of endophytic origin have antiparasitic activity against Plasmodium falciparum.

Three lactones were isolated from the culture medium of the endophytic fungus Xylaria sp. One

was identified as (+)-phomalactone (1). The others were 6-(1-propenyl)-3, 4, 5, 6-tetrahydro-5-

hydroxy-4Hpyran- 2-one (2) and 5-hydroxymellein (3). Compounds 1 and 2 were reported for the

first time as constituents of Xylaria. Also this study was the first report showing the activity of

these lactone compounds against a chloroquine-resistant Plasmodium falciparum strain, Romero et

al., (2008).

Different fungal species have been exploited as an alternative source of plant secondary

metabolites. Endophytic fungi colonize plants internally without apparent adverse effects and do

occur ubiquitously in plants. They are known to produce a number of important secondary

metabolites including anticancer, antifungal, antidiabetic and immunosuppressant compounds e.g.

paclitaxel, torreyanic acid, cytochalasins etc. have been isolated from endophytic sources. The

discovery of Stierle and his co-workers, studies carried out by Strobel and Daisy had raised scope

of using the endophytic fungus as a sustainable alternative source of important plant secondary

metabolites. However our poor understanding of the evolutionary significance of these organisms

and their dynamic interaction with their respective hosts results in failure of exploiting endophytic

fungi in diverse arenas Priti et al., (2009).

Kusari et al., (2009) isolated, identified and characterized an endophytic fungus,

Aspergillus fumigatus from Juniperus communis and L. Horstmann, as a novel producer of

deoxypodophyllotoxin and performed its in vitro antimicrobial assay against a panel of pathogenic

bacteria. The study concluded the production of deoxypodophyllotoxin (found in the host) by the

cultured endophyte which is an enigmatic observation. This demonstrates the horizontal

transmission of genes from the host plant to its endophytic counterpart. It would be interesting to

further study the deoxypodophyllotoxin production and regulation by the cultured endophyte as

well as their scale up process for consistent and dependable production.

Fungal fatty acids

38

Biochemical changes also play a vital role during the growth of fungi. Etten et al., (1965)

made such studies in Penicillium atrovenetum. Changes in the lipid constituents of P. atrovenetum

were studied during the growth and development of this fungus. The total fatty acids increased to a

maximum during the log phase and the major fatty acids were Palmitic, Stearic, Oleic and Linoleic

acids. Younger mycelium contained a much lower percentage (on the basis of total fatty acids) of

Linoleic acid compared to the ungerminated spores. Oleic acid was increasing as well as the

remaining fatty acids like Myristic, Palmitoleic, Pentadecanoic acid increased slightly but Palmitic

acid remained constant. Ergosterol was the only sterol detected. The following changes in lipids

appear to be associated with the development and aging of fungi: (i) the presence of a relatively

high content of non saponifiable lipid and ergosterol in the young mycelium and their later

decrease with age and (ii) a shift from more unsaturated to less unsaturated fatty acids with age,

Etten et al., (1965).

Sumner and Morgan (1969) studied the fatty acid composition of sporangiospores and

vegetative mycelium of temperature-adapted fungi in the order Mucorales. The lipid content and

fatty acid composition of sporangiospores and vegetative mycelium of mesophilic, thermotolerant

and thermophilic fungi in the Mucorales were examined. The spores contained lesser lipids than

the vegetative mycelium. Unusual fatty acids were not present as detected by Gas Liquid

Chromatography in the lipids of spores or mycelium. The fatty acid co positions of spores and

egetati e yceliu ere ualitati ely ery si ilar but spore lipids ere ig ly saturated t an

ycelial lipids. o ering gro t te perature fro 4 to C increased the synthesis of

unsaturated fatty acids in the spores and the mycelium of the thermotolerant and thermophilic

fungi examined.

Stahl and Klug (1996) analyzed cellular fatty acid compositions of fungi and that they can

be differentiated from one another on this basis. Many fungi were found to possess the same fatty

acids but produced different relative concentrations of each. Some fungi differed in both the fatty

acids produced and in the relative concentrations of others. Multivariate discriminant analysis

demonstrated that all of the species included in the study had significantly different (P < 0.001)

fatty acid profiles. Significant differences in fatty acid composition were also found at the

intraspecific level. Both culture temperature and age affected fatty acid composition in the fungi

39

examined but when these factors were held constant, variance in fatty acid composition was not a

problem and fungal fatty acid profiles could be differentiated statistically.

Olsson (1999) investigated the mycorrhizal fungal interactions in soil ecosystems. The

estimation of their biomass is of importance in order to understand their possible role in soil

nutrient processes. He analyzed that the signature fatty acid provides a new and promising tool for

the estimation of fungal biomass in soil and roots. In biomass estimation primarily the

phospholipid fatty acids (PLFAs) are suitable. Through the use of specific PLFAs it is possible to

study interactions between mycorrhizal mycelia and bacteria in soil as well as between AM fungal

mycelia and mycelia of saprophytic and parasitic fungi in soil and in roots. AM fungi in particular

store a large proportion of their energy as lipids and by using the signature fatty acids it is possible

to determine the relation between membrane and storage lipids which could be an indication of

energy storage levels. Various aspects of how the fatty acid signatures can be used for studies

related to questions of biomass distribution and nutritional status of mycorrhizal fungi are

discussed.

Guarro et al., (1999) discussed the developments in fungal taxonomy. Accordingly both the

type of fatty acid present and its relative concentration are useful characteristics for separating

taxa. Until recently these techniques were only rarely used in fungal taxonomy. Although fewer

different fatty acids are produced by fungi than by bacteria, these analyses are increasingly used

for differentiating fungi. Recently Gas Chromatography combined with methods of multivariate

statistical analysis has successfully been used to study the fatty acids of numerous and varied

filamentous fungi including Oomycetes, Zygomycetes, Basidiomycetes and even sterile mycelia

which are useful even at intraspecific level.

For the industrial production of ARA, various studies, such as isolation of a high-potential

strain and optimization of culture conditions, have been conducted. Studies including the

investigation of morphology are important because ARA is accumulated in the mycelia, and thus

cultivation with high biomass concentration is essential for obtaining a high ARA yield.

Combining the results derived from various studies, a high ARA yield was attained in an industrial

fermentor. These ARA production techniques are applicable to the production of other

polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation

technology especially in the field of fungal cultivation, Higashiyama et al., (2002)

40

Trepanier et al., (2005) investigated the dependence of arbuscular-mycorrhizal fungi on

their plant host for Palmitic acid synthesis. Lipids are the major form of carbon storage in

arbuscular-mycorrhizal fungi that fatty acid synthesis by Glomus intraradices and Gigaspora

rosea was studied presently. The growth stimulation of germinating spores by root exudates did

not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by

the fungi in the mycorrhized roots. The data suggest that the fatty acid synthase activity of

arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate

that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-

mycorrhizal fungi.

Analysis and Purification of fungal fatty acids

Purification of 9, 12-cis-hexadecadienoic acid by HPLC and further GC analysis was done

in Trichoderma sp. AM076, isolated from a freshwater sample. It was found to accumulate 9, 12-

cis-hexadecadienoic acid (16: ω4), en gro n it Pal itoleic acid (16:1ω7). Met yl yristate

was the best carbon source for the conversion of Pal itoleic acid to 16: ω4. T e ycelial 16: ω4

content reached 17.4 mg/g dry mycelia (443 mg/L) when the fungus was grown in a medium that

contained 2.0% methyl myristate, 1.5% yeast extract and 2.0% methyl palmitoleate, pH 6.0, for 5 d

at °C it s aking. In bot nonpolar and polar lipids fro t e ycelia, 16: ω4 as detected as

one of t e ajor fatty acids en 16:1ω7 as added. It is probable t at 16:1ω7 is con erted to

16: ω4 t roug t e δ-12 desaturation reaction, Shirasaka et al., (1998)

The lipid content of a source material/sample is traditionally determined gravimetrically by

solvent extractions. The more commonly methods for lipid extraction are Soxhlet method, acid

hydrolysis method, Bligh and Dyer methods. These extraction methods vary in their lipid

extraction efficiency. The total lipid by solvent extraction represents the content of crude fat.

Crude fat is heterogeneous material consisting of a mixture of triacylglycerols, phospholipids, fatty

acids, sterols, waxes and pigments which may also contain non-fat material hence needs to be

purified and often fails to accurately estimate the inhibitory effect on various cancer cell lines

Xiao (2010).

Enormous studies have been made in the development of methods for the analysis of lipids

using High Performance Liquid Chromatography (HPLC). In addition, it could be claimed that

41

there is no type of lipid separation for which Thin Layer Chromatography (TLC) was once

favoured that cannot now be done by HPLC. The latter offers great versatility in that it can be used

in the adsorption, reversed-phase, ion exchange and silver ion modes. It operates at room

temperature, so is particularly suited to molecules containing thermally labile functional groups. It

is possible to isolate the complex lipids from TLC plates and then re-chromatograph them with

more polar solvents (and with silica gel without added binder) to isolate each of the individual

phospholipid classes say with comparable resolution to HPLC. A host of bonded-phases, offering

varying selectivities in specific analyses, are available commercially and many have yet to be

properly explored in lipid applications, Christie and Han (2010).

During the synthesis of FAMEs, the effect of temperature on the reaction rate has shown to

have a significant impact on the transesterification reaction. At higher temperatures, 100ºC initially

showed a faster rate of reaction and reached complete extraction and conversion of TAG to FAME

around 45 min. However after 30 min the reaction rate slowed. At 90ºC the reaction took 60 min to

reach completion and then remained stable for a longer period of time than at 100ºC. While the

reaction does take place more rapidly at 100ºC, at this higher temperature there is evidence that the

FAMEs begin to degrade. Thus a reaction at 90ºC may be more suitable as it is more stable and

there is less risk of losing sample volume. In addition to temperature and biomass/solvent ratio, the

acid concentration became a significant factor when the concentration was lowered. This is

possibly due to the molar ratio of hydrogen ions to triglycerides, Nelson (2010).

Turbidometric growth studies of fungi

A microbroth kinetic model based on turbidity measurements was developed in order to

analyze the growth characteristics of three species of filamentous fungi (Rhizopus microsporus,

Aspergillus fumigatus and Scedosporium prolificans) characterized by different growth rates in

five nutrient media (antibiotic medium 3, yeast nitrogen base medium, Sabouraud broth, RPMI

1640 alone, and RPMI 1640 with 2% glucose). Among the different growth phases distinguished,

the smallest variability in growth rates among the strains of each species was found during the log

phase in all nutrient media. R. microsporus and A. fumigatus grew better in Sabouraud and yeast

nitrogen base medium than in RPMI 1640. None of the media provided optimal growth of S.

prolificans. The germination of Rhizopus spores and Aspergillus and Scedosporium conidia

commenced after 2 and 5 h of incubation, respectively. In conclusion, the growth curves provide a

42

useful tool to gain insight into the growth characteristics of filamentous fungi in different nutrient

media and may help to optimize the methodology for antifungal susceptibility testing (Meletiadis

et al., 2001).

Turbidometric growth curves of different filamentous fungi in the presence of increasing

concentrations of antifungal drugs was studied. 24 clinical mold isolates, including Rhizopus

oryzae, Aspergillus fumigatus, Aspergillus flavus, and Scedosporium prolificans, were tested

against itraconazole, terbinafine, and amphotericin B according to NCCLS guidelines. Exposure to

increasing drug concentrations resulted in prolonged lag phases of the turbidimetric growth curves.

The lag phases of the growth curves at drug concentrations which resulted in more than 50%

growth (for itraconazole and terbinafine) and more than 75% growth (for amphotericin B) after 24

h of incubation for R. oryzae, 48 h for Aspergillus sp., and 72 h for S. prolificans. Using this

system, itraconazole and terbinafine resistance (presence of >50% growth) as well as amphotericin

B resistance (presence of >75% growth) was determined within incubation periods of 5.0 to 7.7 h

for R. oryzae (for amphotericin B resistance incubation for up to 12 h was required), 8.8 to 11.4 h

for A. fumigatus, 6.7 to 8.5 h for A. flavus, and 13 to 15.6 h for S. prolificans while awaiting formal

MIC determination by the NCCLS reference method (Meletiadis et al., 2003).

Biotic and abiotic elicitors as enhancers for metabolite production

Marchand (2005) revealed the ability of fungi to utilize crude glycerol as an alternative to

conventional carbon substrates for growth and lipid production. Screening revealed that 40 of the

61 isolates tested had increased biomass yield compared to glucose, when crude glycerol was

utilized. 29 of these isolates possessed the ability to completely metabolize 14 gL-1

of glycerol

after 7-14 days. The top four candidates belonged to the genera Galactomyces and Mucor. Overall

Galactomyces sp. proved to be better suited for lipid production. In addition to producing biomass

with a high lipid content (up to 45 % w/w), Galactomyces sp. also exhibited high biomass yields

(up to 25 gL-1

). The results obtained in this study compare favourably and in some cases exceed,

other literature reported values for biomass and lipid production using glycerol.

Ahmaed et al., (2006) reported the effects of various process parameters on the production

of Gamma Linolenic acid in submerged fermentation. Seven strains belonging to Mucorales were

taken for the study. Oleaginous endophytic strain belonging to Western Ghats of Kerala produced

43

8% GLA using glucose as sole carbon source. Optimal conditions of 30ºC, 200 rpm for 7 days

yielded maximum dry biomass and GLA respectively. Also combination of yeast extract, corn

steep liquor and bakers yeast in ratio of 1:1:1 serve as better organic nitrogen source for increased

GLA and lipid production.

Muhid et al., (2008) studied the effect of metal ion concentrations on lipid and Gamma

Linolenic acid production in Cunninghamella sp. Effects of different concentrations of Magnesium

sulphate, Ferric chloride, Zinc sulphate, Copper sulphate and Manganese sulphate were examined.

Mg2+

, Fe2+

and Zn2+

showed significant effect on lipid accumulation by the fungi. In particular Zn2+

showed 74% increase in GLA content of the fungi. Hence the study suggests that critical

concentrations of metal ions for optimal production of lipids are required that might interfere in

pathways involving lipid biosynthesis.

Tauk et al., (2009) studied lipid formation and Gamma linolenic acid production by Mucor

sp. and Rhizopus sp. grown on vegetable oil. The fungal strains were tested in bioscreen automated

system to select the best nutritional source. Submerged cultivation was carried out in media

containing sole carbon/nitrogen source. Increased growth of fungi was observed in media

containing vegetable oil with higher concentration of lipids. Results revealed higher amount of

GLA was obtained with Mucor circinelloides in cultures containing sesame oil.

Raina et al., (2011) have shown in their studies that enhancement of productivity was

established with several fold increase that addition of oligosaccharide (oligomannuronate and

oligoguluronate blocks) resulted in a 50% increase in penicillin G yield for Penicillium

chrysogenum. Environmental abiotic and biotic stress factors have been proved to effect variety of

responses in microbes. Elicitors as stress factors induce or enhance the biosynthesis of secondary

metabolites added to a biological system. They are classified into various groups based on their

nature and origin: physical or chemical, biotic or abiotic. Carbohydrates as biotic elicitors

(oligosaccharides, oligomannuronate, oligoguluronate and mannan Oligosaccharides) have also

been used widely in small amounts (mg/ml) as elicitor molecules in bacterial and fungal

fermentations for overproduction of commercially important secondary metabolites.

Bioactivity assay and Apoptotic studies

44

Effects of fatty acids and inhibitors of eicosanoid synthesis on the growth of a human

breast cancer cell line in culture revealed results that Linoleic acid stimulated MDA-MB-231 cell

growth with an optimal effect at a concentration of 0.75 µg/ml, whereas oleic acid produced

growth stimulation at 0.25 µg/ml but was inhibitory at higher concentrations. Docosahexaenoic

acid exhibited a dose-related inhibition of cell growth at concentrations ranging from 0.5 to 2.5

µg/ml and eicosapentaenoic acid was less effective. Similar inhibitory effects occurred with other

saturated fatty acids, Rose and Connolly (1990).

Rodrigues and Hasse (2000) performed antimicrobial assays with the secondary

metabolites produced by endophytic fungi from Spondias mombin (Anacardiaceae). Few of the

isolated endophytes were chosen for preparation of culture broth extracts: Guignardia sp.

Phomopsis sp. and Pestalotiopsis guepinii. Extracts were separated by chromatographic methods

and tested for biological activities. The crude extracts were tested against 14 organisms including

actinomycetes, Gram-negative and Gram-positive bacteria, yeast and filamentous fungi. All fungal

extracts inhibited actinomycete growth. Guignardia sp. was active against Escherichia coli,

Staphylococcus aureus, Saccharomyces cerevisiae, Geotrichum sp. and Penicillium canadensis.

Culture extracts of P. guepinii were active against S. cerevisae, while strains of Phomopsis sp.

showed a pronounced antifungal effect against Cladosporium elatum, Mycotypha sp. and S.

cerevisae.

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death

induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of

an ordered cleavage of the DNA backbone which is separated and visualized as an electrophoretic

pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and

double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA 200-800 kb

or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are

produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH)

depletion in several types of mammalian cells. Reactive oxygen species (ROS) mediated DNA

fragmentation is enhanced by polyunsaturated fatty acids including Arachidonic acid or their

hydroperoxides leading to necrosis. Mitochondrial dysfunction on decrease of trans-membrane

potential accumulation of ROS membrane permeability transition and release of apoptotic factors

during apoptosis or necrosis has been implicated. This review refers to the correlation of

45

chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion and the

possible mechanisms of oxidative stress-induced cell death, Higuchi (2004).

Lotufo et al., (2005) evaluated the anticancer potential of 11 plants used in Bangladeshi

folk medicine. The extracts were tested for cytotoxicity using the brine shrimp lethality assay, sea

urchin eggs assay, hemolysis assay and MTT assay using tumor cell lines. The extract of Oroxylum

indicum showed the highest toxicity on all tumor cell lines as well as on the sea urchin eggs. The

extract of Aegle marmelos exhibited toxicity on all used assays but in a lower potency

than Oroxylum indicum. The study concludes that only the extracts of Oroxylum indicum, Moringa

oleifera and Aegles marmelos could be considered as potential sources of anticancer compounds

among the tested plant extracts. Further studies are necessary for chemical characterization of the

active principles and more extensive biological evaluations.

Discovery of Oleic acid as the major component of olive oil that is responsible for a

healthy Mediterranean diet was mentioned and the prevention of breast cancer by Oleic acid was

examined. One study indicated that the protection of olive oil against breast cancer may be due to

Oleic acid components rather than to the acid itself. Oleic acid is a monounsaturated fatty acid with a

symmetrically placed double bond. Its IUPAC name is cis-9-octadecenoic acid its lipid shorthand name

is 18:1 cis-9 and the CAS registry number is 2027-47-6, David Tin Win (2005).

Karagoz et al., (2007) demonstrated the cytotoxic activity of Astragalus chrysochlorus

crude extracts mixed with different solvents viz. hexane, chloroform, ethylacetate, 80 % ethanol

and water extracts prepared from roots and stems of Astragalus chrysochlorus. The cytotoxic

activity was tested on Vero (V) cells using the MTT assay. Viability of cell cultures was evaluated

in presence and absence of the extracts. Different and random combinations of plant part (stem,

root) solvent were checked where they found the chloroform-root extract exhibited the most

effecti e cytotoxic acti ity at 00 μg/ l (70.3 %) and t e exane-root and water-stem extracts of

Astragalus chrysochlorus ere not cytotoxic at 00 μg/ l.

Phongpaichit et al., (2007) studied on the biological activities of extracts from endophytic

fungi isolated from Garcinia plants. Sixty five crude extracts from 51 selected endophytic fungi

46

were tested for various bioactivities. 80% of the fungal extracts showed antimycobacterial

(76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1%

against NCI-H187 and 12.7% against KB cells) and cytotoxicity to Vero cells (40.0%). Molecular

methods based on internal transcribed spacer rRNA sequence analysis was used to identify 15

bioactive isolates as Aspergillus sp, Botryosphaeria sp, Curvularia sp, Fusicoccum sp, Guignardia

sp, Muscodor sp, Penicillium sp, Pestalotiopsis sp and Phomopsis sp. These results again prove

that endophytes are potential sources of various bioactive natural products.

Aly et al., (2008) produced bioactive metabolites from the endophytic fungus Ampelomyces

sp. isolated from the medicinal plant Urospermum picroides. Extracts of cultures of Ampelomyces

sp. exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells.

Chromatographic separation yielded 14 natural products including pyrone and sulfated

anthraquinones that were identified based on H1 and

13C NMR and mass spectral studies. Few

compounds exhibited strongest cytotoxic activity against L5178Y cells while few showed

antimicrobial activity against the Gram-positive pathogens, Staphylococcus aureus, S. epidermidis

and Enterococcus faecalis. The study further indicates the production of bioactive natural products

by the endophyte in their host under in-situ conditions.

Xu (2008) have found that the C. militaris extract can inhibit growth of MCF-7 human

breast cancer cells in a dose and time-dependent manner. In addition to the apoptotic genes the

levels of the methyltransferase gene DNMT1 and DNMT3a transcripts were also suppressed in

MCF-7 cells incubated with the C. militaris extract. Methylation in some tumor-suppressor genes

may potentially lead to regained expression of these genes and subsequent inhibition of cancer cell

growth and its extract inhibits human breast cancer cell growth through an apoptosis cascade by

inducing pro-apoptotic and suppressing antiapoptotic marker gene expression. C. militaris extract

reduced DNA methylation through the suppression of methyltransferase transcripts leading to the

recovery of tumor-suppressor genes and eventually inhibiting tumor cell growth.

Banu and Kumar (2009) did preliminary screening of endophytic fungi from medicinal

plants in India for antimicrobial and antitumor activity. 16 endophytic fungal isolates tested were

found to exhibit antitumor activity in the yeast cell-based assay. The screening of antimicrobial

activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi was carried out on

47

isopropanol extracts prepared from 121 isolates of endophytic fungi isolated from medicinal plants

in India that includes H. indicus. Sensitivity was found to vary among the microorganisms.

Bacillus subtilis, Saccharomyces cerevisiae and Alternaria sp. were susceptible to extracts from

three, two and two isolates of endophytic fungi, respectively. None was found effective against

Salmonella typhimurium. 16 endophytic fungal isolates tested were also found to exhibit antitumor

activity in the yeast cell-based assay.

Molecular studies

A t ird gene (δ-9-3) encoding a fatty acid δ-9-desaturase was isolated from the oil-

producing fungus Mortierella alpina. The predicted protein of 512 aa shared 53% sequence

identity it t e t o fatty acid δ-9-desaturases, ole1p and ole2p containing three histidine boxes,

four putative transmembrane domains and a C-terminal cytochrome b5 fusion that are typical of

most fungal membrane-bound fatty acid desaturases. However unlike the M. alpina ole1 and ole2

genes, t e δ-9-3 ORF failed to complement the Saccharomyces cerevisiae ole1 mutation. GC-MS

analysis of fatty-acid-supple ented ole1 yeast transfor ants containing t e δ-9-3 gene indicated

that this enzyme had negligible activity with endogenous Palmitic acid (16:0) as substrate and

moderate activity (30–65% desaturation) with endogenous Stearic acid (18:0). Yeast transformants

overexpressing any one of the three M. alpina fatty acid δ-9-desaturase genes or the S. cerevisiae

ole1 gene produced low amounts of Hexacosenoic acid [26:1(n-9)], a fatty acid that is not

normally present in yeast cells. Conversely high levels of desaturase in the endoplasmic reticulum

membrane of these yeast transformants may increase the availability of suitable monounsaturated

substrates for fatty acid elongation, MacKenzie et al., (2002).

Molecular studies have been carried out where gene clusters (lol-1 and lol-2) have been

identified for the production of loline alkaloids in the endophyte Neotyphodium uncinatum,

mutualistic symbiont with the grasses. LOL-1 which is a 25 kb region contains nine genes and

designated in order lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1 and lolE-1 where

as lol-2 contained the homologs lolC-2 through lolE-2 in the same order and orientation. lol-C

expression was decreased through RNAi interference and that loline-alkaloid accumulation in

culture (P < 0.001) was compared to the vector only controls thereby indicating involvement of

lol-C in biosynthesis of lolines. Hence the relationships of lol gene products indicate that the

48

pathway has evolved from various different primary and secondary biosynthesis pathways,

Spiering et al., (2005).

Fungal endophytes of the genera Epichloe and Neotyphodium form symbioses with grasses

of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some

strains produce the ergopeptine alkaloid ergovaline which is implicated in livestock toxicosis

caused by ingestion of endophyte infected grasses. Cloning and analysis of a nonribosomal peptide

synthetase (NRPS) gene from N. lolii revealed a putative gene cluster for ergovaline biosynthesis.

All genes in the cluster were highly expressed in planta but expression was very low or

undetectable in mycelia from axenic culture. This work provides a genetic foundation for

elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot

alkaloid compounds and the regulation of their synthesis in planta, Fleetwood et al., (2007).

MATERIALS AND METHODS

1. COLLECTION OF PLANT SAMPLES

Selection of host plants from appropriate sites and obtaining fresh plant material is

important for studying the occurrence and distribution of fungal endophytes.

1.1 Host and site collection

T o edicinal plant sa ples ere c osen and collected fro t e Irula tribe o en’s

welfare society (ITWSS), Thandarai (12º39'32"–12.6

º39'32"N:79.6

º2'45"- 80

º2'45"E), with an

average annual temperature 29ºC, Uthiramerur taluk, Kancheepuram district near Chengalpet

which is located in South Chennai. The samples were collected every 3 months during a year (June

– Aug, Sep – Nov, Dec – Feb, Mar - May) and the collection was repeated for two years.

1.2 Collection of Plant samples