interaction of a medicinal plant coleus forskohlii...

Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 24 -

STATE-OF-ART


2. STATE OF ART 2.1. Introduction to Medicinal Plants

World is endowed with a rich wealth of medicinal plants. Medicinal herbs are

known as sources of phytochemicals, or active compounds, that are widely sought after

worldwide for their natural properties. Herbs have always been the principal form of

medicine in India and presently they are becoming popular throughout the developed world.

It is estimated that around 70,000 plant species, from lichens to towering trees, have been

used at one time or another for medicinal purposes. The herbs provide the starting material

for the isolation or synthesis of conventional drugs (Purohit and Vyas 2007). Nearly 25,000

effective plant based formulations are used in folk medicine by rural communities in India

(Ramakrishnappa 2002). Both plant species and traditional knowledge are important for the

herbal medicine trade and the pharmaceutical industry, whereby plants provide raw

materials and the traditional knowledge is the prerequisite information (Tabuti et al. 2003).

2.2. Importance of Medicinal Plants for Society

Medicinal plants have curative properties due to the presence of various complex

substances of different composition, which are found as secondary plant metabolites in one

or more parts of these plants. These plant metabolites according to their composition are

grouped as alkaloids, glycosides, corticosteroids, essential oils, etc. The alkaloids form the

largest group which includes morphine and codein (poppy), quinine (Cinchona), reserpine

(Rauwolfia), aconitine (Aconite) and a large number of others. Glycosides form another

important group represented by digoxin (Foxglove), barbolin (Aloe) etc. Some essential oils

like valerian kutch and peppermint also possesses medicating properties and are used in the

pharmaceutical industry (Purohit and Vyas 2007).

The essential oils or aromatic oils are steam volatile, odoriferous substances, mainly

composed of terpenoids. The steam volatization distinguishes them from other fatty oils.

These are the intermediate or final metabolic products and found in glands and special

secretary cells. In some cases the entire plant produces essential oil while in certain cases

only a particular part such as leaves (Eucalyptus), flower (rose), bark (cinnamon), wood

(sandal) and many more produce essential oil. Besides many benefits of essential oil to

plants and people, it also possesses antibacterial properties and is used for imparting aroma

to pharmaceutical preparations to ward- off unpleasant odor and makes them palatable.

Some essential oils are used as therapeutic and purifying agents to combat diseases and as


bacteriocide also used for stomach ache, expectorant, anti- inflammatory agents, as diuretic

and for maintenance of state of equilibrium (Shiva et al. 2002).

2.3. Family Lamiaceae

Lamiaceae has no poisonous members and includes a number of medicinal or sub-

medicinal plants of great value. Lamiaceae, the mint family of flowering plants, with 236

genera and more than 7000 species is the largest family of the order lamiales. They mostly

exhibit aromatic or bitter aromatic, stimulant and astringent properties; and they are used as

tonics, emmenagogues, diaphoretics and antispasmodics (Kirtikar and Basu 2005).

Members of the Lamiaceae have been used since ancient times as sources of spices and

flavorings (Hirasa and Takemasa 1998) and for their pharmaceutical properties (Bais et al.

2002). It is generally accepted that the medicinal properties of this family are due to

secondary metabolites such as phenolic compounds (including flavonoids and

phenylpropanoids) as well as anthocyanins (Phippen and Simon 1998, 2000; Kahkonen et

al. 1999). Important phenolic compounds within the Lamiaceae include rosmarinic acid

(RA), an ester of caffeic acid (CA), which is commonly recognised to have a number of

biological activities, predominantly as an antioxidant but also antibacterial, antiviral and

anti-inflammatory (Petersen and Simmonds 2003). Antioxidants have been used in the food

processing industry for more than 50 years (Cuvelier et al. 1994), and natural antioxidants

such as RA have recently gained recognition because of major concerns about toxic side

effects of synthetic antioxidants like BHT (butylated hydroxytoluene) and BHA (butylated

hydroxyanisole) in food (Pizzale et al. 2002; Sacchetti et al. 2004). Species of Lamiaceae

are also valued for their pharmaceutical properties; for example, the aromatic oils produced

in their leaves are used as antioxidants (Chang et al. 1977; Gang et al. 2001; Etten et al.

1994).

Medicinal properties exhibited by members of family lamiaceae are Mentha

arvensis, Plectranthus amboinicus, Coleus forskohlii, Ocimum basilicum, Origanum

vulgare and many more. Mentha arvensis is an important industrial crop for the production

of menthol, which is extensively used in cosmetics, pharmaceutical, food and flavoring

industries (Gupta et al. 2002). Origanum vulgare has long been recognized as a culinary

herb and medicinal plant with beneficial effects on the digestive and respiratory systems and

antiseptic, antispasmodic, carminative and cholagogue properties (Morone-Fortunato and


Avato 2008). Sweet basil (Ocimum basilicum) belonging to the Lamiaceae has been

traditionally used for the treatment of many ailments, such as headaches, coughs and

diarrhea (Phippen and Simon 1998; Javanmardi et al. 2002; Copetta et al. 2006; Toussaint

et al. 2007). Plectranthus amboinicus Lour. Spreng., also known as Indian borage is an

important medicinal plant largely used in Indian siddha medicine and the leaves are used for

the treatment of urinary diseases, epilepsy, chronic asthma, cough, bronchitis, and malarial

fever, in addition to acting as a powerful aromatic carminative (Rajeshkumar et al. 2008).

2.4. Genus Coleus

More than 150 species belong to genus Coleus, a member of the family Lamiaceae.

Some species especially those with showy colorful foliage, are grown as ornamentals all

over the world. In India, tubers of some coleus species, C. tuberosus and C. forskohlii are

eaten as vegetables and pickles, leaves of C. amboinicus are used as spices, used as

medicines as it is active against skin problems and worms (Petersen 1994). Coleus

(Solenostemon rotundifolius) is a minor tuber crop grown mainly in the homesteads as a

vegetable. It is commonly known as ‘koorka’, ‘cheevakizhangu’ or Chinese potato. Tubers

are preferred for its particular aromatic flavor and sweetness (Archana and Swadija 2000).

In India, the major medicinal species of Coleus is the tuberous C. forskohlii, C.

amboinicus, C. blumei, C. zeylanicus, C. malabaricus and C. scutellaroides and other

species are mainly used to treat dysentery and digestive disorders (De Souza et al. 1983;

Kurian and Sankar 2007).

2.4.1. Coleus forksholii and it’s Significance

2.4.1.1. Origin, Geographical Distribution and Species Status

Coleus forskohlii (willd.) Briq. syn. C. Barbatus ((Andr.) Benth) is an aromatic

herbaceous species of medicinal importance. Indian sub- continent is considered as the

place of origin of C. forskohlii (Valdes et al. 1987; Patil et al. 2001). It grows wild in the

sub-tropical warm temperate climates of India, Nepal, Burma, Sri Lanka and Thailand.

Apparently, it has been distributed to Egypt, Arabia, Ethiopia, tropical East Africa and

Brazil (Willemse 1985). In India, the plant grows wild in the Himalayan region, from the

Shimla hills extending through the Kumaon and Garhwal hills, at an altitudinal range of

600-2300m, in the Parasnath hills (Bihar) and in Gujrat and Western Ghats (Chandel and

Sharma 1997). It is commonly seen on dry, barren hills, wastelands and agricultural fields


throughout the tropical regions of South India, North eastern India and Andaman and

Nicobar islands (Kurian and Sankar 2007).

C. forskohlii Briq., a medicinal plant, is a member of the mint family, Lamiaceae. It

is indigenous to India and is recorded in Ayurvedic Materia Medica under the Sanskrit

name ‘Makandi’ and ‘Mayani” (Shah 1996). The taxonomic position of C. forskohlii is as

follows:

Kingdom - Plantae

Division - Magnolophyta

Class - Magnoliopsida

Order - Lamiales

Family - Lamiaceae

Genus - Coleus

Species - forskohlii

The genus Coleus was first described by Loureiro in 1790 and the generic name was

derived from the Greek word ‘COLEOS’ meaning sheath. All the species of Coleus have

four didynamous, dedinate stamens, and the filaments of the stamens unite at their base to

form a sheath around the style. The species name forskohlii was given to commemorate the

Finnish botanist, Forskel. The genus Coleus consists of 150 species and the following

species viz., C. amboinicus, C. forskohlii, C. spicatus and C. malabaricus occur naturally

(Kavitha et al. 2010). Some species especially those with showy colorful foliage, are grown

as ornamentals all over the world. Related species of Coleus with medicinal properties

include C. ambonicus, C. zeylanicus and C. blumei (Kurian and Sankar 2007).

2.4.1.2. Genetic Base with Chromosome Ploidy

Reddy (1952) reported that C. forskohlii is diploid with n =14. Riley and Hoff

(1961) has reported that the chromosome numbers in C. forskohlii in South African

dicotyledons is diploid with basic chromosome number n = 16. Bir and Saggoo (1982,

1985) reported that Central Indian collections have basic number of n = 17, while South

Indian collections have n = 15 and concluded that variability in base number of various

members of the family could be due to aneuploidy at generic level which ultimately leads to


morphological variations. It is reported that populations from different eco-geographic areas

vary greatly in their morphology (Kavitha et al. 2010).

2.4.1.3. Botanical Description

C. forskohlii is a perennial herbaceous plant that grows to about 45 -60 cm tall (Fig.

1). It has four angled stems that are branched and nodes are often hairy. The entire plant is

aromatic. Leaves are 7.5 to 12.5 cm in length and 3 to 5 cm in width, usually pubescent,

narrowed into petioles. Inflorescence is raceme, 15 – 30 cm in length; flowers are stout, 2 to

2.5 cm in size, usually perfect and calyx hairy inside. The ovary is four loculed and stigma

is two lobed and the flower is cross-pollinated by wind or insects. Fruits are nutlets (Kavitha

et al. 2010). The root is thick, fibrous and radially spreading. Roots are tuberous,

fasciculated, conical fusiform, straight, orangish within and strongly aromatic. C. forskohlii

is the only species of the genus to have fasciculate tuberous roots (Fig.1C). The leaves and

tubers have quite different odours. However, the growth habit of C. forskohlii is strikingly

variable being erect, procumbent or decumbent. Similarly, the root morphology in different

populations is also fascinatingly diverse, being tuberous, semi tuberous or fibrous (Kavitha

et al. 2010).

2.4.1.4. Cultivation Practices

C. forskohlii thrives well in red, sandy loam soils with a pH ranging from 5.5 to 7.

Humid climate with relative humidity between 83- 95 per cent and a temperature of 10 to

25°C is ideal for the crop. It requires an annual rainfall of 100 to 160 cm, necessarily

between June-September (Shah and Kalakoti 1996). It is propagated by seeds as well as

vegetatively by terminal stem cuttings. Seed propagation is difficult and slow whereas

propagation by terminal stem cutting is easy and economical (Fig. 2). When the cuttings are

one month old and have produced sufficient roots, they are transplanted to the main field.

The best period for planting is during June/July and September/ October. Regular care about

watering, weeding and plant protection should be taken (Kavitha et al. 2010). The crop

responds well to organic and inorganic fertilizers. A combination of 40 kg N, 60 kg P2O5

and 50 kg K2O per ha is optimum for obtaining the maximum fresh (120 t/ha) and dry

(3.982 t/ha) tuber yield. Half the dose of N, the whole P and whole K may be applied as the

basal dose followed by the remaining half N, 30 days after planting as top dressing (Kavitha

et al. 2010).


The leaf eating caterpillars, mealy bugs and root knot nematodes are the important

pests that attack this crop. The plant is susceptible to root rot and wilt caused by the fungal

pathogen Fusarium chlamydosporum and studies have shown the AM fungus Glomus

fasiculatum and Pseudomonas fluorescens were the most effective

Fig. 1: Coleus forskohlii. A: Growing in field condition; B: Full Plant; C: Typical tuberous roots

B C

A

A

CB


C

A

C

B

Fig. 2: Cultivation of C. forskohlii. A: Cuttings of C. forskohlii to induce rooting; B: 1 month old rooted cutting in green house; C: Rooted cutting transplanted in field

C

B

A

C


treatments that reduced the severity of root rot and wilt of C. forskohlii by 56-65 per cent

and 61-66 per cent, respectively, under lower and higher levels of pathogen F.

chlamydosporum (Singh et al. 2009). The wilt caused by Fusarium chlamydosporum is a

very serious soil-borne disease but inoculation with Trichoderma viride and Glomus

mosseae gave the best result in controlling the disease (Boby and Bagyaraj 2003).

Senthamarai et al., (2006 a, b) studied in details about nematode- fungal disease complex

involving Meloidogyne incognita and Macrophomina phaseolina. They have evaluated the

biocontrol agents against management of Meloidogyne incognita (root knot nematode) in C.

forskohlii. Pseudomonas fluorescens at the rate of 2.5Kg/ha showed increased plant growth

and reduced root knot nematode population both in soil and the root. Soil application of

Trichoderma viride at the rate of 2.5kg /ha recorded increased plant growth and reduced

nematode population compared to control followed by P. fluorescens (Senthamarai et al.

2006c). The root rot caused by Macrophomina phaseolina affects the tuber yield up to 100

per cent and application of bioformulation viz., Trichoderma harzianum and zinc sulphate

exerted maximum reduction in root rot incidence (Kamalakannan et al. 2006). The crop is

ready for harvest 4 1/2 to 5 months after planting. The plants are uprooted, the tubers

separated, cleaned and sun dried.

On an average, a yield of 800 to 1000 kg/ha of dry tubers may be obtained.

However, if proper cultivation practices were applied, yield of up to 2000 to 2200 kg/ha of

dry tubers could be obtained from cultivation of C. forskohlii (Kavitha et al. 2010).

2.4.1.5. Active Ingredients and Economic Importance

C. forskohlii being aromatic, whole plants including roots, flowering shoots and

leaves are aromatic parts. Roots are the source of an active principle forskolin (coleonol),

which is a diterpenoid and used as drug (Fig. 3). Although diterpenoids are found in almost

all parts of the plant, the roots are the main source (Chandel and Sharma 1997). Whole plant

and roots contain 0.05 and 0.1 per cent forskolin, respectively. Besides, roots also contain

coleosol and colenone. Leaves contain a diterpenoid methylene quinine, coleon, barbatusin

and cyclobutatusin. Barbatusin has inhibitory action against lung carcinoma and lymphatic

leukemia (Kurian and Sankar 2007). Other secondary compounds found in C. forskohlii are

monoterpenes, monoterpene glycosides, sesquiterpenes and phenolic glycosides (Ahmed

and Vishwakarma 1988; Ahmed and Merotra 1991; Petersen 1994).


C. forskohlii is widely used in different countries for various ailments. In Egypt and

Africa, the leaf is used as an expectorant, emmenagogue and diuretic. In Brazil, it is used as

a stomach aid and in treating intestinal disorders (Valdes et al. 1987). It is used as a

condiment in India and the tubers are prepared as pickle and eaten (Shah et al 1980; Kavitha

et al. 2010). In traditional Ayurvedic systems of medicine, C. forskohlii has been used for

treating heart diseases, abdominal colic, respiratory disorder, insomnia, convulsions,

asthma, bronchitis, intestinal disorders, burning sensation, constipation, epilepsy and angina

(Ammon and Muller 1985). The plant is also used for veterinary purposes (De Souza and

Shah 1988). Forskolin is also used in the preparation of medicines that suppresses hair

graying and restoring grey hair to its normal color (Keikichi et al. 1988). Forskolin is also

valued for antiallergic activity (Gupta et al. 1991). Roots are hypotensive and spasmolytic

and are given to children in constipation. Its decoction has tonic effect and is a wormicide.

Root paste mixed with mustard oil is used against boils. Ground root is externally applied to

eczema and other skin diseases. Forskolin, isolated from roots is a broncho dialator and is

used in treatment of congestive heart failure. It is effective against thrombosis and is

employed in glaucoma therapy, owning to its adenylated cyclase stimulant activity (Kurian

and Sankar 2007; Chandel and Sharma 1997).

Forskolin possesses positive inotropic and blood pressure lowering activity through

intravenous administration, is a CNS depressant, bronchodilator (Lichey et al. 1984), serves

nerve regeneration and lowers intraocular pressure (Caprioli and Sears 1983; Meyer et al.

1987; Chandel and Sharma 1997).

This indigenous species, besides being used as a medicinal plant, is used as a potent

source of essential oil (Patil et al. 2001). The essential oil present in tubers has very

attractive and delicate odor with spicy note (Misra et al. 1994). Essential oil has potential

uses in food flavoring industry and can be used as an antimicrobial agent (Chowdhary and

Sharma 1998).

The principle mechanism by which forskolin exerts its hypotensive activity is by

stimulation of adenylate cyclase and thereby increasing cellular concentration of the second

messenger cyclic AMP (cAMP). Forskolin directly activates almost all hormonesensitive

adenylate cyclases in intact cells, tissues and even solubilised preparation of adenylate

cyclase (Kavitha et al. 2010).


2.4.1.6. Phytochemistry

2.4.1.6.1. Forskolin

The tuberous root extracts of C. forskohlii contain minor diterpenoids viz.,

deactylforskolin, 9 - deoxyforskolin, 1, 9 -deoxyforskolin, 1, 9 - dideoxy - 7 -

deacetylforskolin in addition to forskolin (7β - acetoxy - 8, 13-epoxy-1α, 6 β,9α -

trihydroxylabd-14-en-11-one) (Ammon and Kemper 1982; De Souza and Shah 1988).

Forskolin was discovered in the year 1974 and was initially referred to as coleonol. After

the identification of other coleonols and diterpenoids the name was later changed to

forskolin (Saksena et al. 1985). Shah et al. (1980) reported that forskolin occurred

exclusively in C. forskohlii and could not be detected in six other Coleus species viz., C.

amboinicus, C. blumei, C. canisus, C. malabaricus, C. parviflorus and C. spicatus and six

taxonomically related Plectranthus species viz., P. coesta, P. incanus, P. melissoides, P.

mollis, P. rugosus and P. stocksii. Studies carried out using one hundred samples belonging

to species of Coleus, Orthosiphon and Plectranthus of the sub family Ocimoideae at Japan

also revealed the absence of forskolin in all the samples. Mathela et al. (1986) had identified

seven monoterpenes and nine sesquiterpene hydrocarbons and five oxygenated compounds

in the steam distillate from the roots of C. forskohlii.

Second generation forskolin derivatives viz., Δ5-6-deoxy-7-deacetyl-7-methyl amino

carbon forskolin (HIL 568), a potential antiglaucoma agent and 6-(3-dimethylamino

propionyl) forskolin hydrochloride (NKH 477), a potential cardio tonic agent were

developed (Hosono et al. 1990).

Tandon and his colleagues isolated antihypertensive labdene diterpenoid 13-epi-9-

deoxycoleonol (13-epi-9deoxyforskolin) from C. forskohlii and the stereo structure of the

diterpenoid ascertained by various 2D NMR techniques (Tandon et al. 1992). The structure

of two new minor diterpenes 1,9 dideoxy coleonol-B and 1- acetoxy coleosol, isolated from

Fig. 3: Structure of forskolin


the roots of C. forskohlii have been shown to be 7- hydroxyl-6-acetoxy,13-epoxy labd-14-

en-11-one and 1-acetoxy-6,9-dihydroxy,13-epoxy labd-14-en-11-one, respectively, mainly

through the interpretation of 2D NMR data and X ray analysis ( Roy et al. 1993). Shen et

al. (2002) isolated two new diterpenoids, forskolin G and H from the chloroform extract of

the roots of the C. forskohlii and based on the spectroscopic data their structures were

identified as 1α-hydroxy-6β,7β-diacetoxy-8,13-epoxylabd-14-ene-11-one and 1α,6β-

diacetoxy-8,13-epoxylabd-14-ene-11-one. Newer compounds are being identified from the

root extracts of C. forskohlii. Two new diterpenoids forskolin I (1 α, 6 β diacetoxy- 7 β, 9 α

-dihydroxy-8, 13-epoxylabd-14- en-11-one) and J, (1 α, 9 α -dihydroxy-6 β, 7 β diacetoxy-

8, 13-epoxylabd-14-en-11-one) were isolated from C. forskohlii plants collected in Yunnan

Province (Shen and Xu 2005).

Recently, two more new labdane diterpene glycosides, forskoditerpenoside A, B

were also isolated from the ethanol extract of the whole plant (Shan et al. 2007). This was

the first report about the occurrence of glycosides derived from labdane diterpene in the

nature and these compounds showed relaxative effects on isolated guinea pig tracheal

spirals in vitro. Later, three new minor labdane diterpene glycosides, forskoditerpenoside C,

D and E and a novel labdane diterpene forskoditerpene A from the ethanol extract of the

whole plant of C. forskohlii were isolated (Shan et al. 2008). Forskoditerpenoside C, D and

E showed relaxative effects on isolated guinea pig tracheal spirals in vitro and an unusual 8,

13-epoxy-labd- 14-en-11-one glycoside pattern. Forskoditerpene A is the first known

labdane derivative with a spiro element. Forskolin is in great demand in Japan and European

countries for its medicinal use and related research purposes.

2.4.1.6.2. Essential Oil

Essential oil from roots contains 3-decanone, bornyl acetate, a sesquiterpene

hydrocarbon, β-sesquiphellandrene, γ-eudesmol as major constituents (Misra et al. 1994;

Singh et al. 2002a). Singh et al. (2002a) has reported during flowering accumulation of

camphor is highest (23.3 per cent). They have reported seven new compounds were detected

from the leaf oil, which constituted 60-97 per cent of the oil. Kerntopf et al. (2002) reported

the major constituents of oil from leaves, stem and roots as α-pinene (22.2 per cent), β-

phellandrene (26.1 per cent) and (z) β ocimene respectively, in the plants grown in Brazil.


2.4.1.7. Extraction and Separation

2.4.1.7.1. Forskolin

The active compound, forskolin, a labdane diterpenoid is extracted from tubers of

medicinal plant C. forskohlii. The tubers are harvested at 75 to 85 per cent moisture level

on wet basis and stored at less than 12 per cent moisture after drying. Sun drying required

longer period than mechanical drying and recorded the lowest recovery of forskolin. Tubers

mechanically dried at 40°C with tuber slice thickness of 0.5 cm and packed in polyethylene

lined gunny bag retained the highest amount of forskolin (Kavitha et al. 2010). Different

rapid, precise methods for the quantitative estimation of forskolin such as thin layer

chromatography (TLC), gas liquid chromatography (GLC) and high pressure liquid

chromatography (HPLC) have been developed by Inamdar et al. (1980). Later, thin layer

and high performance liquid chromatographic (HPLC) methods are employed. HPLC

method is found to be more rapid and less sensitive than GLC and used to monitor variation

in forskolin content in different germplasm (Inamdar et al. 1984). A monoclonal antibody

specific for forskolin has been developed for affinity isolation of forskolin and it has been

used for extremely sensitive quantification of forskolin in plant tissues at different stages of

development (Yanagihara et al. 1996).

Srivastava et al. (2002) reported the extraction of powdered drug (1gm) by refluxing

for 5 m on water bath with 5ml benzene, then filtered and filtrate was used for analysis,

Mersinger (1988) reported the extraction of cell material by harvesting, freeze-drying and

extracting twice with dichloromethane for 30 m under reflux. Inamdar (1984) reported the

extraction of dried and finely powdered roots (1g) of C. forskohlii with benzene (3 x 50ml)

at 70°C for 2 h. Reddy et al. (2005) reviewed various techniques in details used for the

extraction of forskolin from C. forskohlii.

Nuclear magnetic resonance data and a gas chromatography-mass spectral method

are also used for forskolin quantification (Demetzos et al. 2002). Mukherjee et al. (1996)

performed the HPLC analysis of the extracts and standard solutions, by injecting 10µl of the

each standard solutions and extracted with Hamilton syringe, using C-18 column (Tracer

Analitica, Nucleosil- 100, 25cmX0.4cm) with Photodiode Array (SPD-M10A VP model)

detector. Sasaki et al. (1998) reported the HPLC method with minor changes. Reversed-

phase liquid chromatography with a photodiode array detector at 210 nm is successful in the


qualitative and quantitative evaluation of forskolin in plant material and in market products

claiming to contain forskolin (Schanebera and Khan 2003). A simple, safe, rapid and

economical reverse phase high performance liquid chromatography (RP-HPLC) method

using activated charcoal as an adsorbent in column is developed for the isolation of high-

purity forskolin (Saleem et al. 2006).

2.4.1.7.2. Essential Oil

Detection of essential oils in C. forskohlii is done with the help of Gas

Chromatography- Mass Spectroscopy (GC-MS). The materials from which essential oil to

be extracted (leaves, inflorescences etc.) are subjected to Clevenger’s apparatus from which

colorless oil is collected (Singh et al. 2002a; Khare et al. 2007)

2.4.1.8. Forskolin Content in C. forskohlii

The plant C. forskohlii is valued as a source for forskolin, owing to its unique

pharmacological properties. The presence of yellowish to reddish brown cytoplasmic

vesicles in cork cells of C. forskohlii tubers is unique character of this plant and these

vesicles store secondary metabolites including forskolin (Abraham et al. 1988). This has

been further confirmed by another group of scientists from University of Mumbai, Mumbai.

They found epidermis of the leaf of C. forskohlii showed presence of yellowish to reddish-

brown glands, which are a characteristic feature of this plant. They established that these

yellowish and reddish-brown masses are of diagnostic importance for this drug plant and

can be used for its characterization. Quantification of forskolin in different tissues indicated

that terpenoids are more concentrated in the woody layer (Narayanan et al. 2002).

There is a wide variation in morphology, essential oil content and yield parameters

among the genotypes of C. forskohlii (Patil et al. 2001). Chromatographic analysis of C.

forskohlii extracts from Brazil, Africa and India revealed that plants from each country

produced different compounds in variables quantities and the differences were attributed to

genetic or climatic factors (Tandon et al. 1979). Vishwakarma et al. (1988) attempted to

screen 38 genotypes collected from various locations to identify the potential genotypes for

forskolin. Content of forskolin varies substantially with different genotypes, from 0.01-0.44

per cent.

The demand for forskolin was mainly satisfied by large scale and indiscriminate

collections of C. forskohlii from wild habitats. Since C. forskohlii up to now is the only


known plant source of forskolin (Petersen 1994; Kavitha et al. 2010), this has lead to a

severe depletion of the plant (Vishwakarma et al. 1988) and C. forskohlii is listed as one of

the endangered plant species in India (Sharma et al. 1991). Today, large scale field

cultivation of C. forskohlii is used in India to produce large amounts of plant material for

the isolation of forskolin.

The major hurdle faced at present is that the level of forskolin is very low and it

seems difficult to produce economically. Moreover, the growth rhythm of the plant is

comparatively slow and the alkaloid accumulation pattern is influenced by environmental

and/or geographical conditions (Chandel and Sharma 1997).

2.4.1.9. In Vitro Propagation of C. forskohlii

Tissue culture is one of the most important applications of modern biotechnology in

horticulture. Traditional micropropagation techniques allow rapid production of high

quality, disease free (Raaman and Patharajan 2006) and uniform planting material in

relatively short period of time. It offers several distinct advantages not possible with

conventional propagation techniques (Rajasekharan and Ganeshan 2002). Plant tissue

culture relies on growing plants on nutrient rich growth substrates devoid of microbes,

which results in the production of plantlets without any mutualistic symbiosis (Dolcet-

Sanjuan et al. 1996). In vitro propagation is useful for mass multiplication and germplasm

conservation of any plant species. C. forskohlii being succulent in nature responds well to in

vitro propagation and various explants viz., nodal segments, shoot tip, leaf etc., are

effectively used (Kavitha et al. 2010). Sharma et al. (1991) reported that nodal segments as

explants on MS medium supplemented with Kinetin (2.0 mg/L) and IAA (1.0 mg/L) are

rooted well and their plantlets were established successfully under field conditions. Sen and

Sharma (1991) had reported that shoot multiplication of C. forskohlii was obtained in vitro

within 20-25 days from the shoot tip explants of 30 day old aseptically germinated seedling

using 6-benzylaminopurine (2.0 mg/L). Flowers of this plant had been used for

micropropagation studies and shoots as well as root formation were observed in Murashige

and Skoog’s (MS) medium supplemented with naphthalene acetic acid (0.5mg/L) and

kinetin (2.0mg/L) after 32 days of growth (Suryanarayana and Pai 1998). They also reported

that flowers were a better alternative to regeneration from callus. Reddy et al. (2001)

developed a plant establishment protocol from leaf derived callus and found that the in vitro


raised plants produce comparable quantity of forskolin with that of wild plants.

Bhattacharyya and Bhattacharya (2001) found that complete plantlets of C. forskohlii were

developed within 35-40 days by culturing shoot tip explants in MS medium containing 0.57

µM IAA and 0.46 µM kinetin through direct multiplication at the rate of 12.5 shoots per

explant The significance of the above protocol was the formulation of growth regulators

which affected very fast multiplication of the plant in less time that is, one-third time less of

the previously known methods. Leaf explants of C. forskohlii induced callusing when

cultured on MS media supplemented with Benzene amino purine (1 mg/L) and naphthalene

acetic acid (2 mg/L). Regeneration of shoot-lets was observed after 7 weeks of initial

culture (Anbazhagan et al. 2005).

2.4.1.10. In vitro Conservation of C. forskohlii

The population of C. forskohlii is becoming low due to its large scale indiscriminate

collection which has lead to its rapid depletion from wild population listing it as vulnerable

plant in India (Gupta 1988; Sharma et al. 1991 and Bhattacharyya et al. 2001). Therefore,

the conservation of such rare and endangered plant species has become imperative. C.

forskohlii is mainly propagated vegetatively to maintain clonal genotype. At present, the

most common method to preserve the genetic resources of vegetatively propagated plants is,

as whole plant in the field. But there are several serious limitations with field gene banks

mainly due to attacks by pests and pathogens, exposure to natural disasters etc. In addition,

distribution and exchange from field gene banks is difficult because of the vegetative nature

of the material and the greater risks of disease transfer (Bhattacharyya et al. 2001). For this

reason in vitro conservation and encapsulation technique is needed. Sharma et al. (1995)

had reported in vitro shoots of C. forskohlii remained viable for 18 months when stored at

18°C using polypropylene caps. Encapsulation technique and in vitro storage protocols for

C. forskohlii were developed by Bhattacharyya et al. (2001).

2.4.1.11. In Vitro Forskolin Production in C. forskohlii

Studies on tissue culture methods for forskolin production was carried out because

of the relatively modest content of forskolin in the plant have limited its development as a

drug (Mukherjee et al. 2000a). Forskolin was identified in shoot differentiating culture,

micropropagated plants and root organ suspension by TLC and HPLC. Forskolin produced

by shoot differentiating culture was similar to that of the micropropagated plants whereas


root organ suspension showed only traces of forskolin (Sen et al. 1992). Krombholz et al.

(1992) reported that root cultures of C. forskohlii initiated from primary callus or (Indole

Butryic acid) IBA-treated suspension cultures and maintained on Gamborg's B5 medium

containing IBA (1mg/L) produced forskolin and its derivatives in amounts ranging from

500 to 1300 mg/kg dry weight, corresponding to about 4 to 5 mg/L. Mersinger et al. (1988)

reported that to start the biosynthesis of secondary metabolites, cell aggregates were

transferred in an induction medium. Within two cultivation periods of 14 days the amount

of forskolin increased up to 0.2-1g/kg dry cell weight.

Sen et al. (1993) found that forskolin was identified by TLC and HPLC in 60 days

old shoot differentiating cultures, 30 days old micropropagted plants and root organ culture.

Further this group found that highest amount of forskolin (0.09 per cent) was raised after 60

days of untransformed cultures of C. forskohlii (Sen et al. 1993). Similarly, Tripathi et al.

(1995) were also successful in production of forskolin from callus culture; the kind and

level of phytohormones, glycine, casein hydrolysate and sucrose content of the medium

differently influenced the growth and product formation. Agrobacterium tumefaciens

mediated tumor tissue and shooty tetatomas of C. forskohlii were culture in vitro. Forskolin

was detected in timorous callus (0.002 per cent), rhizogenic callus (0.011 per cent) and root

cultures (0.014 per cent), but not in shooty teratomas (Mukherjee et al. 1996). Forskolin

production was observed in callus cultures from leaf, stem and root origin as well as roots of

in vitro grown plants by HPTLC in C. forskohlii (Malathy and Pai 1999). Studies revealed

that casein hydrolysate significantly enhanced forskolin content in the rhizogenic timorous

line of C. forskohlii (Mukherjee et al. 2000b). The treatment of cell cultures of C. forskohlii

with 50µ M of ancymidol an inhibitor of gibberelin biosynthesis enhanced the

bioproduction of forskolin by 150 per cent using production medium in six well plates as

culture vessel (Mamtha et al. 2002). Mukherjee et al. (2003) found that increased forskolin

yield was obtained in transformed root, rhizogenic calli and cell suspension cultures of C.

forskohlii when treated alone with various concentrations of auxins, auxin conjugates and

gibberellic acid.

2.4.1.12. In Vivo Forskolin Production

The arbuscular mycorrhizal symbiosis is a mutualistic association formed between plants

and a wide variety of fungi from the phylum Glomeromycota (Schuessler et al. 2001;


Schuessler 2002; Varma 2008). The endotrophic AMF are ubiquitous soil microbes

constituting an integral component of terrestrial ecosystems forming symbiotic associations

with plant root systems of over 80 per cent of all terrestrial plant species, including many

horticultural important plants. The plant hosts of AMF are mostly angiosperms, some

gymnosperms, pteridophytes, lycopods and mosses (Smith and Read 1997). In general, the

symbionts trade nutrients, and the AMF obtains carbon from the plant while providing the

plant with an additional supply of phosphorus. The AM symbiosis is associated with a range

of additional benefits for the plant including the acquisition of other mineral nutrients, such

as nitrogen and resistance to a variety of stresses. As a consequence, the AM symbiosis is of

tremendous significance to life on this planet, in both natural and agricultural ecosystems. It

has also been known for several years that different species of AMF can contribute to higher

production and yield of essential oils in plants with medicinal virtues such as mint, ocimum

and many more (Sirohi and Singh 1983; Copetta et al. 2006). Boby and Bagyaraj (2003)

found forskolin concentration in roots of C. forskohlii was very much enhanced by dual

inoculation with G. mosseae and Trichoderma viride. Their work was the first report of an

increase in forskolin concentration in the roots of C. forskohlii because of inoculation of

microbes. Coleus plants raised in presence of the arbuscular mycorrhizal fungus Glomus

bagyarajii, showed an increase in plant growth and forskolin content over those grown in

the absence of AM fungi (Sailo and Bagyaraj 2005).

2.5. Arbuscular Mycorrhizal Fungi

Intensive applications of agrochemicals have lead to severe agricultural problems

like soil acidification, contamination of ground water as well as atmosphere and many more.

Application of mycorrhizae to soil is one of the alternatives to usage of chemical fertilizers

as it is nature friendly. The term "mycorrhizae" refers to symbiotic association of fungus

with roots of higher plants. Symbiotic association between plant and fungus provides

improved means of fighting tough physical conditions, enriching soil, increasing health, and

decreasing dependence on chemical fertilizers.

The arbuscular mycorrhizal symbiosis is a mutualistic association formed between

plants and a wide variety of fungi from the phylum Glomeromycota (Schuessler et al. 2001,

Schuessler 2002). The arbuscular mycorrhizal (AM) fungi are ubiquitous soil microbes

constituting an integral component of terrestrial ecosystems forming symbiotic associations


with plant root systems of over 90 per cent of all terrestrial plant species. The plant hosts of

AM Fungi are mostly angiosperms, some gymnosperms, pteridophytes, lycopods and

mosses (Smith and Read 1997). Arbuscular mycorrhizal fungi (AMF) can form symbioses

(arbuscular mycorrhizas) with the majority of land plants (Smith and Read 1997). When in

symbiosis, AM fungi promote plant water and nutrient uptake, especially of insoluble soil

phosphate (Pi) fraction (Clark and Zeto 2000; Marschner and Dell 1994). The fungi in

return benefit from the supply of carbohydrates derived from photosynthesis (Harrison

1999). AM fungi are thus biotrophic, and carbon compounds may primarily flow from host

to fungus via living arbuscules (Becard and Piche 1989). The benefits of mycorrhizal

associations arise from the nutrient transport between the plant roots and fungal hyphae.

The carbon source is transported from the plant to the fungus, whereas fungal hyphae serve

as a fine link between the roots and the rhizosphere improving supply of the plant with

inorganic nutrients (Harrison 1999; Herrmann et al. 2004; Koide and Mosse 2004). By

linking plant roots with their mycelium, AM extend the roots absorptive capacity. Fungal

hyphae are thinner and branch more frequently than plant roots, providing more flexibility

in nutrient access. These associations are mutualistic symbioses, resulting from plant and

fungal host co-evolution (Mucciarelli et al. 2003).

The formation of mycorrhizal association significantly changes the morphology and

physiology of roots and plants leading to altered root exudation. The changes in root

exudates affect the microbial diversity around the roots, forming the “mycorrhizosphere”.

The mycorrhizosphere is the zone of soil where the physical, chemical and microbiological

processes are influenced by plant roots and their associated mycorrhizal fungi. A major

difference in the rhizosphere around the non-mycorrhizal roots and mycorrhizosphere is the

presence of extramatrical hyphae of mycorrhizal fungi. These extramatrical hyphae extend

well beyond the roots into the bulk soil and are an important source of carbon to the soil

organisms (Varma 1999). The mycorrhizal hyphae increase the soil aggregation and

increase root exudation favoring microbial growth. So far seven types of mycorrhizae have

come into general use over the years on the basis of morphology and anatomy but also of

either host plant taxonomy or fungal taxonomy (Srivastava et al. 1996; Smith and Read

1997). These are: ectomycorrhiza, endomycorrhiza or arbuscular mycorrhiza, ericoid


mycorrhiza, arbutoid mycorrhiza, monotropoid mycorrhiza, ect-endomycorrhiza and

orchidaceous mycorrhiza.

2.5.1. History of Arbuscular Mycorrhizal Fungi

The history of research on arbuscular mycorrhiza is excellently reviewed by Koide

and Mosse (2004). Arbuscular mycorrhizas may have been described as early as 1842 by

Nageli, but most of Nageli’s drawings only remotely resemble the arbuscular mycorrhiza.

Trappe and Berch in 1985 and Rayner (1926–1927) cite other early observations of the

symbiosis during the period 1875–1895. Frank in 1885 gave the name “mycorrhiza” to the

peculiar association between tree roots and ectomycorrhizal fungi. As early as 1889,

Schlicht had already observed the basic anatomical relationships between host and fungal

tissues. Janse in 1897 called the intramatrical spores “vesicules” and in 1905. Gallaud called

the other commonly observed intracellular structures “arbuscules”. Thus the name

“vesicular-arbuscular mycorrhiza” was established and persisted until recently. Gallaud

observed that the arbuscules were located in the inner cortex. Gallaud made very accurate

observations of the arbuscule and concluded, for example, that it is entirely surrounded by a

host membrane, which was later confirmed by Cox and Sanders in the year 1974 using

transmission electron microscopy (Koide and Mosse 2004).

Light and electron microscopical studies of arbuscular mycorrhizas were facilitated

by the finding in 1950 of the Centro di Studio sulla Micologia del Terreno by Peyronel in

Torino, Italy (Bonfante 1991; Koide and Mosse 2004). There, in the year 1968 Scannerini

and Bellando first noted that a space between the host membrane and the fungal wall

contained materials of host origin, probably unconsolidated components of host cell wall. In

2001, Schuessler et al. used molecular data to establish the relationships among arbuscular

mycorrhizal fungi and between arbuscular mycorrhizal fungi and other fungi. The group of

arbuscular mycorrhizal fungi was elevated to the level of phylum (Glomeromycota), which

was shown to be as distinct from other fungi as the Ascomycota are from the Basidiomycota

(Koide and Mosse 2004).

2.5.2. Structure of AM Fungi

On the basis of structure formed by these fungi in colonized roots, the mycorrhizal fungi

may be, Ectomycorrhizas (ECM), Arbuscular mycorrhizas (AM), Ericoid mycorrhizas,

Arbutoid mycorrhizas, Monotropoid mycorrhizas, Ecto-endomycorrhizas, Orchidaceous


mycorrhizas and Jungermannioid mycorrhizas. Today, despite the large number of plant

species forming AM associations worldwide, only two major morphological types have

been defined: The Arum and the Paris type, respectively. In the Arum-type the fungal

symbiont spread in the root cortex via intercellular hyphae. Short side branches penetrate

the cortex cells and produce arbuscules. The Arum-type is commonly described in fast

growing root systems of crop plants. In the Paris-type, the hyphae develop intracellular coils

and spread directly from cell to cell within the cortex. Arbuscules grow from these coils.

Co-occurrence of Arum- and Paris type morphologies of AM is found in cucumber and

tomato.

Arbuscules are relatively short-lived, at least in the Arum-type mycorrhiza and the

hyphae are comparatively long-lived (Smith and Dickson 1991). The arbuscules

progressively degenerate, whilst the plant cell remains alive, which is a difference compared

to many plant pathogenic fungi which causes plant cell death. Dickson (2004) surveyed 12

plants colonized by six species of arbuscular mycorrhizal fungi to explore the diversity of

Arum and Paris mycorrhizal structures. The survey indicated that there was a continuum of

mycorrhizal structures ranging from Arum to Paris depending upon both the host plant and

the fungus. The time course showed that the total colonization increased and the

establishment of the various mycorrhizal structures did not appear to change greatly over

time. He concluded that the morphological structures in individual plants could be grouped

into classes that were more diverse than Arum and Paris (Smith and Smith 1997). Eight

classes were recognized, forming a structural sequence between Arum and Paris type. These

are: classic arum with intercellular hyphae (IH), and arbuscules in cortical cells, arum with

IH and paired arbuscules in adjacent cortical cells, distinct individual arbuscules on IH and

PH (penetrating hyphae), distinct individual arbuscules but on PH, distinct individual

arbuscule on PH and IH in outer layers of root, arbusculate coil (AC) and hyphal coils (HC)

in inner cortex, and IH in outer layers of root, Arum and Paris (both arbuscules and

arbusculate coils in cortical cells), Paris (arbusculate coils and hyphal coils in cortical

cells).

Arum- type mycorrhizas were formed by all three fungi, (Glomus caledonium, G.

intraradices and Gigaspora rosea) in Flax, (Linum usitatissimum) with paired arbuscule

(Smith et al. 2004) as shown previously (Dickson et al. 2003). Medicago truncatula formed


Arum-type AM with the two Glomus species (Burleigh et al. 2002). The Paris-type AM

formed in plant/fungus combination was capable of phosphate transfer, even though there

were very few arbuscule coils. This is consistent with mycorrhizal effects on phosphate

uptake in Australian weed Asphodelus fistulosus via Paris-type AM colonized by G.

coronatumin (Cavagnaro et al. 2003). At low phosphate, A. fistulosus showed very marked

positive responses to colonization, both in phosphate uptake and growth. Both responses

decreased with increased phosphate supply. De Grandcourt et al. (2004) studied

mycorrhizal colonization, growth, phosphorus content, net photosynthesis and root

respiration on seedlings of two-co-occurring species (Dicorynia guianansis and Eperua

falcata) grown at three soil phosphorus concentration with or without inoculation with

arbuscular mycorrhiza seedlings of both species and were found to unable in absorbing

phosphorus in the absence of mycorrhizal association. They exhibited Paris-type

mycorrhizal associations. Regarding phosphorus acquisition, the two species belong to two

different functional groups, D. guianensis being an obligate mycotrophic species.

2.5.3. Functions of AM Fungi

AM fungi are known to improve the nutritional status of plants as well as their

growth and development, and confer resistance to drought and soil saline condition. These

fungi also play an indispensable role in hydratic status of the plant and on soil aggregation

as well as increasing the reproductive potential, improving root performance and providing

a natural defense against invaders, including pests and pathogens (Singh et al. 2000).With

over 130 species of AM fungi recognized and classified and the wide host range they

inhabit, there exists a wide variation in the ways they benefit the host, which in turn are

related to the extent of the colonization of host roots by the fungus. The extent of the root

colonization varies with several soil and climatic factors apart from the host involved.

However, these fungi show a preferential colonization to hosts and thus the extent to which

the host benefit depends of the fungal species involved in the symbiosis (Miller et al. 1987).

The key beneficial functions of AM symbiosis can be summarized as follows:

2.5.3.1. Plant Establishment and Development of Superior Root System: Colonization

of a plant root by AMF can alter the morphology of a root system in a structural, spatial,

quantitative and temporal manner (Atkinson et al. 1994; Norman et al. 1996). The AMF

colonized roots are highly branched, i.e., the root system contains shorter, more branched,


adventitious roots of larger diameters and lower specific root lengths (Berta et al. 1993;

Atkinson et al. 1994).

2.5.3.2. Increased Photosynthesis Efficiency: Most of the studies suggested that AMF

symbiosis helps in increasing the rate of photosynthesis, storage of photosynthates and

export at the same time (Augé 2001). AMF associations have been shown to improve

photosynthetic efficiency by improving P nutrition in plants (Marschner 1995), owing to an

effect of phosphorus status on CO2 assimilatory reactions. It has been shown that

chlorophyll concentration in AMF treated plants is higher than their nonmycorrhizal

counterparts (Giri et al. 2003; Kapoor and Bhatnagar 2007).

2.5.3.3. Increased Water Conducting Capacity: Arbuscular Mycorrhizal Fungi (AMF)

can reduce the negative impact of water stress on plants (Smith and Read 1997; Augé

2001). Mycorrhizal plants are shown to possess high water potentials (Kapoor et al. 2008).

2.5.3.4. Enhanced Nutrient Uptake: These fungi increase the surface area of roots and

thus help in absorbing some diffusion-limited nutrients (P, Zn, Cu etc.). AMF enhances the

plant growth as a result of the improved phosphate nutrition of the host plant. Fungi obtain

carbon from the plant while providing the plant with an additional supply of phosphorus.

This has been confirmed by the use of isotropic traces (Bolan 1991).The inoculation of AM

and other beneficial soil microorganisms significantly increased the biomass of different

medicinal plants (Sena and Das 1998; Kothari et al. 1999).

2.5.3.5. Enhances Plant Tolerance to Environmental Stresses: It obtains increased

protection against environmental stresses (Sylvia and Williams 1992), including drought

(Subramanian et al. 1995), cold (Charest et al. 1993; Paradis et al. 1995), salinity

(Hilderbrandt et al. 2001) and pollution (Tonin et al. 2001; Turnau et al. 2001).

2.5.3.6. Protection from Harmful Soil Borne Pathogens: AM fungi tend to reduce the

incidence of root diseases and minimize the harmful effect of certain pathogenic agents

(Azcon-Aguilar and Barea 1996; Slezack et al. 1999).

2.5.3.7. Enhance Tolerance to Transplantation Shock Experienced by Micro

propagated Plant Species at the Time of their Transplantation to the Field: AM

inoculation of tissue cultured plantlets have been reported to lessen transplantation shock

during acclimatization, thus increasing plant survival and establishment rates (Estrada- Luna

et al. 2000; Padilla et al. 2006; Binet et al. 2007). The benefits associated with the use of


AM inoculation for ‘in vitro’ raised plantlets have been reported in several horticultural and

forest tree species (Rai 2001; Sharma et al. 2007; Kapoor et al. 2008).

2.5.3.8. AM in Forestation of Arid Lands: Arid regions compromise approximately one-

fifth of the earth’s land area and contain a large fraction of the known energy and mineral

reserves. Restoration of forest land devasted for resource extraction is an immediate priority

and a challenging task for arid land ecologists. AM fungi are widespread in forest trees and

this symbiosis can be manipulated to enhance productivity in forestation programs. The AM

fungi change the supply of mineral nutrients from soil thereby modifying soil fertility,

mycorrhizosphere and aggregation of soil particles (Goltapeh et al. 2008).

2.5.3.9. AM Fungi Promote Growth, Fitness and Conservation of Endangered Plants:

Many endangered plant species live in symbiosis with AMF. It may have multiple positive

effects on plant growth, productivity, health, and stress relief. Rare plants often occur in

specialized and also endangered habitats and might utilize specialized or unique AMF.

Selected inocula of AMF could be used to promote growth of endangered plants before the

proper and more effective indigenous AMF are characterized. AMF can be applied in field

sites to protect endangered plants. Endangered plants could be grown as greenhouse cultures

together with appropriate fungi, and, at the relevant developmental stage, they could be re-

planted into native sites to prevent extinction and to preserve plant community ecology

(Bothe et al. 2010).

2.5.4. Biotechnological Applications of AM Fungi and Constraints

AM Fungi have been found to enhance biomass, improve pathogen, heavy metal, salinity

resistance, and stimulate photosynthesis as well as influence the level of secondary

metabolites in plants (Smith and Read 2007). As a consequence, the AM symbiosis is of

tremendous significance to life on this planet, in both natural and agricultural ecosystems.

Mycorrhizal research have intensified to develop safe bioherbicides and to produce

compounds for industrial and pharmaceutical applications (Shearer 2002). The

biotechnological use of AMF was proposed for agricultural (Hamel 1996), endangered

plants (Gemma et al. 2002; Sharma et al. 2007; Zubek et al. 2009, Bothe et al. 2010),

medicinal plant species (Kapoor et al. 2002a, b, 2007; Copetta et al. 2006; Khaosaad et al.

2006; Toussaint 2007; Toussaint et al. 2007; Zubek and Błaszkowski 2009; Jurkiewicz et

al. 2010), as well as plants applied in restoration processes of destroyed habitats (Turnau


and Haselwandter 2002). AM fungi promotes flowering in many economically important

plants and ornamental crops (Long et al. 2010). Test with commercial AMF inocula showed

that mycorrhizal colonization increased the shoot P/K concentrations and the number of

flowers of Pelargonium peltatum (Perner et al. 2007). The knowledge of AM Fungi

interactions with plants is important, as not only the selection of appropriate plant

species/cultivar/ecotype but also well-selected microbial consortia could be essential for the

success of restoration, plant maintenance, or cropping (Turnau and Haselwandter 2002;

Copetta et al. 2006; Toussaint 2007; Zubek et al. 2009).

The most extensively studied AM Fungi are species of the genera Glomus,

Gigaspora and Scutellospora. Despite the numerous important role and ecological function

of AM fungi, mass pure inoculum production and axenic cultivation of this group of

symbiotic fungi are not possible till date. These fungi cannot grow like any other fungi apart

from their host (Obligate photosymbionts). This is the greatest bottleneck for the progress

towards the understanding of the molecular communication between the symbiotic partners

(Singh et al. 2000). In contrast, many ericoid and ectomycorrhizal fungi can be grown in

pure culture, but their host spectrum is restricted to the Ericaceae or to woody plants

(Molina et al. 1992; Varma et al. 1999). Because of the absence of an authentic pure culture

of AM fungi, the commercial production is the greatest blockage in use and their application

in mycorrhizal biotechnology (Singh et al. 2000).

2.6. Endophytes

Endophytes are microorganisms that live within living plant tissues and do not cause

any visible symptoms due to their presence. Many fungi colonize the cortex of the living

roots without causing disease, including pathogenic or necotrophic fungi with latent phases

as well as beneficial fungi that offer protection against pathogens (Brundrett 2006). When in

association with host endophytes can have many effects on their host such as enhancement

of growth, stress tolerance, disease suppression (Schulz 2006). Endophytes also produce

unusual secondary metabolites of plant importance (Bandara et al. 2006; Tian et al. 2004,

Schulz 2006). The colonization and propagation of endophytes and their secondary

metabolites inside the plants may be critical for these effects. These facts indicate that

endophytes can be potential biological control agents and will play an important role in

ecological agriculture (Tian et al. 2004). The non-mycorrhizal microbes such as


Phialocephala fortinii, Cryptosporiopsis spp, dark septate endophyte (DSE),

Piriformospora indica, Fusarium spp and Cladorrhinum foecundissimum have been shown

to improve the growth of their hosts after root colonization (Schulz 2006).

2.7. Piriformospora indica- Model Symbiotic Fungus

Scientists have discovered an endophyte named Piriformospora indica, a member of

the Sebacinales. P. indica has received worldwide attention as it promotes the growth of

several plant species as well as it can be axenically cultivable easily on synthetic media in

contrast to obligate biotrophic AM fungi . Originally, this fungus was isolated during the

screening for AM fungi in the the soil samples collected from the rhizosphere of woody

shrubs Prosopsis juliflora and Zizyphus nummularia growing in the Thar Desert of

Rajasthan, India (Verma et al. 1998; Singh et al. 2000). The fungus has been named as

Piriformospora indica based on its characteristic pear shaped chlamydospores (Fig. 4) and

is related to the Hymenomycetes of the Basidiomycota. It is phylogenetically close to

mycorrhizal endosymbionts of orchids and ericoid root (Verma et al. 1998; Varma 1999;

Weiss et al. 2004). The fungus is able to associate with the roots of various plant species in

a manner similar to arbuscular mycorrhizal fungi and promotes plant growth (Varma et al.

1999, 2001; Singh et al. 2003; Shahollari et al. 2004; Pham et al. 2004a). Hence, it provides

a promising model organism for the investigations of beneficial plant–microbe interaction

and enables the identification of compounds, which may improve plant growth and

productivity. The properties of P. indica have been patented (Varma and Franken 1997,

European Patent Office, Muenchen, Germany, Patent number 97121440.8-2104, Nov.

1998). The culture has been deposited at Braunsweich, Germany (DMS number 11827) and

18S rDNA fragment deposited with GenBank, Bethesda, USA (AF 014929).


Coiled Hyphae

A

B

Pear shaped spore

Fig. 4: Piriformospora indica. A: Structure; B: Flourescent spore

(Photographed by Ajit Varma with Confocal Microscope, Beta Model, Jena, Germany )


2.7.1. Phylogenetic Position of P. indica

P. indica is related to the Hymenomycetes of the Basidiomycota and belongs to

order Sebacinales (Fig. 5, 6). Molecular ecology studies, based on rDNA sequences, reveal

that members of Sebacinales are associated with many plant species all over the world.

Sebacinales are divided into two clades, A and B that differ in their ecology (Weiss et al.

2004; Selosse et al. 2007). Clade A consists of ectomycorrhizae and ectendomycorrhizae

species whereas Clade B includes ericoid along with cultivable orchid root colonizing

mycorrhiza species of the complex Sebacina vermifera and P. indica (Weiss et al. 2004;

Desmukh et al. 2006; Selosse et al. 2007). Plants colonized by these fungal species display

improved growth and fitness.

Glomeraceae( -group A)Glomus

Glomerales

Glomeraceae( -group B)Glomus

Basidiomycetes

AscomycetesPiriformospora indica Verma et al

89 84

0.01State-of-Art

Fig.5: Proposed generalised taxonomic structure of the AM and related fungi (Glomeromycota), based

on SSU rRNA gene sequences. Thick lines delineate bootstrap support above 95% lower values are given on

the branches. The four-order structure for the phylogenetic position of P. indica (after Schuessler et al. 2001;

Varma et al. 2001)

As most of the basal taxa of basidiomycetes consist of predominantly mycoparasitic and

phytoparasitic fungi, it appears that Sebacinaceae is the most basal group of Basidiomycetes

that contains mycorrhiza-forming taxa. Mycorrhizal taxa of Sebacinaceae include

mycobionts of ectomycorrhizas, orchid mycorrhizas, ericoid mycorrhizas and

jungermannioid mycorrhizas. Such a wide spectrum of mycorrhizal types in one fungal

family is unique (Weiss et al. 2004; Shahollari et al. 2007). Extrapolating from the known

rDNA sequences in Sebacinaceae, it is evident that there is a cosm of mycorrhizal

biodiversity yet to be discovered in this group (Fig. 5, 6).

Taxonomically, the Sebacinaceae recognized a new order, the Sebacinales (Weiss et al.

2004). The order primarily contains the genera: Sebacina, Tremelloscypha,


Fig. 6: Phylogenetic placement of the P. indica within the Sebacinales. Dendrogram is estimated by maximum likelihood from an alignment of nuclear rDNA coding for the 5’ terminal domain of the ribosomal large subunit. Branch support is given by nonparametric maximum likelihood bootstrap (first numbers) and by posterior probabilities estimated by Bayesian Markov chain Monte Carlo (second numbers). Support values of<50% are omitted or indicated by an asterisk. P. indica and other related groups are indicated by black circle. (Cf. Deshmukh et al. 2006)


Efibulobasidium, Craterocolla and Piriformospora. Like other cultivable species of the

Sebacinales, P. indica forms moniliod hyphae, which look like pearls in a chain. Based on

this phenotype and rDNA sequence analyses, the endophyte is placed in the polyphyletic

genus Rhizoctonia (Selosse et al. 2007; Oelmüller et al. 2009).

2.7.2. Structure and Genome Size of P. indica

P. indica has potency to grow axenically on a number of complex and semi-

synthetic media (Pham et al. 2004b; Peškan-Berghöfer et al. 2004; Oelmüller et al. 2009)

The mycelium is mostly flat and submerged into the substratum. The hyphae are highly

interwoven often showed anastomosis and are irregularly septated. Hyphae are thin walled

and of different diameters ranging from 0.7-3.5µm. In older cultures hyphae were

irregularly inflated, showing a nodose to coralloid shape, mostly coenocytic and septa were

laid infrequently containing more than one nucleus. Chlamydospores are formed from thin

walled vesicle at the tips of the hyphae and appeared singly or in clusters. They were

distinctive due to their pear shaped appearance with 16-25 µm in length and 10-17 µm in

width. The cytoplasm of chlamydospores contained 8-25 nuclei. Young spores have thin

hyaline walls, but at maturity spores walls thickened up to 1.5 µm, which appeared two

layered smooth and pale yellow. Neither clamp connections nor sexual structures could be

observed. The septal pores consisted of dolipores with continuous parenthosomes. The

dolipores were very prominent, with a multilayered cross wall. The parenthosomes were in

contact with the ER membranes, which were mostly found near the dolipore (Verma et al.

1998). Studies done by confocal microscope revealed the outer layer of the spore of P.

indica generated an intensive autofluorescence, which disappeared after germination. This

autofluorescence appeared again after the co-cultivation of P. indica with Arabidopsis root

hair. Since the fluorescence was not detectable in control root hairs, establishment of a

successful interaction between both organisms could be monitored by the fungus-derived

autofluorescence (Pesˇkan- Berghöfer et al. 2004).

The fungus P. indica was shown to possess at least six chromosomes and a genome

size of about 15.4–24 Mb. Sequences of the genes encoding the elongation factor 1-a (TEF)

and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for genome size

estimation through real-time PCR analysis. Results demonstrate that P. indica can be stably

transformed by random genomic integration of foreign DNA and that it posses a relative


small genome as compared to other members of the Basidiomycota (Zuccaro et al. 2009).

Subsequently this fungus has been sequenced.

2.7.3. Host Spectrum of P. indica

P. indica tremendously improves the growth and overall biomass production of a

diverse host including legumes (Varma et al. 1999, 2001), medicinal and economically

importance plants (Rai et al. 2001; Glen et al. 2002; Singh et al. 2002b; Peškan-Berghöfer

et al. 2004; Pham et al. 2004a; Rai et al. 2005; Shahollari et al. 2005, 2007; Prasad et al.

2008a). Pronounced growth promotional effect was seen with terrestrial orchids (Bhatnagar

and Varma 2006) and even Bryophytes (Pham et al. 2004a). The apparent lack of species

specificity suggests that this beneficial symbiosis might be based on general recognition and

signaling processes (Shahollari et al. 2007). The fungus also provides protection when

inoculated into the tissue culture raised plants by overcoming the ‘transient transplant shock

on transfer to the field and renders almost 100 per cent survivals on transplant (Sahay and

Varma 1999; 2000). Interestingly, the host spectrum of P. indica is very much alike AM

fungi. The fungus colonizes the roots and improves the health, vigor and survival of a wide

range of mono-and dicotyledonous plants. Hosts of P. indica encompass tobacco (Sherameti

et al. 2005; Oelmüller et al. 2009), Arabidopsis (Peškan-Berghöfer et al. 2004), species

from the Fabaceae and Rhamnaceae (Varma et al. 2001, Pham et al. 2004a) and Poaceae

(Waller et al. 2005). P. indica colonises many agri- and horticultural species like maize,

orchids, Petunia, snapdragon; tree species like poplar; medicinal plants like Artemisia,

Bacopa, Abrus, Tridax, Chlorophytum, Withania and many more and (Singh et al. 2000,

Kumari 2005; Oelmüller et al. 2009; Gosal et al. 2010). In addition to their intracellular

growth, they also form abundant extracellular mycelia. Hyphae are grown in living root

cells of the host species.


Fig. 7: Biological applications of P. indica. Fungus promotes growth and flowering of medicinal plants. a: P. indica treated plants of Spilanthes

calva; b: Un-treated S. calva; c: Un-inoculated Withania somnifera, d: P. indica inoculated W. somnifera ( Rai et al. 2001)


2.7.4. Applications and Diverse Functions of P. indica

The endophyte P. indica has an encouraging influence on growth and development

on host plants (Fig.7). It also promotes nutrient uptake, allows plants to survive under water,

temperature and salt stress, confers (systemic) resistance to toxins, heavy metal ions and

pathogenic organisms and stimulates growth and seed production (Verma et al. 1998;

Varma et al. 1999, 2001; Sahay and Varma 1999; Oelmüller et al. 2004, 2005; Pham et al.

2004a, b; Peškan-Berghöfer et al. 2004; Kaldorf et al. 2005; Shahollari et al. 2005, 2007,

Sherameti et al. 2005, 2008a, b; Vadassery et al. 2008, 2009a, b; Waller et al. 2005, 2008).

Like AM fungi, P. indica functions as bioregulator,

biofertilizer and bioprotector as well as delays wilting and withering of the leaves. In

addition, it also prolongs life-span of callus tissues. Several studies have demonstrated that

P. indica may be used for phyto-remediation, because it accumulates heavy metals and

prevents their uptake into the plants (Oelmüller et al. 2009).

Kaldorf et al. (2005) demonstrated that when the plantlets of Populus Esch5

explants with roots were inoculated with P. indica, the root biomass and the number of

second order roots increased. However, when the plantlets were exposed to a medium with

pre-grown fungus, plant and root growth was completely blocked. Prolonged incubation of

the plantlets with the fungus caused even colonization of the aerial parts of poplar.

Application of ammonium to the medium leads to bleaching and withering of the plantlets

in the presence of the fungus. Fungal toxin formation or the extension of the colonization to

the shoots may be responsible for the antagonistic interaction. Deshmukh et al. (2006) and

Schäefer et al. (2007) reported that P. indica requires cell death for the proliferation during

mutualistic interaction with barley. They found that the majority of the hyphae were present

in dead rhizodermal and cortical cells. This suggested that P. indica either actively kills

cells or senses cells that undergo endogenously programmed cell death. Thus, the endophyte

interferes with the host cell death program to form a mutualistic interaction with the plants.

More detailed analysis with other plant species are required to find out whether this is a

general phenomenon or specific for barley, a host that strongly interacts with P. indica

(Oelmüller et al. 2009).

The major key applications and functions of P. indica symbiosis can be summarized

as follows


2.7.4.1. Effect of P. indica on Phosphorus (P), Nitrogen (N), Sulphur (S) and Sugar

Metabolism

P. indica seems to mediate phosphorus and nitrogen uptake from the soil (Sherameti

et al. 2005, Varma et al. 2001). Recent work suggested that P. indica stimulates NADH

dependent nitrate reductase activity in the roots of Arabidopsis and tobacco (Sherameti et

al. 2005). P. indica mediates nitrate uptake from the soil, which is in contrast to AMF,

where nitrogen is preferentially absorbed as ammonium. Malla et al. (2004) have shown

that P. indica contains substantial amounts of an acid phosphatase which has the potential to

solublize phosphate in the soil and delivers it to the host plant. The application of different

techniques for characterization of ACPase (Acid phosphatase) in P. indica and Sebacina

vermifera senu which belong to same taxonomic group show similar morphology,

functions, protein profiles and isozyme characterization along with close acid phosphatase

relationship ( Malla et al. 2010).

Shahollari et al. (2005) could demonstrate that growth promotion of Arabidopsis

seedlings is associated with a massive uptake of radio labeled P from the growth medium.

The supply of the fungus with carbon (C) sources, and the faster growth of colonized plants

require the breakdown of starch which is deposited in the root amyloplasts. Thus, one of the

major starch degrading enzymes, the glucan-water dikinase is activated by the fungus

(Sherameti et al. 2005). Recent studies have shown that also the S metabolism is stimulated

by the fungus (Oelmüller et al. 2009).

Achatz et al. 2010 found barley plants colonized with the endophyte P. indica

developed faster, and were characterized by a higher photosynthetic activity at low light

intensities. They reported increased root formation, faster development of ears as well as the

production of more tillers per plant also. The results indicated that the positive effect of P.

indica on grain yield is due to accelerated growth of barley plants early in development,

while improved phosphate supply. Recent work of Yadav et al. (2010) reported the cloning

and the functional analysis of a gene encoding a phosphate transporter from the root

endophytic fungus Piriformospra indica. High amount of phosphate was found in plants

colonized with wild type P. indica than that of non-colonized plants. Their work suggested

that the gene was actively involved in the phosphate transportation and in turn fungus P.

indica helped in improvements of the nutritional status of the host.


2.7.4.2. Protection Against Abiotic and Biotic Stress

Endophytic root colonization by this fungus confers enhanced growth to the host

plant (Varma et al. 1999; Pesˇkan-Bergho¨fer et al. 2004) and provides protection against

biotic and abiotic stresses (Oelmüller et al. 2009).

2.7.4.2.1. Protection against abiotic stress: Drought and salt tolerance

Waller et al. (2005) had shown that P. indica reprogrammes barley to salt stress tolerance,

resistance to diseases and higher yield. Waller et al. (2005) investigated salt stress tolerance

in barley leaves which were exposed to moderate (100 mM NaCl) and high (300 mM NaCl)

salt concentrations in hydroponic culture. The plants showed leaf chlorosis and reduced

growth. The detrimental effect of moderate salt stress was completely abolished by P.

indica, as shown by the fact that infested plants produce higher biomass than do non-

stressed control plants under these conditions. Sherameti et al. (2008a) reported when

Arabidopsis is exposed to mild drought stress, seedlings co-cultivated with the fungus

continue to grow, while the uncolonized controls do not and show symptoms of withering.

When seedlings are first exposed to drought stress and then transferred to soil, many

colonized seedlings reach the flowering stage and produce seeds, while the percentage for

uncolonized seedlings is much lower.

2.7.4.2.2. Protection against Biotic Stress: Resistance against Pathogenic Fungi

It could be also shown that P. indica induces resistance against root and shoot pathogens

(Waller et al. 2005; Serfling et al. 2007; Deshmukh and Kogel 2007; Sherameti et al. 2008

b; Baltruschat et al. 2008; Stein et al. 2008). Disease resistance is provided not only to the

roots but also to the shoots. P. indica induces enhanced resistance of the host against fungal

pathogens, e.g., powdery mildew (Blumeria graminis), root rot (Fusarium culmorum, F.

graminearum), fungal stem base pathogen Pseudocercosporella herpotrichoides, Fusarium

verticillioides and Verticillium dahliae (Waller et al. 2005; Deshmukh and Kogel 2007;

Serfling et al. 2007; Kumar et al. 2009; Fakhro et al. 2009).

2.7.4.3. Role of Hormone

Any kind of growth regulation and interaction of plants with microorganisms

involve phytohormones and these microorganisms improve plant growth by producing

phytohormones. Some research has been done on the role of auxin, cytokinin and ethylene

in P. indica plant interactions. Sirrenberg et al. (2007) and Vadassery et al. (2008) had


carried out research on the role of auxins in P. indica host symbiosis. Sirrenberg et al.

(2007) analyzed Arabidopsis root colonization by P. indica in sterile cultures on MS

medium, when the fungus forms intracellular structures in the epidermal root cells and

causes changes in the root growth, leading to stunted and highly branched root systems.

This appears to be caused by a diffusible factor and can be mimicked by indole-acetic acid.

Vadassery et al. (2008) had reported that the fungus produces relatively high levels of

cytokinins and it’s level is higher in colonized roots compared to the uncolonized controls.

Schäefer et al. (2009 a) observed stage-specific up-regulation of genes involved in

phytohormone metabolism, mainly encompassing gibberellin, auxin and abscisic acid, but

salicylic acid-associated gene expression was suppressed. The changes in hormone

homoeostasis were accompanied with a general suppression of the plant innate immune

system. Their group also indicated that a general plant defense suppression by P. indica and

significant changes in the GA biosynthesis pathway also (Schäefer et al. 2009b). Research

done by Camehl et al. (2010) proposed that ethylene signalling components and ethylene-

targeted transcription factors are required to balance beneficial and nonbeneficial traits in

the symbiosis. The results demonstrated that the restriction of fungal growth by ethylene

signalling components was required for the beneficial interaction between the two

symbionts.

2.7.4.4. Promotes Early and Excessive Flowering

Studies reported that when P. indica interacted with various hosts showed enhanced

number of inflorescence, flower and seed production in the presence of the fungus. In the

case of Spilanthus calva large and kidney shaped inflorescence were observed frequently

with the inoculated ones. Kidney shaped inflorescences were never observed in un-treated

plants. The length of the inflorescence and the number of flowers on inoculated S. calva

plants were also increased compared to the un-treated (Fig.7). Similarly, the number of

flowers on the inoculated plants of Withania somnifera was higher than on un-inoculated

plants (Rai et al. 2001; Pham et al. 2004a). In a pot trial the most commonly used tropical

ornamental plant, marigold (Tegatus erecta) showed early flower maturation as compared to

untreated control (Pham et al. 2004a). Kumari et al. (2003) reported P. indica treated plants

of Brassica juncea were superior in growth leading to early flowering and fruiting. Effects

on reproductive outputs of Nicotiana attenuate by two related fungi, P. indica and Sebacina


vermifera were studied amd it was found that P. indica promoted early flowering in N.

attenuate as compared to control (Barazani et al. 2005; Barazani et al. 2007).

2.7.4.5. Biological Hardening Agent

In vitro propagation of plants is often associated with a high mortality rate during

the ex vitro establishment phase. Studies have demonstrated that the potential of P. indica as

a biopriming agent for better growth and survival of in vitro plants. P. indica colonizes the

roots of tissue culture raised plants of Bacopa monniera and promoted the overall plant

biomass. A biological hardening rendered almost 100% survivals on transfer from

laboratory to the field (Pham et al. 2004a). The fungus had rendered more than 90%

survival rate of micropropagated transferred plantlets of Nicotiana tabacum (Varma et al.

1999). The micro-cloned plantlets of Chlorophytum borivilianum registered more than 95%

establishment in soil following treatment with various bio-inoculants namely; Glomus

aggregatum, Trichoderma harazianum and P. indica whereas species of Azospirullum and

Actinomycetes showed only up to 85% plantlet establishment (Mathur et al. 2008).

Similarly Vyas et al. (2008) reported that in vitro raised plantlets of Feronia limonia were

colonized using P. indica during their in vitro rooting and their ex vitro transfer. More than

90% of such plants survived in the green house condition. The above studies have

demonstrated the role of P. indica in alleviation of transplantation shock and successful

establishment of micro propagated plantlets. P. indica seems to act in two ways: helps the

plant to attain its best performance and buffers the action of stress during acclimatization.

Biotization of micro propagated Chlorophytum sp with the fungus, P. indica and the

bacterium, Pseudomonas fluorescens, improved plantlet survival rate, growth parameters,

field performance, P content and the micronutrient acquisition (Gosal et al. 2010).

With the discovery of P. indica, fungal root endophytes which can also be axenically

cultivated on economically viable synthetic media have given a new hope to scientists

worldwide to understand the genetic and physiological aspects of mycorrhizal partners

(Pham 2004 b). This axenically cultivable property of P. indica makes it suitable for mass

scale inoculums production for application in agro-forestry and horticulture as well as

promises to serve as an in vitro fungal partner in study of in vitro system of mycorrhization.

The in vitro system of mycorrhization has proved to be a valuable tool to study the

fundamental and practical aspects of host fungus symbiosis, complementing the in vivo


experiments. Dual cultures of host plant and symbiotic fungus are potentially valuable

research tool to study the genetic and physiological activity of the infected and un-infected

plants that can be compared without the interference from rhizosphere organisms (John et

al. 1981; Rai 2001; Giomaro et al. 2005). Axenically produced mycorrhizal plants have

been used to study the effect of externally supplied organic and inorganic phosphate sources

on the incidence, extent and anatomy of infection and also to measure the rate of movement

of phosphate ions via the external mycelium of an infected root system. Plants growing in

this way can be used to examine changes in the host brought about by Arbuscular

Mycorrhizal (AM) infection which are not attributed to the presence of any other

microorganism. Under axenic conditions special attention should be given to components of

the media since they control the physiology of the host plant and consequently influence

host fungal relationships (Bressan 2002).

2.8. Interaction of AM Fungi with Medicinal Plants for Secondary Metabolite

Production

Arbuscular Mycorrhizal (AM) fungi have been used to enhance the plant growth and

yield of medicinal crops and to help maintain good soil health and fertility that contributes

to a greater extent to a sustainable yield and good quality of the products. Utilization of

mycorrhizal biofertilisers in the cultivation of medicinal and aromatic plants is of current

interest. The interest of scientists in research of medicinal plants and mycorrhizae have

gained thrust in recent years due to the higher cost and hazardous effects of heavy doses of

chemical fertilizers as well as commercial importance of active ingredients of medicinal

plants. However, these fungi show a preferential colonization to hosts and thus the extent to

which the host benefits depend of the fungal species involve in the symbiosis (Miller et al.

1987).

Plants produce a high diversity of biologically active secondary metabolites of

economical importance (Dixon 2001; Papadopoulou 1999). Some of these compounds are

synthesized and stored during normal growth and development (Etten et al. 1994), while

others are absent in healthy plants, accumulating only in response to pathogen attack or

stress conditions (Etten et al. 1994). Elicitors are defined as molecules that stimulate

defense or stress-induced responses in plants (Etten et al. 1994). But due to low content of

secondary metabolite compounds in whole plant, the endangered status of many plants due


to their overexploitation, commercially unfeasible chemical synthesis, and geographical and

genotypic variations have resulted in development of alternate biotechnological means to

produce these compounds Although an extensive array of secondary metabolites have been

produced by plant cell culture technology as alternative strategy but, to date; limited

commercial success is a major concern in this area (Baldi et al. 2008). Improvement of

secondary products accumulation in plant is of great importance in medicinal plants

cultivation industry due to its great commercial importance.

Plant–fungus interaction can be used as an alternative method to enhance

accumulation of these phytochemicals as most of these are produced due to activation of

defense related biosynthetic pathways. Therefore, co-culture system is assumed to be a

meaningful and effective tool to biotic elicitation of secondary metabolite production in

plants upon symbiotic fungi infection. These interactions are very complex and may be very

specific to a given combination of the plant and the fungus, as there are about 250,000

species of higher plants and as many as 1.5 million species of fungi (Grayer and Kokubun

2001). Moreover, it is well known that mostly large groups of terrestrial plants ubiquitously

harbor both endophytic fungi and mycorrhizal fungi in their inner tissues. Therefore, Zhi-lin

et al. (2007) presumed that plants will achieve superior outcomes through dual inoculation

with mycorrhizal and endophytic fungi; probably aboveground and below-ground plant

parts establish two types of symbiotic associations and result in increasing microbial genetic

diversity in plant tissues. During the establishment of the arbuscular mycorrhizal (AM)

symbiosis, a range of chemical and biological parameters is affected in plants, including the

pattern of secondary plant compounds.

Medicinal plants in India were originally reported to be non-mycorrhizal, probably

due to the presence of various secondary metabolites (Mohankumar and Mahadeven 1988).

However, roots of field-grown garlic were found to be colonized by arbuscular mycorrhizal

(AM) fungi (Shuja and Khan 1977) and this observation has been supported by many

workers from Asia who found the roots of various medicinal plants to be mycorrhized (Rao

et al. 1989; Laksman and Raghavendra 1990; Sullia and Sampath 1990; Sharma and Roy

1991; Srivastava and Basu 1995; Ratti and Janardhananm 1995). Mycorrhizal colonization

induces many changes in plant physiology and was found to influence the level of

secondary metabolites which may depend on root colonization by AMF (Abu-Zeyad et al.


1999; Fester et al. 1999; Copetta et al. 2006; Khaosaad et al. 2006; Kapoor et al. 2007). It is

well demonstrated through many research work that AMF can influence phytohormone

levels of jasmonate (Hause et al. 2002), carotenoids (Fester et al. 2002), phenols (Zhu and

Yao 2004) and phenolic acids (Jurkiewicz et al. 2010). In addition, the association with

AMF has altered essential oil yield and quality of several plants. The mechanism by which

AM Fungi trigger changes in phytochemical concentration in plant tissues can be

multidirectional and is not quite clear yet (Toussaint 2007). Firstly, the modification of

compounds produced in roots may be the consequence of signaling mechanisms between

symbionts and plant response to AM Fungi colonization (Larose et al. 2002; Toussaint

2007). As it was found in the studies conducted by Larose et al. (2002), several flavonoids

of both stimulating and depressing effect on the AM Fungi development were produced in

different quantities at different stages before and during AM Fungi colonization of

Medicago sativa L. cv. Sitel roots. In addition, also an alkaloid, trigonelline is suggested to

be a regulatory factor during early signal events in the establishment of AM Fungi.

Medicinal plants like Catharanthus roseus, Zingiber officinale, Foeniculum vulgare,

Artemisia annua L and many more are interacted with various AM fungi to reveal its

influence on the medicinal plants. Castanospermine is one of the constituents of the plant

Castanospermum australe A. Cunn. and C. Fraser which has potential to inhibit the AIDS

virus. A positive correlation was found between AM fungal infection and the

castanospermine content of seeds of field-grown trees. The AM fungi increased the growth

and P contents of plants and the yield of castanospermine in the leaves, irrespective of the P

treatment. No significant difference in the production of castanospermine was found

between P treatments when G. margarita was used as inoculum (Abu-Zeyad et al. 1999).

Larose et al. (2002) found flavonoid levels in roots of Medicago sativa were modulated by

the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal

fungus (AMF). Based on their observations they could show that flavonoid accumulation in

M. sativa roots (i) was induced before root colonization, pointing towards the presence of a

fungal-derived signal,

(ii) depended on the developmental stage of the symbiosis and (iii) depended on the root-

colonizing arbuscular mycorrhizal fungus. The data presented indicated not only a time-

specificity of the flavonoid accumulation during the mycorrhizal association, but also an


arbuscular mycorrhizal fungal-specificity. The possible functions of the flavonoid pattern

changes were also discussed (Larose et al. 2002).

Stimulated hyphal growth and an intense hyphal branching with the formation of

many clusters of short and curled hyphae was noted when an in vitro pre-symbiotic system

between mesquite [Prosopis laevigata (Willd.) M.C. Johnst], a semi-arid leguminous plant

and pregerminated spores of Gigaspora rosea Nicol. & Schenck was established. HPLC of

the methanol extract of the roots in contact with Gigaspora rosea spores showed a

significant 1.8-fold increase in trigonelline concentration relative to the control treatment. In

contrast, there was no change in trigonelline concentration in the aerial parts of P. laevigata.

Trigonelline may be a regulatory factor during early signal events in the establishment of

the arbuscular mycorrhizal symbiosis in P. laevigata (Rojas-Andrade et al. 2003).

Intervention of AMF with the medicinal plants not only enhanced the active

constituents of the plants but also the essential oils contents. Kapoor et al. (2002a) observed

that inoculation with AMF Glomus macrocarpum and G. fasciculatum increased

significantly the concentration of limonene and α-phellandrene, respectively; relative to

non-mycorrhizal control plants of Anethum graveolens L. In Coriandrum sativum, Kapoor

et al. (2002b) also observed enhanced concentration and quality of essential oils on

mycorrhized coriander plants. Similar results were noticed by this group in Foeniculum

vulgare also. Two arbuscular mycorrhizal (AM) fungi G. macrocarpum and G. fasciculatum

significantly improved growth and essential oil concentration of Foeniculum vulgare Mill.

However, AM inoculation of plants along with phosphorus fertilization significantly

enhanced growth, P-uptake and essential oil content of plants compared to either of the

components applied separately. Among the two fungal inoculants, G. fasciculatum

registered the highest growth at both levels of phosphorus used with up to 78 per cent

increase in essential oil concentration of fennel seeds over non-mycorrhizal control. The

essential oil characterization by gas liquid chromatography revealed that the level of anethol

was significantly enhanced on mycorrhization. Mycorrhizal plants produced higher number

of umbels as compared to non-mycorrhizal plant (Kapoor et al. 2004).

Another important medicinal plant is annual wormwood (Artemisia annua L.) which

produces an array of complex terpenoids including artemisinin, a compound of current

interest in the treatment of drug resistant malaria. The effects of inoculation by two


arbuscular mycorrhizal (AM) fungi, Glomus macrocarpum and Glomus fasciculatum, either

alone or supplemented with P-fertilizer, on artemisinin concentration in A. annua were

studied by Kapoor et al. (2007). The concentration of artemisinin was determined by

reverse-phase high-performance liquid chromatography with UV detection. The two fungi

significantly increased concentration of artemisinin in the herb.

Additional studies were conducted to study the effect of P nutrition and AMF on the

bioactive compounds of medicinal plants. Arpana and Bagyaraj (2007) conducted field

experiments to find out the influence of inoculation with the arbuscular mycorrhizal (AM)

fungus Glomus mosseae and the plant growth promoting rhizomicroorganisms (PGPRs)

Trichoderma harzizmum singly and in combination on growth and yield of kalmegh

(Andrographis paniculata). Studies have concluded that inoculation with G. mosseae and T.

harzizmum not only improved growth, biomass yield, and phosphorus nutrition and

andrographolide concentration of kalmegh but also helped in saving 25 per cent of the

phosphorus fertilizer application. A field study was conducted to evaluate the effectiveness

of arbuscular mycorrhizal fungi (AMF) and different phosphorus levels for increasing

biomass yield and ajmalicine content in a medicinal plant Catharanthus roseus. The plants

treated with 150 and 200 kg P2O5/ha along with AMF had the maximum plant height,

number of leaves, root biomass, phosphorus content, root colonization, spore count and

ajmalicine content, 120 days after planting when compared with the control plants

(Karthikeyan et al. 2008)

Studies have been undertaken to investigate the effects of four arbuscular

mycorrhizal fungi (AMF) and an assemblage (Mixture) of all four isolates on growth,

development and oleoresin production of micropropagated Zingiber officinale (Silva et al.

2008). It revealed that AMF and phosphorus addition significantly increased shoot height

relative to control plants. Results suggested that the screening and inoculation of arbuscular

mycorrhizal fungi in ginger plant is a feasible procedure to increase the oleoresin production

of Z. officinale and consequently increase the aggregate value of ginger rhizome production

(Silva et al. 2008). An investigation has been made about the response of arbuscular

mycorrhizal (AM) fungus G. fasciculatum on selected medicinal plants of Ocimum sanctum,

Catharanthus roseus, Coleus forskholii and Cymbopogon flexuosus. The percentage of AM

association is 85 and the intensity of formation of vesicles and arbuscules are 70 and 30 per


cent, respectively in AM inoculated C. roseus plants. The total dry matter production (shoot

and root dry weight), protein and total chlorophyll contents are seen to increase in AM

inoculated in all the four medicinal plants. The percentage increase is more in C. roseus,

followed by C. flexosus when compared to un-inoculated plants (Karthikeyan et al. 2009).

A further important study was done on Inula ensifolia L. (Asteraceae); a valuable

xerothermic plant species with potential therapeutic value, when inoculated under

laboratory conditions with different strains of arbuscular mycorrhizal fungi (AMF) showed

AMF species specificity in the stimulation of thymol derivative production in the roots of

the host plant. Studies showed that there was an increase in thymol derivative contents in

roots after Glomus clarum inoculation and at the same time the decreased production of

these metabolites in the G. intraradices treatments. A multilevel analysis of chlorophyll a

fluorescence transients (JIP test) permitted an evaluation of plant vitality, expressed in

photosynthetic performance index, influenced by the applied AMF strains, which was found

to be in good agreement with the results concerning thymol derivative production (Zubek et

al. 2010). JIP test is a biophysical method of testing where test translates the polyphasic

chlorophyll-a fluorescence transient OJIP exhibited by plants upon illumination to

biophysical parameters of the photosynthetic machinery, evaluating plants' vitality (Strasser

et al. 2000, 2004).

2.8.1. Interaction of AM Fungi with Members of Family Lamiaceae for Secondary

Metabolite Production

Mucciarelli et al. (2003) characterized peppermint growth and terpene production of

in vitro generated plants (Mentha piperita) in response to inoculation with a leaf fungal

endophyte, employing both in vitro and in pot cultures. Peppermint plants were studied by

means of morphometric, biochemical and image analysis and leaf essential oils were

analyzed by gas chromatography-mass spectrometry. They reported that the endophyte

induced profound effects on the growth of peppermint, which responded with taller plants

bearing more expanded leaves. The observed increase of leaf dry matter over leaf area

suggested a real improvement of peppermint metabolic and photosynthetic apparatus. Root

architecture was of the herring bone type, showing greater dry biomass percentage over the

total. A sustained lowering of (+)-menthofuran and an increase of (+)-menthol percentage

concentrations were found in plants from both in vitro and pot cultures. Similar type of


research were done on Mentha arvensis L. (mint) where mycorrhizal colonization

significantly increases oil content and yield relative to non-mycorrhizal plants (Gupta et al.

2002). Freitas et al. (2004) also observed that inoculation with AMF resulted in increments

of 89 per cent in the essential oil and menthol contents of mint.

Copetta et al. (2006) studied the effect of arbuscular mycorrhiza (AM) for

production of essential oils in aromatic plant (sweet basil). The essential oils of basil are

widely used in the cosmetic, pharmaceutical, food, and flavoring industries. The effects of

colonization by three AM fungi, Glomus mosseae, Gigaspora margarita, and Gigaspora

rosea on shoot and root biomass, abundance of glandular hairs, and essential oil yield of

Ocimum basilicum L. var. Genovese were studied. Plant phosphorus content was analyzed

in the various treatments and no differences were observed. The AM fungi induced various

modifications in the considered parameters, but only Gi. rosea significantly affected all of

them in comparison to control plants or the other fungal treatments. It significantly

increased biomass, root branching and length, and the total amount of essential oil

(especially α-terpineol). Increased oil yield was associated to a significantly larger number

of peltate glandular trichomes (main sites of essential oil synthesis) in the basal and central

leaf zones. Further more, Gi. margarita and Gi. rosea had increased the percentage of

eugenol and reduced linalool yield. From various experiments Copetta et al. (2006) arrived

at the conclusion that G. rosea increased concentration of camphor and alpha terpineol,

while plants treated with Gigaspora margarita significantly decreased eucalyptol, linalool,

and eugenol contents relative to control. In addition, Glomus mosseae did not alter the

proportion of the aforementioned compounds. Results showed that different fungi can

induce variable effects in the same plant and that the essential oil yield can be modulated

according to the colonizing AM fungus. Another study was done again on active ingredients

of sweet basil by Toussaint (2007). The potential of three arbuscular mycorrhizal fungi

(AMF) to enhance the production of antioxidants (rosmarinic and caffeic acids, RA and CA,

respectively) was investigated in sweet basil (Ocimum basilicum). After adjusting

phosphorus (P) nutrition so that P concentrations and yield were matched in AM and non-

mycorrhizal (NM) plants scientists demonstrated that Glomus caledonium increased RA and

CA production in the shoots. Glomus mosseae also increased shoot CA concentration in

basil under similar conditions. Although higher P amendments to NM plants increased RA


and CA concentrations, there was higher production of RA and CA in the shoots of AM

plants, which was not solely due to better P nutrition. Therefore, AMF potentially represent

an alternative way of promoting growth of this important medicinal herb, as natural ways of

growing such crops are currently highly sought after in the herbal industry (Toussaint

2007).

Family Lamiaceae is well known for its essential oil contents. One of its members,

Oregano plant is valued for its essential oil contents. Investigation on essential oils content

of oregano were done by Khaosaad et al. (2006) and Morone-Fortunato and Avato (2008).

The effect of root colonization by Glomus mosseae on the qualitative and quantitative

pattern of essential oils (EO) was determined in three oregano genotypes (Origanum sp) by

Khaosaad and his group in 2006. In two genotypes the leaf biomass was increased through

mycorrhization. Root colonization by the arbuscular mycorrhizal fungus (AMF) did not

have any significant effect on the EO composition in oregano; however, in two genotypes of

oregano the EO concentration significantly increased. As EO levels in P treated plants were

not enhanced, so they were of the view that the EO increase observed in mycorrhizal

oregano plants is not due to an improved P status in mycorrhizal plants, but depends directly

on the AMF–oregano plant association. Moreover, the research work clearly demonstrated

that the positive effect of mycorrhization is highly dependent on the genotype of the plants

and is not a general characteristic of oregano (Khaosaad et al. 2006). The effects of

arbuscular mycorrhizal (AM) symbiosis on morphological and metabolic variations of

regenerated oregano plants were investigated at different growth stages. AM greatly

increased parameters such as plant leaf area, fresh and dry weight, number of spicasters and

verticillasters in infected plants. An increase of the gland density, especially on the upper

leaf epidermis, was also observed following the physiological ageing of the tissues. The in

vitro plants of Origanum vulgare ssp. hirtum described in this study provided a qualitatively

and quantitatively good source of essential oils that have a chemical profile comparable to

that of the control mother plants with carvacrol as the main compound (Morone-Fortunato

and Avato 2008)

A study was conducted under greenhouse nursery condition on the efficacy of seven

indigenous arbuscular mycorrhizal (AM) fungi in the improvement of growth, biomass,

nutrition and phytochemical constituents, namely total phenols, ortho dihydroxy phenols,


flavonoids, alkaloids, tannins and saponins, in the roots and leaves of Indian borage

Plectranthus amboinicus (Lour) Spreng (Rajeshkumar et al. 2008). Studies showed the

extent of growth, biomass, nutritional status and phytochemical constituents enhanced by

AM fungi varied with the species of AM fungi inhabiting the roots and leaves of P.

amboinicus seedlings. It was observed that Gigaspora margarita was the best AM symbiont

for P. amboinicus used in the experiment. Further consideration of the ability for higher root

colonization, plant biomass, biovolume index, and mineral and phytochemical constituents

suggested that a clear and specific relationship existed between a particular species of

fungus and the plant (Rajeshkumar et al. 2008).

2.8.2. Interaction of P. indica with Medicinal Plants

The medicinal plants Spilanthes calva and Withania somnifera were inoculated with P.

indica, a plant growth-promoting root endophyte, in nurseries and subsequently transferred

to the field. A significant increase in growth and yield of both plant species was recorded

relative to un-inoculated controls. Shoot and root length, biomass, basal stem, leaf area,

overall size, number of inflorescences, flowers and seed production were all enhanced in the

presence of the fungus (Fig. 7). Net primary productivity was also higher than in control

plants. The results clearly indicated the commercial potential of P. indica for large-scale

cultivation of S. calva and W. somnifera (Rai et al. 2001)

Spilanthes calva commonly known as ‘toothache plant’ or ‘virus blocker’ is well known for

enhancing the immunity. Research had been carried out by Rai et al. (2002) to study the

influence of P. indica on the antifungal principle of medicinal plant S. calva. An antifungal

efficacy was shown by aqueous and petroleum ether extracts of S. calva against Fusarium

oxysporum and Trichophyton mentagrophytes. The petroleum ether extract of S. calva was

more effective than the aqueous extract in inoculated as well as un-inoculated plants. The

antifungal activity of the plant was enhanced due to the increase in slight spilanthol content

after inoculation of P. indica (Rai et al. 2002).

Rai and Varma (2005) found that the symbiotic fungus P. indica enhances the growth of

Adatodha vasica as earlier reported with S. calva and W. somnifera (Rai et al. 2001). They

observed a profuse proliferation of roots of A. vasica after inoculation of P. indica, which

was not observed in previous experiments in Rai et al. 2001. Root-colonization of A. vasica

by P. indica increased with time from 53 per cent after 2 months to 95 per cent after 6


months. More over they also found the fresh and dry weight of shoots and roots of A. vasica

inoculated plants was higher than that of the corresponding controls. It has been reported

that P. indica also promoted the plant growth and biomass of medicinal plants Tridax

procumbens, Abrus precatorius and Solanum nigrum. Early flowering and fruit settings

were also noted in fungus inoculated plants (Kumari 2005).

Baldi et al. (2008) developed cell suspension cultures of Linum album from

internode portions of in vitro germinated plant in Gamborg’s B5 medium supplemented

with 0.4 mg naphthalene acetic acid/l. The highest biomass was 8.5 g/l with

podophyllotoxin and 6-methoxypodophyllotoxin at 29 and 1.9 mg/l, respectively after 12 d

cultivation. They were able to successfully co-culture L. album cells with axenically

cultivable arbuscular mycorrhiza-like fungi, P. indica and Sebacina vermifera, for the first

time. These enhanced podophyllotoxin and 6-methoxypodophyllotoxin production by about

four- and eight-fold, respectively, along with a 20% increase in biomass compared to the

control cultures. On dual culture of Artemisia annua with P.indica both in vivo and invitro,

it was been found that P. indica enhanced the performance of the treated plants and

artemisian content was also increased by 2.5 folds in leaves of treated plants (personal

communication).

Research was carried out by Prasad et al. (2008b) on in vitro cultures of Bacopa

monniera with symbiotic fungus P. indica. The fungus treated plants showed enhanced

growth in comparison to non-treated plants. Extensive root colonization was also observed

in root cells of treated plants. Hyphae was present on the surface and occupied the root

cortex at inter- and intra cellular levels. Fungus treated plants produced several fold more

anti-oxidant activity, bacosides and plant biomass. Microbial biotization with dual microbes

namely P. indica and Pseudomonas fluorescens enhanced survival of Chlorophytum spp up

to 91.2 per cent over uninoculated control (78.8 per cent), on transfer from laboratory to

green house. Biotized field grown plants exhibited increase in root length, number of lateral

roots, shoot dry weight, leaf length, number and dry weight of fleshy roots in dual

inoculation which were significantly better over single as well as un-inoculated control.

Plants inoculated with P. indica exhibited maximum chlorophyll content while maximum P

content was observed in dual inoculated plants, which was at par with P. indica alone even


at low phosphorus. Higher saponin content was observed with both, P. indica alone as well

as dual inoculations (Gosal et al. 2010).

Recent work of Dolatabadi et al. (2010) demonstrated that P. indica and Sebacina

vermifera inoculation of Foeniculum vulgare (fennel) significantly increased oil yield as

compared to non- inoculated control plants. Their work revealed through GC and GC-MS

studies that the level of anethol was also enhanced with P. indica and S. vermifera

inoculation.

interaction of a medicinal plant coleus forskohlii...

Documents