interaction of a medicinal plant coleus forskohlii...
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Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 24 -
STATE-OF-ART
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 25 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 26 -
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
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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
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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
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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).
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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
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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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 32 -
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).
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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).
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 34 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 35 -
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.
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 36 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 37 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 38 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 39 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 40 -
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;
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 41 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 42 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 43 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 44 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 45 -
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,
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 46 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 47 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 48 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 49 -
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).
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 50 -
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 )
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 51 -
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,
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 52 -
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)
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 53 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 54 -
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.
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 55 -
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)
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 56 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 57 -
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.
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 58 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 59 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 60 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 61 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 62 -
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.
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 63 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 64 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 65 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 66 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 67 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 68 -
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,
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 69 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 70 -
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
Interaction of C. forskohlii with P.indica for Secondary Metabolites Evaluation - 71 -
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.