Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

A thorough review of literature has shown that P. indica has enormous potential for growth promotion of diverse groups of plants by colonization of host roots (Singh et al. 2000; Rai et al. 2001; Malla et al. 2002, Rai and Varma 2005; Oelmuller et al. 2009; Achatz et al. 2010). The fungus is of utmost biotechnological importance and is multifunctional (Singh et al. 2003: Kumar et al. 2011). P. indica is similar to arbuscular mycorrhizal fungi in many respects (Varma and Schuepp 1994; Varma et al. 1999, 2001; Singh et al. 2000; Rai and Varma 2005). But, unlike arbuscular mycorrhizal fungi, it can be cultured on artificial medium (Varma et al. 1999, 2001; Pham et al. 2004). Thus, this revolutionary endophytic fungus functions as a plant growth promoter and biofertilizer in nutrient-depleted soils. P. indica promotes the growth of plants and improves their productivity, increases the drought tolerance, delays the wilting of leaves, prolongs the ageing of callus tissue, enhances the uptake of phosphate from the soil and relieves the stress conditions caused by acidity, desiccation and heavy metal toxicity (Oelmuller et al. 2009; Yadav et al. 2010; Camehl et al. 2010; Kumar et al. 2011). Plants treated with P. indica resulted in increase in overall growth, flowering, nutrient uptake and enhancement in secondary metabolites production in plants (Dolatabadi et al. 2011a, b; Das et al. 2012). Pronounced growth promotional effect was recorded with terrestrial orchids also (Prasad et al. 2004). For more information readers may refer to Chap. 1.

Medicinal plants are of great importance because of their healing properties due to the presence of various active principles and different secondary metabolites (Rai 1994). A large number of remedies have been offered to mankind by the plant kingdom, of which many are provided by aromatic plants (Wyk and Wink 2004). Over three quarters of the world population rely mainly on medicinal plants and plant extracts for health care. More than 30 % of the entire plant species, at one time or other, was used for medicinal purposes. The rural folks and tribals in India as well as in other developing countries even now depend largely on the surrounding plants/forests for their day-to-day needs. Medicinal plants are being looked upon not only as a source of health care but also as a source of income. The value of medicinal plants related trade in India is of the order of 5.5 billion US dollar and is further increasing day by day. The international market of herbal products is estimated to be US $ 62 billion. India shares in the global market of medicinal plants trade is less than 0.5 % (http://www.agricultureinformation.com/forums/questions-answers/34618-cultivation-medicinal-plants-india-government-support.html, dated 17th April 2012). It has been estimated that in developed countries such as United States, plant drugs constitute as much as 25 % of the total drugs, while in fast developing countries such as China and India, the contribution is as much as 80 %. Thus, the economic importance of medicinal plants is much more to countries such as India than to rest of the world. These countries provide two-third of the plants used in modern system of medicine, and the health care system of rural population depends on indigenous systems of medicine (Chandel and Sharma 1997; Lambert 1998; Kavitha et al. 2010). Traditional Indian Ayurveda medicines have a 70 % share of the formal medicine market in India (Lambert 1998).

India is one of the world’s 12 biodiversity centres with the presence of over 45,000 different plant species. India’s diversity is unmatched due to the presence of 16 different agro-climatic zones, 10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of specific species). Of these, about 15,000–20,000 plants have remarkable medicinal value. However, only 7,000–7,500 species are used for their medicinal values by traditional communities (Kurian and Sankar 2007, http://megforest.gov.in dated 26th April 2012). There are about 45,000 medicinal plant species in India, with hot spots in the region of Eastern Himalayas, Western Ghats and Andaman and Nicobar Island. The officially documented plants with medicinal potential are 3,000, but traditional practitioners use more than 6,000. In rural India, 70 % of the population depends on the traditional type of medicine, the Ayurveda (http://medicinalpltsinindiamp, dated 17th April 2012). Hence, the medicinal and aromatic plants are of immense importance for welfare of the mankind. Taking into consideration, the above facts, it is the need of the hour to employ techniques which may enhance the growth and yield of economically important plants. In this context, arbuscular mycorrhizal fungi and other endophytes like P. indica play a crucial role (Oelmuller et al. 2009; Harman 2011).

It has been extensively studied that symbiotic fungus P. indica promoted growth and enhanced active ingredients of the medicinal as well as economically important plants by forming association with their roots. This novel fungus has been also established as an agent for biological hardening of tissue culture-raised plantlets (Sahay and Varma 1999, 2000; Oelmuller et al. 2009; Gosal et al. 2010). So far, more than 148 plants have been interacted with the fungus and documented. This include plants such as Centella asiatica, Coriandrum sativum, Artemisia annua, Spilanthes calva, Arabidopsis thaliana, Cajanus cajan, Arachis hypogaea, Mimosa pudica, Cicer arietinum, Allium cepa, Hordeum vulgare, Zea mays, Saccharum officinarum, Withania somnifera, Solanum lysopersicum, etc. (Bagde et al. 2010). This chapter provides an overview of case studies of selected medicinal plants and their interaction with the wonder fungus P. indica.

2 Interaction of P. indica with Different Medicinal Plants

A large number of medicinal plants like Spilanthes calva, Withania somnifera, Bacopa monnieri, Coleus forskohlii and others were inoculated with the P. indica in pots as well as in fields to study its influence on the host plants. The symbiotic fungus promoted the overall growth and development of all the medicinal plants tested so far. It also enhanced percentage flowering and fruit development in medicinal plants like Coleus forskohlii, Spilanthes calva and Withania somnifera. It also promoted enhancement of secondary metabolites contents in medicinal plants inoculated with P. indica. Details of case studies are discussed from Sect. 9.2.1 to 9.2.13.

2.1 Interaction with Artemisia annua

Artemisia annua is also known by many names like sweet wormwood, sweet sagewort or annual wormwood. It is a common type of wormwood which is native to temperate Asia but now found throughout the world (Kapoor et al. 2007). The plant has traditionally been grown in China for medicine and more recently in Europe for its aromatic leaves which are used in flavouring beverages. It has fern-like leaves, bright yellow flowers and with camphor-like scent (Fig. 9.1a). Its height averages about 2 m tall, and the plant has a single stem, alternating branches and alternating leaves which range 2.5–5 cm in length. It secretes many medicinal compounds including “artemisinin” which is well antimalarial drug (Kapoor et al. 2007). P. indica showed growth promotion in A. annua when inoculated in seedlings stages under field condition (Fig. 9.1c,d; Tables 9.1a and 9.1b).

Fig. 9.1
figure 00091

Field grown plants of Artemisia annua (a) at flowering stage (b) Seeds; Five months old plants (c) Control plants without P. indica showing yellow-brown leaves, (d) Plants inoculated with P. indica showing dark green leaves

Full size image
Table 9.1a Interaction of Artemisia annua seedlings with P. indica (field trial expt.)
Full size table
Table 9.1b Interaction of Artemisia annua seedling with P. indica (field trial expt.)
Full size table

P. indica promoted the growth of the tissue culture-raised plantlets when interacted in vitro. It showed higher rate of shoot and root development (Fig. 9.2a, b). Similarly, it helped in biohardening of tissue culture-raised plantlets when transferred to the natural environmental conditions. Inoculated plants showed a higher survival rate than the uninoculated control (uninoculated) plants. It was also reported that interaction with fungus not only increased the biomass but also the Artemisinin (an antimalaria drug) content.

Fig. 9.2
figure 00092

Effect of P. indica on tissue culture-raised plantlets with high rate of shoots and root development. (a) Shoots without P. indica, (b) shoots with P. indica, (c) roots without P. indica, (d) roots with P. indica

Full size image

2.2 Interaction with Tridax procumbens

Tridax procumbens Linn. belongs to family Asteraceae, found throughout India and is employed as indigenous medicine. It is reported for treatment of variety of ailments including jaundice (Kumari 2005; Bhagwat et al. 2008). It is commonly known as “Ghamra” and in English popularly called “coat buttons” because of the appearance of its flowers. It has been extensively used in Indian traditional medicine as anticoagulant, antifungal and insect repellent, in bronchial catarrh, diarrhoea and dysentery. Moreover, it possesses wound healing activity and promotes hair growth. Antioxidant properties have also been demonstrated (Bhagwat et al. 2008). Interaction of T. procumbens with P. indica promoted the growth and biomass as depicted in Fig. 9.3 and Table 9.2 (Kumari 2005).

Fig. 9.3
figure 00093

Effect of P. indica on plant growth and biomass of T. procumbens. (a) Plants without fungus, (b) plants with fungus

Full size image
Table 9.2 Comparison of different growth parameters in control and T. procumbens plants inoculated with P. indica
Full size table

2.3 Interaction with Abrus precatorius

Abrus precatorius is a plant of the Fabaceae family. It is also known as Indian liquorice, jequirity, crab eye, Glycyrrhiza glabra, among others. The plant grows widely in fairly dry climates of tropical and subtropical regions, such as India, Sri Lanka, Nigeria and the West Indies. Plant pacifies vitiated “pitta”, “vata”, inflammation, vitiligo, skin disease, wounds, ulcer, alopecia, asthma, stomatitis and fever (Kumari 2005). The principal active ingredients present are abrin, abraline, choline, precatorine, abricin and abridin. Studies showed positive influence of the fungus P. indica on Abrus precatorius (Fig. 9.4 and Table 9.3).

Fig. 9.4
figure 00094

Effect of fungi on plant growth and biomass of A. precatorius (a) without P. indica, (b) with P. indica

Full size image
Table 9.3 Comparison of different growth parameters in control and A. precatorius plants inoculated with P. indica
Full size table

2.4 Interaction with Bacopa monnieri

Bacopa monnieri commonly known in India as Brahmi is an important Ayurvedic medicinal plant belonging to Scrophulariaceae family. In the traditional system of medicine, Brahmi is a reputed nervine tonic; it is also used to treat asthma, insanity, epilepsy, hoarseness, enlargement of the spleen, snake bite, rheumatism, leprosy, eczema and ringworm and as a diuretic, appetitive and cardiotonic. The main active ingredient of B. monnieri is believed to be the bacosides (Prasad et al. 2004).

Tissue culture technology could play an important role in the clonal propagation, germplasm conservation and improvement of B. monnieri. Shoot regeneration has been reported from the distal ends of 1–12 mm long internode segments of B. monnieri cultured on growth regulator-free medium, longer internode being more conducive to regeneration (Fig. 9.5). P. indica is documented to promote plant growth and protects the host against root pathogens and insects. P. indica promoted the plant growth and enhanced antioxidant activity as well as the active ingredient bacoside by several folds (Prasad et al. 2004). Plant treated with P. indica also showed significant increase in number of leaves, number of branches, root and shoot length as depicted in Fig. 9.5 and Table 9.4.

Fig. 9.5
figure 00095

In vitro grown plants of B. monnieri. (a) Plants without P indica, (b) plants with P. indica

Full size image
Table 9.4 Effect of P. indica on growth of B. monnieri in 90 days time on Kaefer semisolid medium. Control: Without P. indica
Full size table

It was found that there was an inherent problem with the micropropagated plants during the time of transplantation from the laboratory to field. It was noticed that the rate of survival was very low, up to 40 % in the field conditions. The salient reason could be the “transient transplant shock” that resulted in the stunted growth. For employing the technique, inoculation of micropropagated plantlets with active cultures of AMF or mycorrhiza-like fungi appears to be critical for their survival and growth. These pre-acclimatized plantlets when transferred to field, overcome the transient transplant shock, and were able to cope up with changed environment of transplantation which also helps in their successful establishment. Pre-establishment of mycorrhiza in the host roots also helps in the development of the synergistic effect with other rhizosphere microflora that competes in the ecosystem for successful survival (Prasad et al. 2004).

2.5 Interaction with Coleus forskohlii

Coleus forskohlii (willd.) (Briq. syn. C. Barbatus (Andr.) Benth) is an aromatic herbaceous species belonging to the family Lamiaceae. It is native to India and is recorded in Ayurvedic Materia Medica under the Sanskrit name “Makandi” and “Mayani” (Shah 1996; Patil et al. 2001). It grows wild in the subtropical warm temperate climates of India, Nepal, Burma, Sri Lanka and Thailand. It is also found in Egypt, Arabia, Ethiopia, tropical East Africa and Brazil (Willemse 1985; Chandel and Sharma 1997; Kurian and Sankar 2007). C. forskohlii is a perennial plant that grows to about 45–60 cm tall. It has four angled stems that are branched, and nodes are often hairy. Leaves are 7.5–12.5 cm in length and 3–5 cm in width, usually pubescent, narrowed into petioles. Inflorescence is raceme, 15–30 cm in length; flowers are stout, 2–2.5 cm in size, usually perfect and calyx hairy inside. Upper lip of calyx is broadly ovate. The blue or lilac corolla is bilabiate. The root is typically golden brown, thick, fibrous and radially spreading. Roots are tuberous, fasciculated, 20 cm long and 0.5–2.5 cm in diameter, conical fusiform, straight, orangish within and strongly aromatic. C. forskohlii is the only species of the genus to have fasciculated tuberous roots (Kavitha et al. 2010). The entire plant is aromatic, and whole plant C. forskohlii (roots, flowering shoots and leaves) have commercial importance. The plant contains 0.05–0.1 % forskolin/g fresh weight which is a diterpenoid and used as drug. Roots are the major source of forskolin (coleonol), although diterpenoids are found in almost all parts (Chandel and Sharma 1997). Forskolin from C. forskohlii is an activator of adenylate cyclase and thus leads to the production of second messenger cyclic AMP (cAMP) (Kavitha et al. 2010). Leaves also contain diterpenoid methylene quinine, coleonol, barbatusin and cyclobutatusin. Barbatusin is used against lung carcinoma and lymphatic leukaemia (Kurian and Sankar 2007). Other secondary compounds found in C. forskohlii are monoterpenes, monoterpene glycosides, sesquiterpenes and phenolic glycosides (Ahmed and Viswakarma 1988; Ahmed and Merotra 1991; Petersen 1994). In the traditional Ayurvedic 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 greying and restoring grey hair to its normal colour (Keikichi et al. 1988). Furthermore, forskolin is valued for anti-allergic activity (Gupta et al. 1991). Roots are hypotensive and spasmolytic and are given to children in constipation. 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; Kavitha et al. 2010). 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 an attractive and delicate odour with a spicy note (Misra et al. 1994). The essential oil has potential use in the food flavouring industry and can be used as an antimicrobial agent (Chowdhary and Sharma 1998). There is a wide variation in morphology, essential oil content and yield parameters among the genotypes of C. forskohlii (Patil et al. 2001). Vishwakarma et al. (1988) screened 38 genotypes collected from various locations to identify the potential genotypes for forskolin. The content of forskolin varied substantially with different genotypes, from 0.01 to 0.44 % on the fresh weight basis.

C. forskohlii was interacted with root endophytic fungus P. indica that mimics AMF under field condition. Growth of C. forskohlii in the presence of P. indica resulted in an enhancement in aerial growth and biomass production of medicinal plants. The height, number of branches, average length of the branches and number of leaves and leaf area of P. indica-treated 6-month-old plants was significantly increased compared to the untreated control plants (Table 9.5a and Fig. 9.6). Although the fungus also promoted the number (22 %) and lengths (32 %) of the roots, they looked fibrous and tough in texture (Table 9.5a). This demonstrates that the initial promotion of rooting shifted towards the development of fibrous root structures in the presence of the fungus. Consequently, after 6 month on the field, the overall weights of colonized roots were lower than the weight of the uncolonized controls (Table 9.5b). Also the thickness of colonized roots was dramatically reduced. As a result, shoot growth was promoted, and root growth was retarded by P. indica when medicinal plants C. forskohlii when treated with P. indica. Flowering occurred earlier and more vigorously in P. indica-colonized plants. Colonized plants flowered at least 7 days earlier than the untreated controls. After 180 days, 31 % of non-treated but 81 % of P. indica-treated plants flowered. Moreover, the number and length of the inflorescence were significantly higher in colonized plants compared to their untreated counterparts (Das et al. 2012).

Table 9.5a Influence of P. indica on plant length (cm), number of branches, average length of branches (cm), number of leaves, leaf area (cm2), number of roots and root thickness (cm) of field grown C. forskohlii plants (growth period: 6 months)
Full size table
Fig 9.6
figure 00096

Influence of P. indica on 6-month-old C. forskohlii in field condition. Photographed after 6 months. Figure represents plant morphology as a result of interaction between P. indica on C. forskohlii under field condition. Each polythene bag contained 2.5 kg of unsterile sand, field soil and compost (1:1:0.25 w/w). The fungal inoculum was 2 % (w/v). Each bag contained 30-day-old rooted plant cuttings. Irrigation was done on alternate days using underground water. -Pi: Plants without inoculum of P. indica; +Pi: Plants treated with fungus

Full size image
Table 9.5b Influence of P. indica on plant biomass (growth period 6 months)
Full size table

2.6 Interaction with Adhatoda vasica

Adhatoda vasica Nees, commonly known as Malabar nut belongs to family Acanthaceae. It is an evergreen shrub. The plant is used for the preparation of medicine for asthma, bronchitis and other pulmonary disorders. It is also used as antiarthritis, antiseptic, antimicrobial, expectorant, sedative and antituberculosis (Dey 1980; Singh and Jain 1987). Glycodin®, a well-known product used for the cure of bronchitis, is extracted from the leaves of the plant. In Ayurveda, a number of medicines are manufactured by this plant. There is a pressing need of rapid multiplication of this plant due to increasing demand by pharma industries.

Rai and Varma (2005) studied the role of P. indica in growth promotion of A. vasica. The cuttings of A. vasica were inoculated with P. indica to assess the growth promoting property of P. indica. They reported that P. indica enhanced the growth of A. vasica (Fig. 9.7). The authors further observed profuse proliferation of roots of A. vasica after inoculation of P. indica. Root colonization of A. vasica by P. indica augmented with time from 53 % after 2 months to 95 % after 6 months. There was a significant enhancement in the growth rate of the plants inoculated with P. indica. The growth was very fast up to 2 months (Fig. 9.8) and slowed down thereafter. At each observation, growth was significantly higher for the plants inoculated with P. indica as compared to the control plants.

Fig. 9.7
figure 00097

Effect of P. indica on A. vasica (a) Plants without P indica, (b) Plants with P. indica

Full size image
Fig. 9.8
figure 00098

Growth response of A. vasica (in cm) after inoculation with P. indica, control plants are without P. indica

Full size image

The fresh and dry weight of shoots and roots of A. vasica-inoculated plants was higher than that of the corresponding controls (Fig. 9.9). This suggests that P. indica is an appropriate endophyte for fast growth of the plants. After additional trials and evaluation of active principles or secondary metabolites production, P. indica may be recommended for growth enhancement of A. vasica.

Fig. 9.9
figure 00099

Fresh and dry weight of A. vasica after 6 months after interaction with P. indica, control plants are without P. indica

Full size image

2.7 Interaction with Withania somnifera and Spilanthes calva in Field Trials

W. somnifera is also known as Indian ginseng and belongs to the family Solanaceae. There are more than 91 pharmaceutical products manufactured from the roots of this plant and are potential sources of a promising drug for cancer (Devi 1996). S. calva is a member of Asteraceae and is called as “toothache plant” and is well known for enhancing immunity (Rai et al. 2001). The plant has anti-ageing property and cures diseases of gums like pyorrhoea and is also useful in toothache. The leaves of this plant stimulate salivation which is due to an active chemical known as “spilanthol”. Because of high medicinal value, there is an increasing demand for these plants in national and international market (Rai et al. 2001). Therefore, there is a greater need to augment the growth and secondary metabolites of these plants. This inoculation of beneficial microorganisms like arbuscular mycorrhizal fungi or other growth promoting endophytes serves the purpose.

Pronounced growth response in W. somnifera and S. calva after inoculation with P. indica was noted (Figs. 9.10a, b and 9.11a, b). The data revealed that the plants treated with P. indica were superior in development compared to control plants (uninoculated with P. indica). A significant increase in the shoot length was observed in the inoculated plants. The microscopic examination of stained root samples revealed a high colonization of S. calva and W. somnifera by P. indica in 62 and 73 % root length, respectively (Fig. 9.12).

Fig. 9.10
figure 000910

Pronounced growth response in W. somnifera after inoculation with P. indica: (a) control (without P. indica), (b) inoculated with P. indica

Full size image
Fig. 9.11
figure 000911

Pronounced growth response and flowering in S. calva after inoculation with P. indica. (a) Control (without P. indica), (b) inoculated with P. indica

Full size image
Fig. 9.12
figure 000912

Influence of P. indica on shoot and root length and on per cent root colonization of S. calva and W. somnifera in a field trial. The control plants were treated with an equal amount of autoclaved mycelium

Full size image

The basal and leaf area of treated plants was also increased (Table 9.6). Interestingly, in inoculated S. calva plants, some large, kidney-shaped heads were observed among the normal round heads. These kidney-shaped heads were never observed in control plants. The length of the inflorescence and the number of flowers in inoculated S. calva were also increased (Table 9.6; Fig 9.11a, b). Similarly, in the inoculated plants of W. somnifera, the number of flowers was higher (Table 9.6) as compared to controls. In both medicinal plants, seed count was higher as compared to controls.

Table 9.6 Influence of P. indica on morphology and growth of host plants (S. calva and W. somnifera) 90 days after inoculation in a field trial. The control plants were treated with equal amount of autoclaved mycelium; control: without P. indica
Full size table

2.8 Interaction with Turmeric (Curcuma longa L) in a Field Trial

Turmeric (Curcuma longa L.) is a perennial crop of Zingiberaceae family, widely cultivated in India and other parts of the world. In medieval Europe, turmeric became known as Indian saffron, since it was widely used as an alternative to the far more expensive saffron spice (Ruby et al. 1995). The plant has wide spectrum of medicinal properties and commercial applications due to presence of the well-known bioactive component curcumin. The microbial bioinoculants such as, phosphate solubilizing bacteria (PSB) and arbuscular mycorrhizal fungi (AM fungi) were used for the improvement of the crop, which are shown by the enhancement in the productivity (Eigner et al. 1999). It is a very important medicinal plant and extensively used in Ayurveda, Unani and Siddha medicine as the treatment for various diseases (Dasgupta et al. 1969). It is also used as a food additive (spice), preservative and colouring agent in many countries such as China and Southeast Asia. The turmeric is also used in Ayurveda for the treatment of sprains and swelling caused by injury (Ammon et al. 1992).

The present study focused to understand the interaction of the P. indica with turmeric plants. The impact of P. indica as bio-inoculant on growth and yield of turmeric was assessed by morphological parameters of the turmeric plant both in treated (with P. indica) and untreated (control) plants in field trials. It was observed that the plants of turmeric treated with P. indica demonstrated remarkable growth as compared to the control (Fig. 9.13). There was enhancement of 12 % in yield of treated turmeric plants. The rhizomes of the plant treated with P. indica were found to be healthier and thicker than the control (Fig. 9.13).

Fig. 9.13
figure 000913

Effect of the P. indica on the field grown plant and rhizomes of turmeric (Curcuma longa L.) (a) Plants without P. indica, (b) plants treated with P. indica. Control: plants without P. indica, P. indica: inoculated with P. indica

Full size image

2.9 Estimation of Total Alkaloids and Withanolides in Inoculated and Control Plants

Quantitative determination of total alkaloids, withanolides and withaferin A was made by TLC densitometry. Ashwagandha roots were extracted with methanol (20 ml × 3), filtered and evaporated. The extract thus obtained was defatted with n-hexane (10 ml × 3) and then extracted with 1 % sulphuric acid (5 ml × 3), basified with ammonia, extracted with chloroform (10 ml × 3), dried over anhydrous sodium sulphate, filtered, evaporated and weighed for total alkaloid content. The sulphuric acid insoluble was extracted with diethyl ether (10 ml × 3), dried over anhydrous sodium sulphate, filtered, evaporated and estimated as crude withanolide. The total withanolides and alkaloid contents increased after inoculation of P. indica. This suggests that P. indica can be used for enhancement of active principles (Table 9.7).

Table 9.7 Percentage of alkaloid content in inoculated Withania somnifera (with P. indica) and uninoculated plants
Full size table

2.10 Enhancement of Antimycotic Activity in Spilanthes calva due to Increase in Active Principles after Inoculation of P. indica

Plants of Spilanthes calva inoculated with P. indica and uninoculated (control) exhibited antifungal activity against Fusarium oxysporum (opportunistic human pathogen) and Trichophyton mentagrophytes (potential human pathogen). A significant antimycotic activity in aqueous and petroleum ether extracts of S. calva was recorded. Petroleum ether extract of S. calva was more effective than aqueous extract in inoculated as well as uninoculated plants. The minimum inhibitory concentration of F. oxysporum was recorded as 125 μl/ml when treated with aqueous extract of inoculated plants, whereas 250 μl/ml aqueous extract of inoculated plant was needed to inhibit the growth of T. mentagrophytes. F. oxysporum was found to be more sensitive than T. mentagrophytes. The value of MIC for F. oxysporum was very high (1,000 μl/ml) when aqueous extract of uninoculated plant was tested. In addition, aqueous extract of uninoculated plant of S. calva did not inhibit the growth of T. mentagrophytes which is a potential fungal pathogen causing skin and nail infections in human beings (Rai et al. 2004).

Petroleum ether extract of inoculated plants of S. calva showed a remarkable antifungal activity. Extract (62.5 μl/ml) was sufficient to inhibit the growth of 1 ml spore suspension of F. oxysporum, whereas in extract of uninoculated plants the MIC value reached to 500 μl/ml of spores. Similarly, for T. mentagrophytes, 125 μl extract of inoculated plants was enough to arrest the growth, while MIC value was quite high (1,000 μl/ml) in case of petroleum ether extract of uninoculated plants.

The extract of inoculated plants of S. calva showed significantly higher activity compared to uninoculated plants. Additionally, extract of petroleum ether was found to be more active than aqueous extract which shows synthesis of higher quantity of spilanthol in inoculated plants. The enhancement of spilanthol was revealed by the chemical analysis of the roots of the plant (Fig. 9.14).

Fig. 9.14
figure 000914

Estimation of spilanthol in roots of Spilanthes calva. Control: plants without P. indica, P. indica: plants treated with P. indica

Full size image

2.11 Interaction with Safed Musli (Chlorophytum sp.)

Chlorophytum sp., which is commonly called as “Safed Musli”, is an important medicinal herb belonging to family Liliaceae, having fleshy roots of medicinal importance for the cure of physical weakness, diabetes and arthritis. The dried fasciculed storage roots of this herb, popularly known as “Musli”, have strong aphrodisiac, antistress and immuno-modulatory properties due to the presence of steroidal saponins and polysaccharides. The continued collection of these medicinal plants has resulted in the fast depletion of its population, may be due to low rate of multiplication through vegetative means and shy flowering behaviours (Mathur et al. 2008; Gosal et al. 2010).

Chlorophytum plants when inoculated with the fungus P. indica, after 30 days of growth in green house, significant increase in root length and the number of lateral roots were observed over uninoculated control (Gosal et al. 2010). Survival rate of musli plants as recorded after transplantation in soil in the greenhouse was improved with microbial P. indica biotization (Mathur et al. 2008; Gosal et al. 2010). In vitro plantlets with roots when interacted with P. indica, there was 86 % of plantlets survival in green house, at biohardening stage. An important observation was noted that even the micropropagated un-rooted shoots could form roots under in vivo conditions when treated with P. indica with more than 43 % plantlet establishment (Mathur et al. 2008). Biotization with P. indica alone or in combination with P. fluorescens has led to increase in saponin content (Gosal et al. 2010).

2.12 Interaction of P. indica with Fennel (Foeniculum vulgare)

Fennel (Foeniculum vulgare) a member from the family Apiaceae is one of the most important aromatic plants widely applied in culinary and medicinal preparations. It is generally considered indigenous to the Mediterranean area, but it is also cultivated elsewhere (Russia, India, China and Japan). Fennel is used against digestive disorders such as spasmodic gastrointestinal complaints and bloating. It may be an effective diuretic and a potential drug for the treatment of hypertension, nervous disturbances, paediatric colic and some respiratory disorders due to its antispasmodic effects. Essential oils are mainly concentrated in the fruits and provide their unique aroma and taste. Anethole and fenchone are the most important volatile components of F. vulgare essential oil (Dolatabadi et al. 2011a).

P. indica significantly increased growth of the inoculated of fennel plants in comparison to uninoculated control plants. As well as it significantly increased dry weight of 1,000 fruits in comparison to controls. Not only biomass but the concentration of essential oil increased in pot cultures inoculated with P. indica in comparison to controls (Dolatabadi et al. 2011a). Their work revealed through GC and GC-MS studies that the level of anethole was also enhanced with P. indica inoculation.

2.13 Interaction with Linum album

Lignans constitute a large group of secondary metabolites synthesized by many plants. These compounds are usually formed from two phenylpropanoid units and manifest considerable biological activity. Podophyllotoxin, a lignan with antiviral and antineoplastic activities, is used today primarily as a precursor for the semi-synthesis of established cancer therapeutics such as etoposide, teniposide and Etopophos. Linum album is an herbaceous and medicinal plant that has important lignan such as podophyllotoxin. Podophyllotoxin and 6-methoxy podophyllotoxin have antiviral and anticancer properties (Chashmi et al. 2011). Podophyllotoxin has been obtained by solvent extraction from the rhizomes of plants Podophyllum peltatum and P. hexandrum that belong to the family Berberidaceae. Due to endangered status of its natural source and economically unfeasible chemical synthesis, there is an imperative need to search for alternate ways to produce these lignans by cell cultures. Cell cultures of Linum album are known to produce these lignans with highest productivity (Baldi et al. 2008).

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-day cultivation. They were able to successfully coculture 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.

3 Conclusions

From the studies of the interaction of P. indica with selected medicinal plants, it can be concluded that P. indica acts as a potential colonizer and the plant growth promoter fungus which induced a faster development of the aerial part by promoting early maturation with respect to flowering and biomass. Besides this, it also enhances the absorption of the nutrients by underground roots. The increased growth of P. indica-colonized plants is due to enhanced nutrient uptake such as phosphorus and nitrogen from the soil. P. indica is also involved in the transportation of the phosphate to the host plant. The fungus colonizes both monocots and dicots. The wonder fungus also provided protection when inoculated into the tissue culture-raised plants by overcoming the “transient transplant shock” on transfer to the field or green house and rendered very high survival percentage on transplanted in vitro plants. Finally, the fungus is multifunctional and thus should be used for the growth promotion of medicinally important plants.