The root endophyte fungus Piriformospora indica leads to early ...

RESEARCH PAPER Plant Signaling & Behavior 7:1, 1–10; January 2012;

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2012 Landes Bioscience

The root endophyte fungus Piriformospora indica leads to early flowering, higher biomass and altered secondary metabolites of the medicinal plant, Coleus forskohlii Aparajita Das,1 Shwet Kamal,2 Najam Akhtar Shakil,3 Irena Sherameti,4 Ralf Oelmüller,4 Meenakshi Dua,5 Narendra Tuteja,7 Atul Kumar Johri6,* and Ajit Varma1,* 1

Amity Institute of Microbial Technology (AIMT); Amity University Uttar Pradesh (AUUP); Noida, India; 2Directorate of Mushroom Research; Indian Council of Agriculture Research; Solan, Himachal Pradesh, India; 3Division of Agricultural Chemicals; Indian Agricultural Research Institute; New Delhi, India; 4Institute of General Botany and Plant Physiology; Friedrich-Schiller-University Jena; Jena, Germany; 5School of Environmental Sciences; Jawaharlal Nehru University; New Delhi, India; 6School of Life Sciences; Jawaharlal Nehru University; New Delhi, India; 7Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India

Keywords: Coleus forskohlii, Piriformospora indica, secondary metabolite, p-cymene Abbreviations: AMF, arbuscular mycorrhizal fungi; GC, gas chromatography; MS, mass spectrometry; HPLC, high pressure liquid chromatography; NT, non- colonized plants as controls; T, P. indica colonized plants; FW, fresh weight; DW, dry weight

This study was undertaken to investigate the influence of plant probiotic fungus Piriformospora indica on the medicinal plant C. forskohlii. Interaction of the C. forskohlii with the root endophyte P. indica under field conditions, results in an overall increase in aerial biomass, chlorophyll contents and phosphorus acquisition. The fungus also promoted inflorescence development, consequently the amount of p-cymene in the inflorescence increased. Growth of the root thickness was reduced in P. indica treated plants as they became fibrous, but developed more lateral roots. Because of the smaller root biomass, the content of forskolin was decreased. The symbiotic interaction of C. forskohlii with P. indica under field conditions promoted biomass production of the aerial parts of the plant including flower development. The plant aerial parts are important source of metabolites for medicinal application. Therefore we suggest that the use of the root endophyte fungus P. indica in sustainable agriculture will enhance the medicinally important chemical production.

Introduction Medicinal plants have curative properties due to the presence of various complex substances of different composition, which are known as secondary plant metabolites. Of the great number of remedies offered to mankind by the plant kingdom, many are provided by aromatic plants. C. forskohlii (willd.) Briq. [syn. C. Barbatus (Andr.) Benth] is an aromatic herbaceous species of the family Lamiaceae. The whole plant C. forskohlii (roots, flowering shoots and leaves) have commercial importance. The plant contain 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.1 Leaves also contain diterpenoid methylene quinine, coleon, barbatusin and cyclobutatusin. Barbatusin is used against lung carcinoma and lymphatic leukemia.2 Other secondary compounds found in C. forskohlii are monoterpenes, monoterpene glycosides, sesquiterpenes and phenolic glycosides.3-5 In the traditional

Ayurvedic medicine, C. forskohlii is used for treating heart diseases, abdominal colic, respiratory disorder, insomnia, convulsions, asthma, bronchitis, intestinal disorders, burning sensation, constipation, epilepsy and angina in humans and additionally, this plant is also used for veterinary purposes.6-8 Forskolin is also used in the preparation of medicines that suppresses hair graying and restoring gray hair to its normal color. Furthermore, forskolin is valued for antiallergic activity. Roots are hypotensive as well as 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.1,2,8 This indigenous species, besides being used as a medicinal plant, is used as a potent source of essential oil.9 The essential oil present in tubers has an attractive and delicate odor with a spicy note.10 The essential oil has potential use in the food flavoring industry and can be used as an antimicrobial agent.11 There is a wide variation in morphology, essential oil contents and yield parameters among the genotypes of C. forskohlii.9 A total of 38 genotypes were

*Correspondence to: Atul Kumar Johri and Ajit Varma; Email: [email protected] and [email protected] Submitted: 06/25/11; Revised: 10/14/11; Accepted: 10/19/11 http://dx.doi.org/10.4161/psb.7.1.18472

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screened which were collected from various locations to identify the potential genotypes for forskolin. The content of forskolin varied substantially with different genotypes, from 0.01–0.44% on the fresh weight basis.12 The beneficial influence of AMF on C. forskohlii is well-documented.13,14 The root endophyte fungus P. indica mimics AMF in many morphological, functional and growth promotional aspects.15 P. indica can colonize monocot as well as dicot and it acts as a bioregulator, biofertilizer and bioprotector against root pathogens; it overcomes water stress (dehydration), acidity, desiccation and heavy metal toxicity; it protects from pests, delays the wilting of the leaves, prolongs aging of callus tissues, it enhances secondary metabolites production and also increases the nutritional value of the plant.15-21 In contrast to AMF, P. indica can be easily grown on synthetic media thus therefore can be very useful in sustainable agriculture for crop improvement.19 The present study was undertaken to check the impact of P. indica on overall growth, flowering, nutrient uptake and secondary metabolites production on C. forskohlii. We present evidence that P. indica promotes the performance of C. forskohlii, similar to AMF.

Histological studies of the roots of C. forskohlii colonized by P. indica showed inter- and intra-cellular spread of hyphae and the formation of chlamydospores (Fig. 5). The chlamydospores were separated from each other or arranged in chains with two-to-many spores. Not all spores showed the pear shaped structure but some

Results Plant growth, flowering and root colonization. Growth of C. forskohlii in the presence of P. indica resulted in an increase in aerial growth and biomass production. The height, number of branches, average length of the branches, number of leaves and leaf area of P. indica colonized six-month old plants were significantly increased compared with the non-colonized plants (control) (Fig. 1A, B, C and Fig. 2A). Increases of 41% in length of the branches and of 44% in the number of leaves were observed in P. indica colonized plants as compared with the non-colonized plants. Although the fungus also promoted the number (22%) and lengths (32%) of the roots, they looked fibrous and tough in texture (Fig. 1A, B and Fig. 2A). This demonstrates that the initial promotion of rooting shifted toward the development of fibrous root structures in the presence of the fungus. Consequently, after 6 mo on the field, the overall weights of colonized roots were lower than the weight of the uncolonized controls (Fig. 3). Also the thickness of colonized roots was dramatically reduced (Fig. 1A and Fig. 2A). Flowering occurred earlier and more vigorously in P. indica colonized plants. Colonized plants flowered at least 7 d earlier than the non-colonized plants. After 180 d, 31% of non-treated but 81% of P. indica colonized plants flowered (Fig. 2A and Fig. 4A). Moreover, the number and length of the inflorescence were significantly higher in colonized plants compared with non-colonized plants (Fig. 4B, C and Fig. 2B).

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Figure 1. Influence of P. indica on growth parameters of field grown C. forskohlii plants (growth period: 6 mo). (A) Plant length (cm), average length of branches (cm) and root thickness (cm), (B) Number of branches, number of leaves and number of roots, (C) Leaf area (cm2). Each data represents the mean of two independent replicates (Year 2008 and 2009) and each replicate represents 18 samples. *not significant, however all other data were found significant at p , 0.05 according to Student’s t-test.


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Figure 2. A: Influence of P. indica on six months old C. forskohlii under field conditions. Top panel represents plant morphology as a result of interaction between P. indica on C. forskohlii under field conditions. 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 d old rooted plant cuttings. Irrigation was done on alternate days using underground water. Lower panel represents root morphology of C. forskohlii. B: Inflorescence in C. forskohlii. -Pi: non colonized plants; +Pi: colonized plants. Photographs were taken after six months.

Figure 3. Influence of P. indica on plant biomass (growth period 6 mo) Each data represents the mean of two independent replicates (Year 2008 and 2009) and each replicate represents 18 samples, *significant at p , 0.05 according to student’s t-test. ** not significant



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Figure 5. P. indica root colonization of C. forskohlii at the age of six months.

Figure 4. Influence of P. indica on inflorescences on C. forskohlii plants (growth period 3 mo). (A): Flowering %, (B): Number of inflorescence per plant, (C): Average length of inflorescence (cm) per plant. Each data represents the mean of two independent replicates (Year 2008 and 2009) and each replicate represents 18 samples. In case of P. indica colonized plants flowering %, number of inflorescence and average length of the inflorescence were found increased as compared with the non-colonized plants and this difference fold was found significant at p , 0.05 according to Student’s t-test.

of them also appear to be round or ovoid. We have observed 26% root colonization. Chlorophyll and phosphorus contents. We have observed that the number of leaves and leaf area differed significantly between P. indica colonized plants and non-colonized plants (Fig. 1B, C). Chlorophyll a and b and total chlorophyll contents (mg/g

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fresh weight) were more than 30% higher in the leaves of the colonized plants as compared with the non-colonized plants (Fig. 6). The amount of total phosphate per g dried weight of plant increased from 0.55 ± 0.02 mM to 0.72 ± 0.03 mM in the presence of the fungus and this difference was found significant (p , 0.05) (Fig. 7). Secondary metabolite contents in inflorescence and roots of C. forskohlii. Metabolites contents in the inflorescence were determined by GC-MS and HPLC as described in the materials and methods. The amount of the essential oils p-cymene and nonanal, is increased in the P. indica colonized plants as compared with non colonized plants (Fig. 8). Contents of p-cymene is increased more than 2-folds and was found significant (p , 0.05), however nonanal was marginally increased in the colonized plants as compared with the non-colonized plants and was not found significant. (Fig. 8A, B). In agreement with the observation that the overall weight of the roots is reduced in the presence of the fungus, we also noticed that the forskolin content was reduced in the roots of colonized plants (180 mg/ 100 g fresh weight) as compared with the non-colonized plants (380 mg/100 g fresh weight) (Fig. 8C). Discussion In the present study, P. indica was axenically cultivated and applied to the medicinal plant C. forskohlii under controlled field conditions in order to analyze its potential on morphogenesis and secondary metabolite production of the plant. We observed that P. indica enhanced the growth of the plant as compared with the non-colonized plants which is also reported previously about the growth enhancing capabilities of P. indica and thus support our data.15,22-28 We also observed that P. indica not only induced a faster development of the aerial part of the plant, but also caused early maturation with respect to flowering. Similar results were observed in case of barley plants in pot cultures.28 The possible


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compared with the non-colonized plants under field conditions. It has been suggested recently that P. indica is involved in the transportation of the phosphate to the host plant which takes place by a phosphate transporter (PiPT).19 Further knockdown of PiPT results in the less bio mass of the host plant and less transfer of the phosphate in the knock-down-PiPT-P. indica colonized plants as compared with the wild type P. indica colonized plants. It is likely that a crucial determinant for the growth response of the plant is the delivery of phosphorus to the roots from the fungal hyphae. Contrary to this hypothesis, other workers have reported that phosphate does not play a role in the increased biomass of P. indica-treated barley and Nicotiana attenuata treated plants.28,34 The available data from other plant species, the fungal knock out line for the phosphate transporter19 and the established co-cultivation conditions of C. forskohlii Figure 6. Chlorophyll content (mg/g fresh wt) of leaves of field grown C. forskohlii (growth period: six mo) of P. indica-colonized and non-colonized plants. Each data with P. indica under field conditions described here represents the mean of two independent replicates (Year 2008 and 2009) and each allow addressing a large number of scientific replicate represents six samples. In case of P. indica colonized plants chlorophyll a and b questions relevant for agricultural applications. contents were found increased as compared with the non-colonized plants and this Since P. indica colonizes a large variety of host difference was found significant at p , 0.05 according to Student’s t-test. plants (mono as well as dicot), it is likely that the beneficial symbiosis is based on general and not explanation for the faster development of P. indica colonized roots host-specific signaling events. Plant hormones might be a key to compared with the non-colonized plants during all stages of explain the broad host spectrum of P. indica.35-37 Auxin for growth could be due to the earlier expression of developmentally example has been proposed to be involved in P. indica-induced regulated genes.29 growth stimulation.37-39 In our experiments, there was extensive The increased growth of P. indica-colonized plants could branching of roots treated with fungus, which provides evidence be associated with an enhanced nutrient uptake (especially of that auxin also plays a role in the C. forskohlii/P. indica symbiosis. phosphorus and nitrogen) from the soil, as observed for A study on soyabean demonstrates that exogenously applied auxin mycorrhizal associations. In particular, P. indica seems to promote mediates induction of phosphate uptake,40 which may also play a phosphorous and nitrogen uptake from the soil.30-32 It has been role in this symbiosis. It has been reported that the fungus reported that P. indica colonization resulted in increased phos- produces relatively high levels of cytokinins and it’s concentration phorus levels in Arabidopsis as compared with the non-colonized are higher in colonized roots.39 Recently it was demonstrated that plants.33 In the present study we also observed the increased the restriction of fungal growth by ethylene signaling components contents of phosphate in P. indica colonized C. forskohlii plants is required for the beneficial interaction between Arabidopsis and P. indica.41 The role of phytohormones in P. indica-induced reprogramming of C. forskohlii development needs to be further investigated. Like in mycorrhizal symbiosis42,43 increase in leaf area, higher photosynthetic potential and chlorophyll levels may result in increased carbon assimilation in P. indica-colonized plants, which is the basis for faster development and higher biomass production.28,44 The beneficial effect of P. indica was restricted to the aerial parts of the plant whereas the underground biomass decreased. The number of secondary roots and the root length increased which is consistent with the idea that auxin play a role in this symbiosis. Also plants colonized by AMF have lower root/ shoot ratios45,46 probably because the hyphae take over the nutrient absorbing functions of the roots.46 Interestingly, P. indica strongly induced early and vigorous Figure 7. Total phosphate content of the dried leaves of both flowering in C. forskohlii. Similar findings were also reported treated and non treated C. forskohlii field grown plants (growth period: previously and thus support our data.23,27,34,43,47-52 It was suggested six months). Data significant p , 0.05 that the increase in flower production may be caused by an



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Figure 8. Secondary metabolite contents in inflorescence of Coleus plants colonized by P. indica (T) and non-colonized plants (NT). (A) p-cymene, (B) Nonanal and (C) Forskolin. Estimation of p-cymene and nonanal contents of C. forskohlii was measured as described by Khare et al.77 and forskolin contents was measured as described by Mukherjee et al.78 The inflorescences of C. forskohlii were collected and the oil was obtained by hydrodistillation for 4 h using a Clevengertype apparatus. The material was dried over anhydrous sodium sulfate. Gas chromatography-mass Spectrometry (GC-MS) was performed to analyze the essential oil contents of the samples. GC-MS data were obtained on GC-MS (Thermo Scientific GC; GC Focus model; mass spectroscopy: Model DSQII). Compounds were identified by running five standard samples of essential oil (Sigma) and their mass fragmentation pattern of the peaks was compared with those present in the library MS data (NIST and WILEY). *significant at p , 0.05 according to Student’s t-test.

increase in plant nutrient (especially K+) uptake in combination with a possible hormonal effect.49 Hormones, such as gibberellins that induce the bud production could be transported in faster rates due to higher levels of K+ in the plant.49 Different studies have emphasized the importance of nutrient phosphorus on the impact on bud formation and development, the number of

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flowers, the size of the pollen grain and seed formation. It has been demonstrated in case of tomatoes that phosphorus promotes the formation of flowers and increases the mass of the fruit, seed count and pollen count of the plant as well as the average pollen production of each individual flower.53 Root colonization during the present study was found to be quite low compared with other host of P. indica.18,23,47 We suggest that low colonization rate may be plant-specific or dependent on growth or cocultivation conditions. Nevertheless, this does not appear to be crucial for the symbiosis, since the aerial plant biomass increases under these co-cultivation conditions. This is in agreement with previous findings that the extent of AMF colonization is not necessarily correlated with the effects of the symbionts on plant’s performance.14,54-56 It has been reported that C. forskohlii plants show a better performance in association with Glomus bagyarajii than with other AMF tested.14 Therefore, the interaction fungi also contribute to the benefits of the plant. It is well documented that mycorrhiza influence the level of secondary metabolites production of plants.43,57-63 The forskolin concentration in roots of C. forskohlii was enhanced by dual inoculation with G. mosseae and Trichoderma viride.13 However we found the reduced contents of forskolin in case of P. indica colonized plants as compared with the non-colonized plants. We speculate that this reduction of forskolin contents in P. indica colonized plants is due to the lower amount of root biomass compared with the noncolonized plant. However, we observed that the level of essential oils (p-cymene and nonanal) increased in the aerial parts of the colonized plants as compared with the noncolonized plants, because the fungus stimulates its biomass production. Similarly,20,21 also reported that P. indica inoculation significantly increased oil yield in Thymus vulgaris and Foeniculum vulgare as compared with non-colonized plants. Interestingly, we observed a strong effect on the inflorescence, raising the question


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whether they are synthesized as defense compounds59,64 or simply synthesized because of a better supply of the plant with nutrition.54,55,60-62 In case of AMF symbiosis, stimulation of secondary metabolites is specific for the AMF species.59,64 Thus, the role of P. indica and environmental factors for the production of secondary metabolites need to be analyzed in detail.65-67 Biomass of the aerial parts of C. forskohlii including the levels of the medicinally important secondary metabolites can be substantially stimulated by co-cultivation with P. indica under field conditions. We suggest the use of P. indica in sustainable agriculture for crop yield improvement. Methods Plant material and in vitro culturing. C. forskohlii plants were collected from herbal garden of Amity Centre for Herbal Development, Manesar, Haryana, India. For authentication and accession number (IC 582592) in vitro culture of C. forskohlii was prepared with single node explants of three months old C. forskohlii Briq. plants maintained under field conditions and then were submitted to National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Cuttings of one month old C. forskohlii plants were used for the field experimental studies. Cultivation of fungus. For broth batch culture of P.indica, the mycelium of 4–5 fully grown fungal agar discs (4 mm in diameter) was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of Hill and Kaefer broth medium.68 It was incubated at 28 ± 2°C with constant shaking at 120 rpm on a metabolic shaker (Infors). The fungal mycelium was obtained from liquid cultures after removal of the medium and washed with an excess of distilled water to remove the adhering salts. The prepared inoculums were stored at 4°C until use.69 Plant-fungus co-cultivation. Location. The study was performed under field conditions at Amity Centre for Herbal Development, Amity Education Valley, Manesar, Haryana, India. C. forskohlii was treated with the fungus P. indica in a polythene bag following the “sandwich layer model.”22,70 Substratum. The potting mixture was composed of unsterilized sand, soil and compost (1:1:0.25 w/w). Each polythene bag (25 ¾ 20 cm) was filled with 2.5 kg of the potting mixture. The soil used was sandy loam, pH 7.2 and an electrical conductivity of 0.11 dsm21. The substrate contained phosphorus (15.232 kg/ha), potash (228.48 kg/ha) and organic carbon (0.59%, w/w). The indigenous AMF spore concentration was 20/100 g of soil. Experimental Design. To study the interaction of C. forskohlii with P. indica polythene bags containing a “sandwich layer” of potting mixture with 2% of fungal biomass (w/w) was used. One month-old rooted cuttings of C. forskohlii (about 14 cm long) were obtained, and one cutting was planted into each bag. The experiments were replicated twice in the years 2008 and 2009. Each replicate had 18 plants per treatment. The plants were maintained under field conditions for six months and watered regularly with underground irrigation. The average daily temperature on the field for both years (from July to December 2008 and 2009) was around 31°C and the precipitation was around 655 mm.


Growth parameters. The growth parameters of the aerial plant parts (were-delete) evaluated were shoot length, number of branches, average length of the branches, number of leaves and leaf area, at six months of plants’ growth. The length parameters were measured (in cm) using a thread. The leaf areas (cm2) were calculated with the help of graph paper. Determined parameters for the underground parts of the plant were root number, length and thickness at the time point of six months plants’ growth of plants. Root thickness was measured using Vernier Callipers. Dry weight was determined separately for the shoots and roots at 100°C for 3 d until the weight of the materials were constant. Biochemical parameters. To determine the chlorophyll contents, six months old fresh leaves were harvested from P.indica colonized and non-colonized plants, washed, blot dried and homogenized in ice-cold 80% acetone (Qualigens) with chilled pestles and mortars. Homogenized tissue was centrifuged at 10,000 rpm (Beckman coulter, Avanti J26 XP, Roter J25.50 ) and the supernatant was used to measure the absorbance at 645 and 663 nm, respectively. Chlorophyll a, chlorophyll b and total chlorophyll (mg/g fresh weight) were calculated using the equations described by Arnon.71 Each sample was replicated six times. Inorganic phosphate was determined using acid extraction of dried plant material as described previously.72 For this purpose, leaves of colonized and non-colonized plants were weighed and to this, 40 ml of 5M H2SO4 was added per 20 mg of dried leaf. The samples were ground in a hand held device and then 3 ml of water was added to the mixture. The resulting solution was filtered using a filter (Whatman No. 4) and a subsample, ranging from 20 ml to 1.5 ml (depending on the phosphate concentration) was analyzed for phosphate. The subsample was made up to 1.5 ml with water and to this 0.5 ml of Malachite green reagent was added and total contents were mixed vigorously.73 After 30 min, the A650 of the solution was measured. Standards in the range of 125 nM to 50 mM of phosphate as KH2PO4 were used. P. indica root colonization assessment. The percentage of root colonization was estimated following the method described previously.74,75 Assessment of the colonization by P. indica was done as follows; 10 root samples were selected randomly from the six months old colonized plants. Samples were softened in 10% KOH solution for 15 min, acidified with 1 M HCl for 10 min, and finally stained with 0.02% Trypan blue overnight and were distained with 50% lacto-phenol for 1–2 h prior to observation under a light microscope (Leica type 020–518.500). The distribution of chlamydospores within the root was taken as an index of colonization.18 The percentage of colonization for the inoculated plants was calculated using the formula as described by McGonigle et al.76 Assessment of secondary metabolites. Assay of essential oil of C. forskohlii inflorescences. Estimation of essential oil content of C. forskohlii was performed as described by Khare et al.77 The inflorescences of C. forskohlii were collected, weighed and the oil was obtained by hydro distillation for 4 h using a Clevenger-type apparatus. The material was dried over anhydrous sodium sulfate. It gave 0.2% (v/w) of volatile oil. Gas chromatography-mass Spectrometry (Thermo Scientific GC; GC Focus model; mass spectroscopy: Model DSQII) was performed to analyze the essential


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oil contents of the samples. The injection volume was 1 ml. For GC, the injector temperature was 220°C, Helium was used as carrier gas, and the flow rate was 1.2 ml/min with a split ratio of 1:20; the MS transfer line temperature was 280°C. Oven temperature was programmed from 100–280°C at a rate of 3°C/min, with initial hold of 2 min. For MS analysis, the ion source temperature was 250°C with a mass range of 35–350 at full scan mode. The non polar column TR-5MS (0.25 mm x 0.25 mm x 30 mtr) was used.77 Compounds were identified by running five standard samples of essential oils a Pinene, Limonene, p-Cymene, Nonanal, Cineole (Sigma Aldrich) and comparing mass fragmentation pattern of the peaks with those present in the library MS data (NIST and WILEY) provided with the instrument. Forskolin assay. The forskolin content of the underground parts of the plants was determined by HPLC.78 For this purpose, samples were prepared from six months old P.indica colonized and non colonized plants. The mobile phase was prepared by dissolving 1.2 g octane sulphonic acid sodium salt in 555 mL 1% phosphoric acid. After mixing, 445 ml of acetonitrile was added, mixed again and filtered though a 0.45 mm nylon membrane. The standard solution was prepared with forskolin (Sigma) in methanol. The biological slurry was sonicated and volume was made up (with methanol). A sample solution corresponding to 3 g of fresh weight was transferred to a soxhlet apparatus. Sufficient References 1.

Chandel KPS, Sharma N. Micropropagation of Coleus forskohlii (Willd.) Briq. In: Bajaj YPS, ed. Biotechnology in Agriculture and Forestry. Hi Tech and Micropropagation. Berlin: Springer, 1997: 74-84. 2. Kurian A, Sankar A. Important medicinal plants in trade. In: Peter KV, ed. Medicinal Plants. Vol 2. New Delhi: New India Publishing Agency, 2007: 294-295. 3. Ahmed B, Viswakarma RA. Coleoside, a monoterpene glycoside from Coleus forskohlii. Phytochemistry 1988; 27:3309-3310; http://dx.doi.org/10.1016/0031-9422 (88)80050-5 4. Ahmed B, Merotra R. Coleoside-B: a new phenolic glycoside from Coleus forskohlii. Pharmazie 1991; 46: 157-8. 5. Petersen M. Coleus spp: In vitro culture and the production of forskolin and Rosmarinic acid. In: Bajaj YPS, ed. Biotechnology in Agriculture and Forestry, Medicinal and Aromatic plants VI. Berlin: Springer, 1994: 69-85. 6. Ammon HPT, Muller AB. Forskolin: from an ayurvedic remedy to a modern agent. Planta Med 1985; 46:473-7; http://dx.doi.org/10.1055/s-2007-969566 7. De Souza NJ, Shah V. Forskolin – an adenylate cyclase activating drug from an Indian herb. Econ Med Plant Res 1988; 2:1-16. 8. Kavitha C, Rajamani K, Vadivel E. Coleus forskohlii: A comprehensive review on morphology, phytochemistry and pharmological aspects. J of Med Plants Res 2010; 4:278-85. 9. Patil S, Hulamani NC, Rokhade AK. Performance of genotype of Coleus forskohlii Briq. for growth, yield and essential oil content. Indian Perfumer 2001; 45: 17-21. 10. Misra LN, Tyagi BR, Ahmad A, Bahl JR. Variablity in the chemical composition of the essential oil of Coleus forskohlii genotypes. J Essent Oil Res 1994; 6:243-7. 11. Chowdhary AR, Sharma ML. GC-MS investigations on the essential oil from Coleus forskohlii Briq. Indian Perfumer 1998; 42:15-6.

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amount of methylene chloride was added and the material was extracted at , 50°C. Methylene chloride was dried under vacuum, the material dissolved in methanol and the volume was adjusted to 100 ml with methanol. HPLC (Shimadzu LC 2010) analysis were performed on an Octadecyl Silane 5 mm (4.6 mm x 2510 mm) Nucleosil column with a flow rate of 1.2 ml per min. Forskolin was detected at 205 nm and its retention time compared with that of standard (Sigma). Statistical analysis. Means and standard deviations were calculated for each independent experiment. Statistical analysis was performed using the student’s t test using Sigma Stat32 program Differences were considered to be significant at p , 0.05. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

AV is thankful to Department of Biotechnology (DBT) and Defense Research and Development Organization (DRDO), Govt. of India for partial financial assistance. AKJ is thankful to the Department of Science and Technology (DST), Govt. of India for financial assistance. A capacity-build grant to AKJ from JNU is greatly appreciated. Authors are thankful to Anil Chandra for carrying out the statistical analysis.

12. Vishwakarma RA, Tyagi BR, Ahmed B, Hussain A. Variation in forskolin content in the roots of Coleus forskohlii. Planta Med 1988; 54:471-2; PMID: 17265327; http://dx.doi.org/10.1055/s-2006-962512 13. Boby VU, Bagyaraj DJ. Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants. World J Microbiol Biotechnol 2003; 19:175-80; http://dx.doi.org/10.1023/A:1023238908028 14. Sailo GL, Bagyaraj DJ. Influence of different AMfungi on the growth, nutrition and forskolin content of Coleus forskohlii. Mycol Res 2005; 109:795-8; PMID:16121565; http://dx.doi.org/10. 1017/S0953756205002832 15. Oelmüller R, Sherameti I, Tripathi S, Varma A. Piriformospora indica, a cultivable root endophyte with multiple biotechnological applications. Symbiosis 2009; 49:1-17; http://dx.doi.org/10.1007/s13199-009-0009-y 16. Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, et al. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. PNAS USA 2005; 102:13386-91; PMID:16174735; http://dx.doi.org/ 10.1073/pnas.0504423102 17. Deshmukh S, Hückelhoven R, Schäfer P, Imani J, Sharma M, Weiss M, et al. The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley. PNAS USA 2006; 103:18450-7; PMID:17116870; http://dx.doi.org/10.1073/pnas.0605697103 18. Kumar M, Yadav V, Tuteja N, Johri AK. Antioxidant enzyme activities in plants colonized with Piriformospora indica. Microbiology 2009; 155:780-90; PMID: 19246749; http://dx.doi.org/10.1099/mic.0.019869-0 19. Yadav V, Kumar M, Deep DK, Kumar H, Sharma R, Tripathi T, et al. A Phosphate transporter from the root endophytic fungus Piriformospora indica plays a role in the phosphate transport to the host plant. J Biol Chem 2010; 285:26532-44; PMID:20479005; http://dx.doi. org/10.1074/jbc.M110.111021


20. Dolatabadi HK, Goltapeh EM, Jaimand K, Rohani N, Varma A. Effects of Piriformospora indica and Sebacina vermifera on growth and yield of essential oil in fennel (Foeniculum vulgare) under greenhouse conditions. J Basic Microbiol 2011; a51:33-9; PMID:21259287; http://dx.doi.org/10.1002/jobm.201000214 21. Dolatabadi HK, et al. Effect of Piriformospora indica and Sebacina vermifera on plant growth and essential oil yield in Thymus vulgaris in vitro and in vivo experiments. Symbiosis 2011. b53:29-35; http://dx.doi.org/ 10.1007/s13199-010-0104-0 22. Varma A, Verma S. Sudha, Sahay NS, Franken P. Piriformospora indica, a cultivable plant growth promoting root endophyte. Appl Environ Microbiol 1999; 65:2741-4; PMID:10347070 23. Rai M, Acharya D, Singh A, Varma A. Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 2001; 11:123-8; http://dx.doi.org/10.1007/s005720100115 24. Rai MK, Varma A, Pandey AK. Antifungal potential of Spilanthes calva after inoculation of Piriformospora indica. Mycoses 2004; 47:479-81; PMID:15601453; http://dx.doi.org/10.1111/j.1439-0507.2004.01045.x 25. Peškan-Berghofer T, Shahollari B, Giong P, Hehl S, Market C, Blanke V, et al. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmatic reticulum and at the plasma membrane. Physiol Plant 2004; 122:465-77; http://dx. doi.org/10.1111/j.1399-3054.2004.00424.x 26. Rai MK, Varma A. Arbuscular mycorrhizae—like biotechnological potential of Piriformospora indica, which promotes the growth of Adhatoda vasica. Electron J Biotechnol ISSN 2005; 8:107-12.

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27. Fakhro A, Andrade-Linares DR, von Bargen S, Bandte M, Büttner C, Grosch R, et al. Impact of Piriformospora indica on tomato growth and on interaction with fungal and viral pathogens. Mycorrhiza 2010; 20:191-200; PMID:19789897; http://dx.doi.org/10.1007/s00572009-0279-5 28. Achatz B, von Rüden S, Andrade D, Neumann E, Pons-Kühnemann J, Franken P, et al. Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating early plant development. Plant Soil 2010; 333:59-70; http://dx.doi.org/10.1007/s11104-010-0319-0 29. Waller F, Mukherjee K, Deshmukh SD, Achatz B, Sharma M, Schäfer P, et al. Systemic and local modulation of plant responses by Piriformospora indica and related Sebacinales species. J Plant Physiol 2008; 165:60-70; PMID:18031866; http://dx.doi.org/10. 1016/j.jplph.2007.05.017 30. Varma A. Piriformospora indica: An axenically culturable mycorrhiza-like endosymbiotic fungus. In: Hock B, ed. The Mycota IX, Fungal Associations. Berlin: SpringerVerlag, 2001: 123-150. 31. Sherameti I, Shahollari B, Venus Y, Altschmied L, Varma A, Oelmüller R. The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan–water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor which binds to a conserved motif in their promoters. J Biol Chem 2005; 280:26241-7; PMID:15710607; http:// dx.doi.org/10.1074/jbc.M500447200 32. Kumar M, Yadav V, Singh A, Tuteja N, Johri AK. Piriformospora indica enhances plant growth by transferring phosphate. Plant Signal Behav 2011; 6:723-5; PMID:21502815; http://dx.doi.org/10.4161/psb.6.5. 15106 33. Shahollari B, Varma A, Oelmüller R. Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains. J Plant Physiol 2005; 162: 945-58; PMID:16146321; http://dx.doi.org/10.1016/ j.jplph.2004.08.012 34. Barazani O, Benderoth M, Groten K, Kuhlemier C, Baldwin IT. Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuate. Oecologia 2005; 146:234-43; PMID:16032437; http://dx.doi.org/10.1007/s00442-005-0193-2 35. Schäfer P, Pfith S, Voll LM, Zajic D, Chandler PM, Waller F, et al. Phytohormones in plant rootPiriformospora indica mutualism. Plant Signal Behav 2009; 4:669-71; PMID:19820343; http://dx.doi.org/ 10.4161/psb.4.7.9038 36. Schäfer P, Pfiffi S, Voll LM, Zajic D, Chandler PM, Waller F, et al. Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica. Plant J 2009; 59:461-74; PMID:19392709; http://dx.doi.org/10.1111/j.1365-313X.2009.03887.x 37. Lee YC, Johnson JM, Chien CT, Sun C, Cai D, Lou B, et al. Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin. Mol. Plant-Microb Interact 2011; 24:421-31; http://dx.doi.org/10.1094/ MPMI-05-10-0110 38. Sirrenberg A, Göbel C, Grond S, Czempinski N, Ratzinger A, Karlovsky P, et al. Piriformospora indica affects plant growth by auxin production. Physiol Plant 2007; 131:581-9; PMID:18251849; http://dx.doi.org/ 10.1111/j.1399-3054.2007.00983.x 39. Vadassery J, Ritter C, Venus Y, Camehl I, Varma A, Shahollari B, et al. The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica. Mol Plant Microbe Interact 2008; 21:1371-83; PMID:18785832; http://dx.doi. org/10.1094/MPMI-21-10-1371


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