Influence of arbuscular mycorrhizal fungi on andrographolide concentration in Andrographis paniculata.
Abstract: Andrographis paniculata Nees (Acanthaceae), an annual erect herb, is known for its broad range of pharmacological properties. This study reports the effect of different arbuscular mycorrhizal (AM) fungal inocula on plant growth and percent concentration of andrographolide. The study revealed a significant increase in growth in all the treatments and unsterilised soil (control) compared with sterilised soil (control). Plants grown in unsterilised soil (control) and inoculated with Gigaspora albida enhanced leaf number, shoot and total plant dry weights significantly compared with other treatments and sterilised control. Andrographis paniculata revealed high mycorrhizal efficiency when grown in unsterilised control and Gi. albida. Increased concentration of andrographolide with Gi. albida inoculum confirms host preference in AM symbiosis and also identifies it as an efficient AM fungal inocula for commercial cultivation of A. paniculata.

Keywords: A. paniculata, andrographolide, AM symbiosis, host preference, AM fungal inocula
Article Type: Report
Subject: Mycorrhizas (Health aspects)
Authors: Radhika, K.P.
Rodrigues, B.F.
Pub Date: 03/22/2011
Publication: Name: Australian Journal of Medical Herbalism Publisher: National Herbalists Association of Australia Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 National Herbalists Association of Australia ISSN: 1033-8330
Issue: Date: Spring, 2011 Source Volume: 23 Source Issue: 1
Geographic: Geographic Scope: Australia Geographic Code: 8AUST Australia
Accession Number: 254971771
Full Text: Introduction

Andrographis paniculata Nees (Acanthaceae) commonly known as 'king of bitters' has been used for centuries in Asia to treat gastrointestinal tract, upper respiratory infections, fever, herpes, sore throat and a variety of other chronic and infectious diseases. It is found in the Indian Pharmacopeia and is a constituent in at least 26 Ayurvedic formulae. It is an annual herb, branched, erect, running 0.5-1 metre in height.

Active phytochemicals are extracted from the aerial parts of the plant (leaves and stems). It has a surprisingly broad range of pharmacological effects which includes abortifacient, analgesic, antibacterial, antipyretic, antithrombotic, antiviral, cancerolytic, cardioprotective, choleretic, depurative, digestive, expectorant, hepatoprotective, hypoglycemic, immune enhancement, laxative, sedative, thrombolytic and vermicidal (Jean Barilla 1999).

The primary medicinal component of A. paniculata is andrographolide, a diterpene lactone which is bitter in taste and colourless in appearance. The leaves contain the highest amount of andrographolide (2.39%), while the seeds contain the least amount (Sharma 1992). Medicinal properties include anti-inflammatory, hepatoprotective activities against galactosamine, paracetamol intoxication, cancer therapy and anti HIV activity (Puri 1993).

The mycorrhizal symbiosis represents a series of complex feedbacks between host and fungus that is governed by their physiology and nutrition. The outcome of a mycorrhizal relationship depends on the balance between fungal demands for energy (in terms of carbon based compounds) and the plant need for nutrients (Miller 2002). Despite the widespread distribution and ecological significance of AM symbiosis, only some characterisation of its effects on secondary metabolites has been achieved. Relatively little is known about the effects of AM colonisation on the accumulation of active phytochemicals in shoots of medicinal plants which are often the harvest products and used for human consumption.

Secondary metabolite accumulation in plants in the course of plant symbiotic fungal interaction definitely impels the development of attractive strategies to bring medicinal plants cultivation into a new era for pharmaceutical purposes.

This paper presents the results of a study on the effect of different AM fungal inocula on growth of the plant and percent concentration of andrographolide in A. paniculata.

Materials and methods

Plant material

Uniform size seeds of A. paniculata were germinated on moist filter papers in petri plates in a growth incubator at 26[degrees]C (16 hr photoperiod and relative humidity (RH) 62%). Germination percentage was determined, the seeds considered germinated only after the emergence of 2 mm of radical.

Arbuscular mycorrhizal inoculum

Monospecific cultures of AM fungi prepared from spores isolated from trap cultures were selected for the study. Soil based spore inocula (80-120 spores 100 [g-.sup.1] of soil) of Acaulospora scrobiculata, Gigaspora albida, Glomus fasciculatum, Scutellospora biornata and Scutellospora calospora were used. Unsterilized control consisted of mixed AM fungal spores viz, Acaulospora scrobiculata, A. laevis, Glomus fasciculatum, G. maculosum, G. magnicaule, G. diaphnum, G. multicaule, G. geosporum and G. intraradices. Sand soil (1:3) mix (control) used in the experiment was sterilized for 1 hr at 15 lbs pressure for three consecutive days to eliminate naturally occurring endophytes and other contaminants.

Growing conditions

The experiment was conducted for a period of five months (November 2007- March 2008) in polyhouse at 270C and RH 65%. Throughout the experiment the pots were watered on alternate days and Hoagland solution without P (Hoagland 1938) was added at intervals of 15 days.

Inoculation with AM fungal cultures

Uniform seedlings of germinated A. paniculata were planted in seven trays (22.5 cm x 17.5 cm) with five monospecific cultures of AM fungi, unsterilized (control) and sterilized (control).

Experimental design

A randomised block design experiment was conducted comprised of seven treatments. Each treatment consisted of four replicates. The seven treatments consisted of two controls and five pure monospecific cultures of AM fungi. Control (C1) consisted of uninoculated sterilized soil and control (C2) unsterilized soil. The five AM treatments included Scutellospora calospora (M1), Acaulospora scrobiculata (M2), Glomus fasciculatum (M3), Gigaspora albida (M4) and Scutellospora biornata (M5). After 45 days, seedlings were transferred to pots (15 cm diam.) filled with unsterilized sand soil (3:1) mix. The experiment was terminated after 150 days of growth and A. paniculata plants were harvested and subjected to analysis.

Plant growth measurements

Shoot and total plant dry weights and leaf number were recorded. There were three replicates for each treatment.

Mycorrhizal efficiency index (MEI)

Using plant dry weight, mycorrhizal efficacy in enhancing the growth was calculated by taking the average dry weight of the plant. The MEI was estimated according to Bagyaraj (1994) as the weight of inoculated plant less the weight of uninoculated plant multiplied by 100 divided by the weight of inoculated plant

HPLC analysis for secondary metabolite concentration

Andrographolide concentration from the leaf extracts of A. paniculata were carried out using HPLC analysis (Pholphana 2004). Andrographolide content per plant was obtained by multiplying dry weight of the shoot by its andrographolide concentration.

Statistical analysis

Statistical analyses were carried out by Analysis of Variance (ANOVA) and correlation coefficient using web based Agricultural Statistical Package 2.0 (WASP).

Results

Root colonisation by arbuscular mycorrhizal fungi

Mycorrhizal colonisation was observed in all the roots of plants grown in inoculated and unsterilized soil while no colonisation was observed in uninoculated sterilized soil. Arbuscular and hyphal colonisation was observed in all the treatments whereas hyphal, arbuscular and vesicular colonisation was observed in unsterilized control.

Growth parameters

A significant increase in growth of A. paniculata was observed in all the treatments and in unsterilized soil compared with sterilized soil. At the time of harvesting S. calospora, S. biornata and Glomus fasciculatum inoculated and sterilized control plants were in the vegetative stage, Gi. albida inoculated plants in flowering stage while A. scrobiculata and unsterilized control plants were in the flowering and fruiting stage. Thus early flowering and fruiting was recorded in unsterilized control and A. scrobiculata inoculated plants.

At the time of harvesting (150 days after transplantation), A. paniculata plants inoculated with all AM treatments and unsterilized control showed increased growth compared with sterilized control. However growth responses varied among AM treatments. Plants grown in unsterilized control and those inoculated with Gi. albida and A. scrobiculata separately recorded a statistically significant increase in leaf, shoot and total plant dry weights compared with other treatments and sterilized control (F=0.028, CD=0.439; F=0.023; CD= 0.456; P<0.05) (Table 1). Observations revealed a significant increase in shoot and total plant dry weights of plants grown in unsterilized soil and Gi. albida compared with sterilized soil whereas other AM fungal treatments did not differ significantly from the sterilized soil (Table 1). Maximum leaf number was recorded in plants inoculated with Gi. albida and minimum was recorded in sterilized control. The treatments were significantly different from the control (F=0.000, CD= 5.309, P <0.05 (Table 1).

Mycorrhizal efficiency index

Mycorrhizal efficiency study revealed that unsterilized control had high efficiency (80.00%) in enhancing the growth of A. paniculata followed by Gi. albida (66.10%) while S. calospora had least efficiency (4.76%). Mycorrhizal efficiency varied from 23.07% to 44.44% in other treatments (Fig. 1). Significant weak positive correlation was observed between shoot and total plant dry weights and mycorrhizal efficiency index (r=0.3; r=0.49, P<0.05).

Andrographolide concentration

Concentration of andrographolide in leaf extracts varied among the treatments and the controls. Sharp symmetrical peaks were recorded in all the treatments and the controls. The standard reference andrographolide showed a percent recovery of 92% at a retention time of 15.2 (Fig. 2). The percent recovery of these compounds ranged from 1.98% to 58.53% at a retention time of 15.0 to 15.4 in all the treatments and control. The maximum percent recovery of andrographolide was observed in plants inoculated with Gi. albida (58.3%) whereas least was observed in plants grown in sterilized soil (1.98%) (Table 2 Fig 3-9). Maximum andrographolide content per plant was observed in Gi. albida (31.49) and minimum in sterilized soil (0.19) (Table 3).

Discussion

The present study confirms the benefit of mycorrhizae as biofertilizers on growth, yield, nutrient uptake and increase in andrographolide concentration in A. paniculata. Except for sterilized control, mycorrhizal colonisation was observed in all the AM fungal treatments and unsterilized control. Similar observations were reported earlier (Chiramel 2006). Arum type of arbuscules were observed in roots of all the treatments. Arbuscule represents a dead end in the growth of AM fungi (Bonfante 1995) as they senesce and collapse after 4-10 days of symbiosis (Sanders 1977) and thus the plant cell recovers its original morphology (Jacquelinet-Jeanmougin 1987). In this way cortical cell is able to allow a second fungal penetration and arbuscule formation. The short life span of arbuscules is perhaps because they are digested by the host cell presumably when no longer needed for transfer (Toth 1984).

Mycorrhizal and non-mycorrhizal plants differed significantly in shoot and total plant dry weights. A significant increase in shoot and total plant dry weights was observed in unsterilized control indicating that native AM fungi were more efficient in stimulating plant growth than the inoculated strains. Similar observations have been reported earlier (Bagyraj 1980). Gigaspora albida showed a significant increase in plant and shoot dry weights compared with other treatments but there was no significant difference between plants grown in other AM fungal treatments and sterilized control.

The study also confirms the earlier findings that plant growth varies with plant/fungus interaction as certain combinations of host and fungus are more or less compatible than others (Parke 2000). Growth and mineral nutrition of plants are known to be enhanced by inoculation with AM fungi (Clark 2000). The reasons for species specific responses to AM fungal colonisation and plant benefit are probably mediated by a complex combination of plant and fungal signals based on genetically controlled substances (Gianinazzi 1991).

Mycorrhizal efficiency for A. paniculata varied among the treatments and between the two controls. Plants grown in unsterilized soil recorded high mycorrhizal efficiency compared with all other treatments. The efficiency of Gi. albida in enhancing the growth of A. paniculata was found maximal in comparison with other treatments.

In the present study, the high percent concentration of andrographolide observed in the leaf extracts of A. paniculata inoculated with Gi. albida confirms that AM symbiosis enhances the production of secondary metabolites in medicinal plants. Minimum recovery of secondary metabolite concentration was observed in plants grown in sterilized soil. The highest concentration of andrographolide was observed during the flowering stage of the plant.

The synthesis of secondary metabolites is also dependent on plant age and developmental stage (Maffei 1989). Andrographolide content per plant was maximum in plants inoculated with Gi. albida which confirms host preference by AM fungi. Host preference has been reported in many forest tree species (Rajan 2000) but in few medicinal plant species (Gracy 2005). In the present study the concentration of andrographolide varied with treatments which suggest that AM fungal colonisation is responsible for an increase in secondary metabolites of the plant which confirm earlier reports (Copetta 1996).

Gigaspora albida was found the most efficient arbuscular mycorrhizal fungal inoculum for A. paniculata cultivation. AM symbiosis induces changes in the accumulation of secondary compounds, some of them acting as signal molecules (Akiyama 2002). Enhancement of secondary product accumulation in medicinal plant is of great importance in the medicinal plant cultivation industry.

Acknowledgement

The authors thank Dr CG Naik, Scientist, National Institute of Oceanography (NIO) for providing facilities for carrying out HPLC analysis.

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KP Radhika, BF Rodrigues

Department of Botany, Goa University, Goa 403206 India

Corresponding author email: felinov@gmail.com
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