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Organic Production of Medicinal, Aromatic and Dye-yielding Plants (MADPs). With inputs from FRLHT.
by EcoPort version by Peter Griffee, FAO.

Contributor:Peter Griffee QA and TEM

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ID: 145

19. Utilization of VA Mycorrhizal Fungi and Trichoderma viride on Plant Growth and Drug Content of Micropropagated Bacopa monnieri (L.) Pennell bySowmya R. etal. Abstract: Bacopa monnieri (L.) Pennell, one of the most important medicinal plants, belongs to the family Scrophulariaceae. Normal and micro-propagated plants were inoculated with the VAMs Glomus mosseae and Glomus fasciculatum with or without the phosphate solubilizng microorganism (PSM) Trichoderma viride. Growth and physiological status of normal and micro-propagated plants with their controls were analysed and compared. Micro-propagated plants in association with G mosseae and T. viride were found to be far superior to non mycorrhizal micro-propagated and normal plants in plant growth, carbohydrates, proteins, phenolics and bacoside content. Introduction: The significance of VAM association in enhancing the growth and nutritional status has been well established in several food and horticultural crops (Khan, 1972, Mohandas, 1995, Declerck et al., 1995 and Shashikala et al., 1999). In the last few decades, endomycorrhization during the weaning stages of micropropagated plants has been an area of intense research. (Berta et al., 1990, Wang et al., 1993 and Subhan et al., 1998). Although there are several advantages of micro-propagation that include

higher rates of multiplication, production of disease free planting material and the small amount of space required to multiply large numbers of plants, the major impediments to its success is the high mortality rate of tissue cultured plantlets, either during acclimatization or during transfer to the field due to various factors (Pierik, 1988). Inoculation of VAM fungi during an early stage of acclimatization process has become an alternative strategy for better establishment by improving the plant growth (Gianinazzi et al., 1989). However, little is known about how VAM fungi improve the acclimatization and growth of micro-propagated plantlets (Granger et al., 1983 and Ponton et al., 1990). Though there are reports on beneficial effects of VAM on other crops, a literature search reveals only a few reports on the VAM association with medicinal plants (Sivaprasad et al., 1995). This work is an attempt to study the effect of VAM fungi with and without T. viride on the nutritional status and drug content of micropropagated plants of B. monnieri. Materials and methods: Micro-propagated plants raised in the tissue culture laboratory were maintained in the departmental garden with normal plants. The two species of VAM fungi G. mosseae and G. fasciculatum and T. viride were obtained from the Department of Biotechnology, The Indian Institute of Horticultural Research, Hessarghatta, Bangalore. The fungal inoculum was maintained in pot culture with sterilized sand and soil as a substrate and Rhodes grass as the host for the mycorrhizal fungi. The inoculum containing 100-150 spores/g was added at the rate of 25g/pot in the pot culture. Whereas T. viride, a PSM fungus, was maintained on potato dextrose (PD) agar medium at 4 degrees C. One week old cultures were grown in 100 ml of PD broth in 250 ml flask for 7 days on a rotary shaker. Then it was decanted and suspended in 20 ml of distilled water and brought into uniform suspension by using a blender. Pots were inoculated at the rate of 5 ml/pot. The different treatments used were: I. Control - Normal plants: b) Normal plants inoculated with G. fasciculatum c) Normal plants inoculated with G. mosseae d) Normal plants inoculated with T. viride e) Normal plants inoculated with G. fasciculatum+ T. viride f) Normal plants inoculated with G. mosseae+ T. viride. II. Control - Micro-propagated plants:

b) Micro-propagated plants inoculated with G. fasciculatum c) Micro-propagated plants inoculated with Glomus mosseae d) Micro-propagated plants inoculated with T. viride e) Micro-propagated plants inoculated with G. fasciculatum+ T. viride f) Micro-propagated plants inoculated with G. mosseae+ T. viride. The percent infection of both normal and micro-propagated plants of each treatment and control were estimated according to Phillips and Hayman, 1970. Mycorrhizal spore count was estimated by the wet sieving and decanting method outlined by Gerdman and Nicolson, 1963. The micro-propagated plants inoculated with Glomus mosseae and G. fasciculatum with or without T. viride were compared with treated normal plants and the control using the following parameters: Morphological studies: The surface area of the leaf of 10th node of the control and inoculated normal and micro-propagated plants was measured with the help of an area calculating device. The lengths of the internode from the 1st-10th node were recorded for all treatments. The average of 20 different primary branches was taken for the record of fresh and dry weight. These readings were recorded 6 months after inoculation. Phytochemical studies: (i) The following estimations were carried out 2, 4 and 6 months after inoculation. Chlorophyll pigments were estimated following the Arnon, 1949 method. (ii) Quantitative estimation of total carbohydrates and reducing sugars from leaves, stems and roots of each treatment were carried out according to Mahadevan and Sridhar, 1986. (iii) Total proteins of leaves, stems and roots of each treatment were estimated following the Lowry et al., 1951 method. (iv) The total phenolics of leaves, stems and roots were determined using the standard curve of caffeic acid. (v) Bacoside - The content of whole plants was determined by HPTLC using a calibration curve. The accuracy parameters of this method are: Linear calibration: Y = 952.82 Y + 53.17 (r = 0.99); recovery 98% as

calculated by standard addition experiments; CV = 0.46 N = 3. Results and Discussion: (i) Percent colonization and spore count: The percentage of colonization was recorded at regular intervals of 2.4 and 6 months after inoculation (See Fig. 1). Plants colonized with Glomus mosseae and with T. virideshowed the highest colonization (85%) which was reflected in the higher spore count of about 264 in the rhizosphere soil. The lowest colonization percent and spore count were recorded in normal plants inoculated with T. viride. Similar pot culture studies on Catharanthus roseus showed a positive response to Glomus mosseae and Glomus aggregatum inoculation in respect of percent colonization and spore count. (ii) Effect of treatments on morphological characters: It is well documented that mycorrhizal fungi helps better plant growth (Bhagyaraj and Verma, 1995) Even in micro-propagated plants, the beneficial effects of VAM fungi on growth is well established. Normal and micro-propagated plants showed a better growth response to VAM fungal inoculation with T. viride recorded in terms of surface area of leaves, shoot and root length and fresh and dry weight. Based on Tejavathi and Nagashree, 1998, comparative studies on normal and micro-propagated plants, it was concluded that micro-propagated plants are better in growth response than normal plants. VAM inoculation with PSM in the micro-propagated plants further improved the plant growth. The mean leaf surface area was higher in dual inoculated micro-propagated plants than the control plants and differences increased with time and during acclimatization. This agrees with the observations made by Martins et al., 1997 in micro-propagated {{Castanea striata}e} inoculated with Pisolithus tinctorius. In the present study, there is a positive correlation between percent mycorrhizal colonization and plant growth. The higher shoot elongation found with dual inoculation of VAM and PSM inoculated micro-propagated plants indicates a change in the hormonal balance induced by mycorrhizal symbiosis. (Allen et al., 1980). The potential role of hormones in the mycorrhizal plant is evident at the induction of better plant growth in terms of shoot and root length and surface area of leaves of micro-propagated plants. Leaves and roots of Bouteloua gracilis were reported to have higher concentrations of cytokinins when colonized by Glomus fasciculatum than grown in the absence of VAM fungi

(Miller, 1971 and Allen et al., 1980). It was also suggested that mycorrhizal fungi could affect the plant growth by producing auxins. (Ulrich, 1960). Enhancement in the rate of growth of dual inoculated micro-propagated plants correlates with the increase in fresh and dry weights, which indicates higher photosynthetic rates in inoculated plants than in control plants. This is in agreement with previous reports that the presence of mycorrhizae on plant root systems is correlated with higher net photosynthetic rates (Reid et al., 1983 and Nylund and Wallander, 1989). (iii) Effect of treatments on the biosynthesis of primary metabolites: Primary metabolites are considered the building blocks of whole plants. They are precursors for the biosynthesis of secondary metabolites. Increased chlorophyll content in micro-propagated plants inoculated with Glomus mosseae and T. viride is directly correlated with increased surface area of the leaves and photosynthetic rate in terms of fresh and dry weight (Allen et al., 1980, Reid et al., 1983, Pahwar and Thakur, 1995, Tejavathi and Nagashree, 1998) (See Fig. 2). Similar conclusions were documented in Prosopis cineraria, where increased chlorophyll contents was noticed by Mathur and Vyas, 1995 when treated with mycorrhizal fungi. A typical feature of vesicular-arbuscular mycorrhizal synthesis is that the fungus depends upon the host for the supply of photosynthates (Hayman, 1978). Owing to heteromorphic nature of the VA endophyte, the carbohydrate status of the plants ought to change when roots sustain the fungus growth. Mycorrhizal plants differ from uninfected plants in significantly higher C transformation from shoot to root system (Losel et al., 1979 and Snellgrove et al., 1982). But the carbon drain in the mycorrhizal plants may be compensated with increased C assimilation (Snellgrove et al., 1982). The control micro-propagated plants show more total carbohydrate and reduced sugar content than the normal plants. This is because of an increased photosynthetic rate measured in terms of leaf area, chlorophyll content and better physiological status of micro-propagated plants (Tejavathi and Nagashree, 1998) However, in normal mycorrhizal plants the contents are decreased in roots, stems and leaves. This can be related to the demand for carbohydrates by the fungus for its sustained growth. Further, there is an increase in the total carbohydrate and reduced sugar content in the leaves of the mycorrhizal micro-propagated plants (See Fig. 6-6a, Fig. 7-7a, Fig. 3 and Fig. 4) The drain of carbon compounds to the fungus stops the accumulation of carbohydrates in the leaves which in turn enhances the carbon assimilation through a higher rate of photosynthesis. (Martins et al., 1997).

In contrast, Ocampo and Azcon, 1985) observed an increase in total and reducing sugars in the roots of mycorrhizal wheat plants. Based on their studies, Harley and Smith, 1983, concluded that carbohydrate levels in roots depend on the balance between C demand of the fungus and C status of the host. Finlay, 1992 feels that carbon compounds released by the host are not exclusively used by fungus. The view has further strengthened the suggestion of France and Reid, 1983 who believe that incorporation of ammonium into the carbon skeleton, derived from fungal trehalose and mannitol, results in reverse translocation to the host plant as amino acids. Our observations on the total protein content in inoculated normal and micro-propagated plants with the controls confirms the results of Martins et al., 1997 in chestnut plants and Mathur and Vyas, 1996 in P. cineraria where there is an increase in protein content in the mycorrhizal plants (See Fig. 5). (iv) Effect of treatments on biosynthesis of secondary metabolites: Secondary metabolites play an important role in various interactions between plants and their environment including the symbiotic relationship between plant roots and arbuscular mycorrhizal fungi (Morandi, 1996). The total phenolics showed significantly increased levels in all the parts of the micro-propagated plants inoculated with VAM, either alone or in combination with T. viride. (See Fig. 6). The increased resistance of mycorrhizal plants to various pathogens may be associated with metabolic changes, including enhanced production of phenolic compounds. (Dehne, 1982). Bacopa monnieri comprises mixtures of triterpenoid saponins, bacosides A, B, C and D, that are considered the main compounds of medicinal value. Bacoside A is the main active principle as a memory vitaliser. HPTLC studies conducted in mycorrhizal with or without T. viride treated normal and micropropagated pants with respect to their uninoculated control plants, showed that the bacoside A content in mycorrhizal micro-propagated plants inoculated with T. virideis almost double that in normal plants. (See Fig. 7). Increase in the biosynthesis of secondary metabolites in VA mycorrhizal inoculated plants was also reported by Nemac and Luand, 1990 in Citrus jambhiri, Ionkowa, 1995) in {{Astralogus sp.}e} and Maier et al., 1995 in species of Poaceae. In the present investigation micro-propagated mycorrhizal plants contained 3.96% bacoside A compared to control plants that had only 2.10%. There is increasing evidence that colonization of plants with arbuscular mycorrhizal fungi play a significant role in the biosynthetic pathways of terpenoid metabolism (Dannerberg et al., 1993). The aforesaid data clearly indicates that mycorrhizal fungi can benefit Bacopa in an eco-friendly way without harming its metabolic activity. The increased

biomass and drug content in the mycorrhizal micro-propagated plants with the PSM T. viride is an encouraging aspect that can be exploited by the growers in organic farming by mass propagation of micro-propagated Bacopa in association with VA mycorrhizal fungi. Since the drug 'brahmin' is extracted from whole plants, mycorrhizal micro-propagated plants are a better source than normal plants. At this juncture, pharmaceutics can play a significant role by utilizing the mycorrhizal micro-propagated plants for drug extraction, thereby conserving the natural resources of this taxon.

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