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Organic Production of Medicinal, Aromatic and

Dye-yielding Plants (MADPs). With inputs from
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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 micro-
propagated 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 micro-
propagated 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 micro-
propagated 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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