The Effects of Cyanobacterial (Blue Green Algae) Culture Filtrates on the Biomass and Biochemicals of Withania somnifera Dunal
The present investigation was aimed at studying the
effect of culture filtrates of cyanobacteria (blue green algae) namely
Anabaena ambigua Rao and Oscillatoria foreaui Fremi in different
soil conditions on the growth of Withania somnifera Dunal in terms
of biomass and biochemical characteristics along with active ingredients
of the roots. Experiments conducted in field by replenishing the plant
rhizosphere as foliar spray with increasing quantities of culture filtrates
enhanced the growth of the plant more precisely in sandy loam soil when
compared to red soil. The yield potentials of the roots also increased
with the culture filtrate of O. foreaui along with the other parameters
studied, attributing to the availability of the nutrients from the cyanobacterial
One of the important milestones of biological research in the last millennium
is the understanding that soil microorganisms play a vital role in agriculture.
The subsequent development of biofertilizer technology has coincided with
the energy crisis in the 1970`s and the compulsive need for contemplating
alternate source of energies often than the chemical input. Nitrogen is
an important constituent of amino acids, protein and protoplasts, directly
influence the plant growth and development in terms of both morphological
and biochemical characteristics. Since, nitrogen management is one of
the major factors to attain higher productivity, particularly under limited
water supply, where the use of higher dose of inorganic fertilizers is
restricted, demands the integration of various sources of nitrogenous
fertilizers in a more appropriate way because this not only reduces the
use of inorganic fertilizers but also makes the environment eco-friendly.
A large number of fresh water and marine organisms excrete organic and
inorganic substances in the medium in which they grow. It has been demonstrated
that certain nitrogen fixing blue-green algae liberate appreciable amount
of fixed nitrogen into the medium as polypeptide or free amino acids (Watanabe,
1951; Fogg, 1952) and were suggested as promising components of biofertilizer
because of their ability to fix the atmospheric nitrogen both as free
living organisms and components of symbiotic associations (Vessey et
al., 2005). Besides nitrogen fixation the cyanobacteria contain several
extra cellular products like growth promoters, amino acids, vitamins,
useful enzymes and nutrients like carbohydrates and nitrogen (Singh and
Trehan, 1973; Malliga et al., 2002) resulting in moderate but constant
productivity in fields where nitrogen fertilizer is applied. Hence, the
ideas of utilizing algal biofertilizers as an alternative or supplementary
source of nitrogen have to be promoted for the potential biological system
under low cost production technology. Since, cyanobacterial bio fertilizers
contain (Ravishankar, 2000; Christopher et al., 2007) the nutrients
required by the plants and help to increase the quality of the soil with
a natural microorganism in the environment and that foliar spray could
be used in many different growing medium with excellent production resulting
in its utilization as organic farming in gardening. Because one of the
aspects of current interest in recent years is to use the available natural
resources for the welfare of mankind and in this respect medicinal plants
occupy a priority rank and since cyanobacteria are available naturally
as a major biofertilizer, their exploitation is not completely understood
and is demanding globally.
Study of the medicinal plants and using drugs obtained from natural
products has been an important area of research during the last decade
of the last century.
Potential drug yielding plants and their compounds obtained are being
studied for the pharmacological values. Withania somnifera Dunal
of Solanaceae (also known as Indian Ginseng) is one such important tropical
medicinal plant commonly used in ayurveda (Indian natural therapy) and
other traditional system of medicines. Since more than 91% of pharmaceutical
products are produced from the roots of this plant, there is an increasing
demand for this herb in both national and international markets extending
an enhancement in growth and desired bioactive ingredients. However, sustainable
agricultural practices have been adopted for this plant all around India,
implications of cyanobacterial input especially its extracellular products
(that are available at a cheaper and economical value) have never been
attempted so far. Although, cyanobacteria have been previously shown to
enhance the growth of numerous plants of economic importance, a lacuna
in the information of precise quality and quantity of growth promoting
potential of commercial inoculants are to be standardized to formulate
and facilitate studies on the comparison on potential benefits of cyanobacteria.
Hence, a pioneering attempt has been made in this effort to fill the lacuna
and to enumerate the effects of cyanobacterial culture filtrates for the
effective growth of Withania somnifera.
Anabaena ambigua Rao, (a heterocystous filamentous form) and Oscillatoria
foreaui Fremi, (a non-heterocystous undifferentiated form) were chosen.
Their culture filtrate response on the growth and biomass in terms of
morphology and biochemical characteristics and active ingredients of Withania
somnifera (Ashwagandha in Sanskrit, Amukura in Tamil) a herb extensively
used as anti-inflammatory, anti-oxidant, anti-tumour, anti-stress, immuno-modulator
and rejuvenator showing a positive influence on the endocrine, cardiopulmonary
and central nervous system. The roots are recommended for hiccup, female
disorders, cough, rheumatism and dropsy.
MATERIALS AND METHODS
Culture and culture conditions: Cultures of Anabaena ambigua
Rao (A 100) and Oscillatoria foreaui Fremi (A1340) were obtained
from the Culture Collection of Algae, Centre for Advanced Studies in Botany
(CAS), University of Madras, Chennai. A. ambigua was maintained
in nitrate free BG11 medium and O. foreaui was maintained
in nitrate amended BG11 medium (Rippka et al., 1979)
at 27±1C under fluorescent illumination of 40 μ Em-2
sec-1. Cultures were incubated in growth chamber (12
/12 h light/dark cycle) fitted with Sangmo Weston Ltd., S650 313 F model
automatic timer. Gentle shaking of the cultures was done manually every
day to reduce the clumping of cells. Based on the growth studies (data
not provided), cultures were harvested at the exponential phases (16th
day for A. ambigua and 12th day for O. foreaui) by centrifugation
at 11,424 x g for 20 min in a Beckman centrifuge. The culture filtrates
obtained were further used as foliar sprays to analysis it performance
on the growth and biochemicals of a potential herb.
Site characteristics: Experiment was carried out during the period
1999 to 2000 for two seasons at the experimental farm (Maduravoyal) of
Botany Field Research Laboratory, Centre for Advanced Studies in Botany,
University of Madras (13 04` N latitude and 80 14` E longitude) which
is located at a distance of 13 km East of Chennai. The site contained
sandy loam soil which, belonged to the Series-Hillsdale, Order-Alfisols,
Type-Hapludalfs and Family of Coarse-loamy, mixed and active. The red
soil collected from Poonamallee region belonged to the Series-Ruple, Order-Ultisols,
Type-Rhodudults and Family of Fine, parasesquic and thermic (according
to the US soil taxonomy). Initially, the physico-chemical properties of
both soils were analyzed at the soil test laboratory, Kancheepuram, which
showed pH of 6.1 and 6.5, Electrical conductivity of 0.26 and 0.35 (dS
m-1). The nutrients available were found to be Nitrogen (45
and 59 mg kg -1), Phosphorus (10.4 and 13 mg kg-1)
and Potassium (149 and 165 mg kg-1) in red and sandy loam soils,
Planting material and experimental design: The seeds of Withania
somnifera were obtained from Indian Council of Agricultural Research
(ICAR), Anand, Gujarat, India. The experiment was conducted with 0.1%
mercuric chloride sterilized seeds in both soil types (red soil and sandy
loam soil). Red soil was mixed with sand in the ratio of 2:1, double sterilized
and transferred to pots of size 30x30x20 cm, while sandy loam soil of
the site was directly transferred to same sized pots after double sterilization.
About 10 kg of soil was packed in each bag and 60 mg of seeds was used
for the experiment. The plants were harvested bimonthly up to six months.
Besides, watering the plants every day, culture filtrates of both A.
ambigua and O. foreaui at different quantities of 50, 100 and
200 mL was given individually and regularly as foliar spray on alternate
days until its harvest. Triplicates were maintained for each set and were
compared against the control (only water).
Growth parameters (biomass and biochemical characteristics): The
effect of cyanobacterial culture filtrate on the growth and development
of W. somnifera was measured in terms of shoot and root lengths,
fresh and dry weights, fruit yield and size of leaf lamina, number of
root branches and diameter of the roots per plant along with certain biochemical
characteristics. The amount of total chlorophyll (Arnon, 1949), total
nitrogen (Umbriet et al., 1972), total carbohydrate (Clegg, 1956),
total lipids (Folch and Stoare-Stanley, 1957) and total withanolides-extracted
(Nittala et al., 1981) and quantified (Ramaiah et al., 1984).
Statistical analysis: All the data were subjected to One way Analysis
of Variance (ANOVA) and the significant difference among the means were
compared by Duncan`s new Multiple Range Test (DMRT) at a threshold p-value
of 0.05 to test the differences between the treatments using SPCC/PC +
Student ware statistical software.
RESULTS AND DISCUSSION
Shoot and root lengths: The shoot length of W. somnifera
treated or amended with different quantities (also referred to quantities
in the text) of both culture filtrates showed tremendous increase under
both the soil conditions. With prolonged cyanobacterial culture filtrate
inoculum the growth of the plant seemingly increased and was found to
be higher especially in sandy loam soil by O. foreaui, which showed
the maximum length of 155.15 and 137.54 cm in red soil at the highest
quantity of 200 mL (Table 1). Although, A. ambigua
was in-par with O. foreaui, increased the shoot lengths
to 120.26 and 135.80 cm in red soil and sandy loam soil, respectively
at the highest quantity of 200 mL (Table 1) was not
as effective as that of O. foreaui. However, corresponding to the
shoot length, the root length also increased by the culture filtrates
in both soil types. Though the culture filtrates of both the organisms significantly increased the root length
by two fold, maximum effect was observed by O. foreaui which,
at 200 mL increased to 19, 30 and 54.8 cm especially in sandy loam
soil during the 2nd, 4th and 6th months, respectively. Unlike that of
O. foreaui, A. ambigua, increased to only 42 and 47 cm
in red and sandy loam soils, respectively (Table 2).
Fresh and dry weights: The culture filtrate of cyanobacteria amended
at different quantities proved to be very effective on the biomass in
terms of fresh and dry weights in both the soil types. Significant increase
in biomass of the plant was obtained in sandy loam soil than red soil
with the higher volume of 200 mL culture filtrate of O. foreaui compared
to A. ambigua. Biomass in terms of fresh weight increased to 31.2%
by the culture filtrate of O. foreaui and to 28.9% by the culture
filtrate of A. ambigua against the control in sandy loam soil
(Table 3). Similarly, the dry weight was also increased
to 73.8% by the culture filtrate of O. foreaui and to 71% by the
culture filtrate of A. ambigua against the control in sandy loam
soil compared to red soil (Table 3).
Fruit yield and leaf area index: Co-relating to the fresh and
dry weights, the yield of the fruits measured at the final harvest was
enhanced by the cyanobacterial culture filtrates in both soil types. Significant
yield was achieved by the culture filtrate of O. foreaui, which
showed 64.4 and 61.4% in sandy loam soil and red soil, respectively, while,
less significant yield (p<0.05) was observed with the culture filtrate
of A. ambigua yielding to just 43.9 and 44.7% against the control
in sandy loam soil and red soil, respectively (Table 4).
Likewise, leaf are
||Effect of cyanobacterial culture filtrates on the shoot
length of W. somnifera
||Effect of cyanobacterial culture filtrates on the root
length of W. somnifera
||Effect of cyanobacterial culture filtrates on the biomass
of W. somnifera
|Values are mean±SE of four determinations, *:
Indicates statistical significance at p<0.05 level in relation
||Effect of cyanobacterial culture filtrates on the vegetative
growth of W. somnifera
|Values are mean±SE of four determinations, *:
Indicates statistical significance at p<0.05 level in relation
Effect of cyanobacterial culture filtrates on the
root branches and diameter of W. somnifera
index also significantly increased 63.8 and 62.2% with the highest volumes
of 200 mL culture filtrate, with the culture filtrates of each O. foreaui
and A. ambigua, respectively against the control (Table
Number of root branches and diameter: Corresponding to the growth,
reduction in the number of root branches was influenced by the increasing
quantities of the culture filtrates. The culture filtrate of both
A. ambigua and O. foreaui proved to be very effective at higher
volumes of 200 mL, where they reduced the number from 7 to 3 in red soil
and from 7 to 2 in sandy loam soil during their final harvest at the 6th
month (Table 5). The girth or diameter of the roots
was also influenced by the cyanobacterial culture filtrates, which gradually
increased with increasing volumes of 50, 100 and 200 mL,
||Effects of cyanobacterial culture
filtrates on the biochemical characteristics of W. somnifera
(a) total chlorophyll content (b) total nitrogen content (c) total
carbohydrates and (d) total lipids
respectively. However, the culture filtrate of A. ambigua increased
the root diameter from 4.1 to 6.62 cm in red soil (Table
5). While, O. foreaui effectively promoted it from 4.9 to 7.20
cm in sandy loam soil (Table 5) signifying that the
culture filtrates liberate an abundant quantity of many extra cellular
organic compounds (Vaidya et al., 1970) which have capacities to
chelate with several micronutrient elements.
Biochemical characteristics: The biochemical characteristics analyzed
revealed the effective response of the plant to the culture filtrates
amended. Higher the volume, greater was the output by the plant. In general,
total chlorophyll, total nitrogen and total carbohydrates analyzed showed
manifold increase in plants treated with culture filtrates compared to
the control plants. The plants grown in sandy loam soil recorded the highest
chlorophyll content compared to red soil. Significant contents of 1.053
and 1.174 mg g-1 were recorded with the culture filtrates of
A. ambigua and O. foreaui, respectively (Fig.
1a). The total nitrogen content varied with respect to the soil and
organisms used. The highest content was observed in two combinations (i)
A. ambigua with red soil which showed 4.102 μg g-1
and (ii) O. foreaui in combination with sandy loam with the content
of 4.757 μg g-1. Thus, it was increased by 42% in red
soil and 68.2% in sandy loam soil with the culture filtrates of A.
ambigua and O. foreaui, respectively (Fig. 1b).
With respect to chlorophyll, the content of carbohydrate also increased
||Effect of cyanobacterial culture filtrates on the root
withanolides of W. somnifera
in sandy loam soil by both the organisms. It showed 3.147 and 4.097 μg
g-1 with the culture filtrates of A. ambigua and O.
foreaui, respectively (Fig. 1c). Contemporarily,
the total lipid content was increased manifold by the culture filtrate
of A. ambigua, which, showed 444.50 μg g-1 in red
soil and 423.62 μg g-1 in sandy loam soil, whereas, the
culture filtrate of O. foreaui showed 279.62 and 347.75 μg
g-1 in red and sandy loam soil, respectively (Fig.
1d), which was in accordance with the results of (Kerby et al.,
1987; Katyal and Carter, 1989), according to whom, cyanobacteria apart
from fixing nitrogen, plays a major role in enriching the soil fertility
as well chelating the micronutrients present in the soil to be available
to the plants as such by either forming a consortium or liberating abundant
quantity of many extra cellular organic compounds from its culture as
leachate (Selvarani, 1983; Kumar and Mohan, 1997; Ravishankar, 2000).
Total withanolides of root: In relation to the enhanced growth
of the plants in terms of both biomass and biochemical characteristics,
the total withanolide content of the roots estimated increased with the
culture filtrates of cyanobacteria at highest volumes of 200 mL. The active
principle (withanolides) increased to about 15 and 13%, respectively against
the control in both red soil and sandy loam soils when compared. Although,
both the organisms significantly increased the quantity of withanolide
compared to control, maximum quantification was possible with the plants
treated with the culture filtrate of O. foreaui (0.35 μg g-1
DW) showing the minimum of 4% increase against the plants treated with
the culture filtrate of A. ambigu (0.31 μg g-1
DW) (Table 6).
All parameters studied have proved the potentials of cyanobacteria, which,
besides increasing nitrogen fertility, also benefited the plant by enriching
and increasing their yields by its growth-promoting substances such as
gibberellins (Metting, 1988), whose effect has been proved in paddy (Gupta
and Shukla, 1972), mangroves (Toledo et al., 1995) and some vegetables
like tomato and Cucumber (Ordoz and Pulz, 1996) especially by Arthonema
africanum (Stirk et al., 1999). The nutrient requirements for
the crops are not one and the same for all cultivars due to ecology and
several other management practices (Reddy and Mohammed, 2000) and since
nutrient requirement has become one of the most sparkling priority areas
of research, requires the utilization of bio resources; especially cyanobacteria,
which have the capacity to fix several forms of nitrogen.
Very few attempts made in the past to study the plant growth promoter
nature of extra cellular or water-soluble products of Calothrix
sp., Anabaena sp. and Stratonostoc sp., have proved the
rhizogenous and stimulatory effect on the plant organs of wheat and mulberry
(Koptiyeva and Tantsiurenka, 1971) and against certain chemical fertilizers
(Zeenat and Sharma, 1990), there appears no record as far as the medicinal
plants are concerned, except for Solanum nigrum (Lakshmi et
al., 2005). Since, more direct evidence for hormonal effects of culture
filtrates primarily with pre-soaking of rice seeds, decreased the losses
from sulphate reducing processes attributing to the enhancement of germination
and a faster seedling growth (Christopher et al., 2007), the present
report also evidences similar effect and suggest the favorable role of
cyanobacterial culture filtrates on the tested herb. A significant increase
in sandy loam soil compared to red soil, could also be due to the fact
that more porosity was provided on soil surface creating a way for the
roots to penetrate deep further, thereby increasing the root length as
already suggested by Bisoyi and Singh (1986) and Vessey et al.
Many of our most important drugs are obtained from natural sources particularly
plants and that almost all drugs used in medicine today have been developed
by modifying compounds originally obtained from plants. Therefore, herbs
are important in not only the past and present but in the future. Most
of the medicinal plants are generally wildly distributed and their effective
utilization, proper cultivation with enhanced biomass and potentials becomes
mandatory. Thus, the experiments formulated to optimize the level of nutrient
supply and potentials of cyanobacterial culture filtrates on the yield
potentials and economy of Withania somnifera produced certain interesting
and significant observations. However, the useful interaction of microbial
and plant systems are bound to exist in natural ecosystems and the efforts
of scientists lies in the identification of such systems that can be beneficial
to humankind. The present study is probably one such attempt to make use
of two systems analyzed by the scientists in the last century-the cyanobacterial
biofertilizers and the medicinal plants agrotechniques, which requires
further detailed investigation in future to be commercialized.
The authors are thankful to Prof. N. Anand, Director of Centre for Advanced
Studies in Botany, University of Madras for providing us the field laboratory
facilities and valuable guidance throughout the course of this investigation.
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