African Journal of Biotechnology Vol. 4 (7), pp. 667-671, July 2005
African Journal of Biotechnology Vol. 4 (7), pp. 667-671, July 2005
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2005 Academic Journals
Full Length Research Paper
Response of Glycine max in relation to nitrogen fixation
as influenced by fungicide seed treatment
Anupama Bikrol, Nidhi Saxena and Kiran Singh*
Laboratory of Bioenergetics, Department of Microbiology, Barkatullah University, Bhopal-462026, India.
Accepted 8 March, 2005
Glycine max – Rhizobium interaction is a well known symbiotic association occurring in nature and
responsible for biological nitrogen fixation. Thiram a well-known fungicide has been in practice as seed
dressing in order to prevent fungal colonization. In the present study the effect of various thiram
concentrations is investigated. Thiram concentration beyond 500 g/ml was observed to be highly toxic
with respect to plant growth factors and rhizobial infection to the G. max. The nodulation, nodule dry
weight, nitrogenase activity were observed to be maximum at 100 g/ml of thiram. The study is useful in
determining the threshold concentration of fungicide for soybean seed dressing for effective nitrogen
fixation and crop yield.
Key words: Ascorbic acid, chlorophyll content, Glycine max, nodulation, nodule dry weight, nitrogenase
activity, protein content, thiram.
Most of the terrestrial plants live in symbiosis with root synthetic chemicals including fungicides, biocides,
infecting microorganism. Colonization of roots with insecticides and fertilizers also influence nodulation
Bradyrhizobium species is beneficial because it provides process of biological N2 fixation (Anderson, 1978; Gaur,
N2 in fixed form as to the host and to the soil system 1980; Kundu and Trimohan, 1989).
(Beijerick, 1888b; Saffald, 1888; Fred et al., 1932). The fungicide applied to leguminous plants either as
Several microbial species interacts in both positive and seeds dressing or soil drench reach the soil and may
negative ways among each other (Anderson, 1978, affect the symbiotic relationship. Further, fungicide
Gaur, 1980, Kundu and Trimohan, 1989). Intraspecific applied to another crop may be sufficiently persistent to
interaction prevails within the species of Bradyrhizobium effect nitrogen level (Gaur, 1980). Apart from this,
and results into nodule occupancy (Burton, 1979) in several plant pathogenic microbes affects soybean plant
competitive manner by specific strain (Rennie, 1986). health at different growth stages. The uses of non-
Not only biological factors are involved in root nodulation mercurial fungicide TMTD (Thiram) for seed dressing of
and N2 fixation (Garrett, 1963, Halverson and Stacey, G. max have been practiced in most of the agricultural
1986; Beijerick, 1888b; Saffald, 1888; Fred et al., 1932) operations.
but abiotic components such as soil profile and certain Several deviating observation in relation to
compatibility of Rhizobial strains with fungicides have
been previously demonstrated (Afifi et al., 1969; Rivellin
et al., 1993; Grahm et al., 1980; Guene et al., 2003). In
the present study the effect of thiram on Rhizobium
*Corresponding Author. Tel: +91 0755 2677729, Fax: +91 0755 inoculants with respect to nodulation, N2 fixation, plant
2677729. E-mail: [email protected] factors such as chlorophyll content, ascorbic acid
concentration and protein content was investigated. G.
Abbreviations: Chl, chlorophyll; N2–ase, nitrogenase; TMTD, max is one of the important crops of Madhya Pradesh
thiram; YEMA, yeast extract mannitol agar.
(India) and it becomes necessary to elucidate the role of
668 Afr. J. Biotechnol.
thiram in field conditions effecting productivity of G. max
Pot Experiment Tube Experiment
as well as enhancement of nitrogen content of soil in
MATERIAL AND METHODS
Glycine max (L) merril, variety Punjab-1, seeds from Plant %
Breading Department, R.A.K. Agriculture College, Sehore, Madhya Nodule 80
Pradesh, India, were after pre-sterilization with mercuric chloride
solution (HgCl2). Experiments were performed in black cotton soil dry
obtained from Raisen District of Madhya Pradesh, India. Seeds weight 60
were inoculated in both sterilized and unsterilized soil at pH 8.2.
Apart from this, the G. max was also inoculated on agar media 40
(Gibson, 1963; Thrompton, 1930). Pure culture of Rhizobium
japonicum strain SB 119, obtained from IARI, New Delhi, India, 20
were grown on YEMA (Vincent, 1970). Soybean seeds were
pretreated with varying concentration of thiram ranging from 10 to 0
750 g/ml. All experiments were performed for 75 days followed by
removal of soybean plant for further estimation of nodule number,
0 10 50 100 150 250 500 750
nodule dry weight and N2-ase activity (Hardy et al., 1973; Turner Thiram concentration ( g/ml)
and Gibson, 1980; Subba Rao 1984).
The residual N2 content of soil was estimated by Kjeldahl method
(Ferrai, 1960; Subba Rao, 1979) from the soil obtained upto the Figure 1b. Effect of different concentrations of thiram on nodule dry
depth of 6 cm. After 75 days of incubation of G. max chlorophyll weight of G. max inoculated with R. japonicum in pot and tube
content was measured by the method performed by Arnon (1949) experiments.
and Witham et al. (1971). The ascorbic acid content was also
measured by the method of Harris (1935) and Sadasivam et al.
(1987), and observations were inferred with the help of standard RESULTS
curve of oxalic acid. The protein content of leaves was calculated
by Lowry’s method (Lowery et al., 1975).
Nodulation and Nodule dry weight
The nodule number and dry weight in G. max was
observed in thiram treated and untreated seed inoculum
in soil and synthetic media. Thiram upto 100 g/ml was
found to promote nodule number and dry weight which
was reduced by further increase in thiram concentration
160 and reached zero at 750 g/ml (Figure 1a). The present
Pot Experiment result is similar to earlier findings of Vyas et al. (1990).
140 The increase in nodule number was found to be 6, 8, 19
and 33% in pot experiment and 4, 13, 27 and 36% in
120 tube culture in the presence of 150, 100, 50 and 10 g/ml
thiram, respectively. Similar trend of nodule dry weight
Nodule number (%)
100 was observed in G. max when seeds were pretreated
with varying concentrations of thiram (Figure 1b). In case
of synthetic media the nodule dry weight was found to
increase to a maximum of 40% whereas, 46%
enhancement was observed in soil condition at 100
g/ml of thiram. A fall in nodule dry weight was observed
at 250 and 500 g/ml of thiram concentration, which was
40 30 and 70% in case of pot experiment, and 39 and 80%
in case of tube experiment (Figure 1b).
0 10 50 100 150 250 500 750
There were no nodules appearing at 750 g/ml of thiram
Thiram concentration ( g/ml) and also the N2–ase activity was observed only upto
500 g/ml thiram in both pot and tube experiments.
Figure 1a. Effect of different concentrations of thiram on nodule Increase in N2–ase activity in pot experiment was 12, 10
number of G. max inoculated with R. japonicum in pot and tube and 8% and in tube experiment it was 25, 18 and
experiments. 15% at thiram concentrations of 100, 50, 10 g/ml,
Bikrol et al. 669
respectively (Figure 2). After 100- g/ml thiram a sharp Pot Experiment Tube Experiment
decline in nitrogenase activity occur and no activity was 120
observed beyond 500 g/ml thiram. The result was B
consistent with the previous phenomenon of formation of 100
nodule under similar conditions of thiram treatment in
both pot and synthetic media. 80
Pot Experiment Tube Experiment acid 40
% 80 0 10 50 100 150 250 500 750
Thiram concentration ( g/ml)
0 120 Pot Experiment Tube Experiment
0 10 50 100 150 250 500 750
Thiram concentration ( g/ml) 100
Figure 2. Effect of different concentrations of thiram on nitrogenase C
in root nodules of G. max inoculated with R. japonicum in pots and % Protein
tube experiments. 60
% Chlorophyl lcontent
0 10 50 100 150 250 500 750
80 Thiram concentration( ug/ml)
40 Figure 3c.
Figure 3. Effect of different concentrations of thiram on (a)
0 chlorophyll, (b) ascorbic acid and (c) protein contents in leaves of
0 10 50 100 150 250 500 750 G. max inoculated with R. japonicum in pot and tube experiments.
Thiram concentration ( g/ml)
Pot Experiment chlorophyll a Pot Experiment chlorophyll b
Pot Experiment chlorophyll a+b Tube Experiment chlorophyll a concentration and leaf protein content (Figures 3a,b,c).
Tube Experiment chlorophyll b Tube Experiment chlorophyll a+b The increase in chl a with respect to control in pot
experiment was 4, 3.46 and 2.466%, and in tube
experiment it is 6, 4.28 and 1.26% at thiram
Figure 3a. concentrations of 100, 50 and 10 g/ml, respectively.
However reduction was noticed at thiram concentrations
of 150 g/ml and higher. Increase in total chlorophyll
Effect of thiram on plant factors content in both pot and tube experiments were 3.5, 2.0,
and 1.28% in pot, and 5.5, 3.45 and 1.0% in tube
Response of G. max in relation to plant factors were experiments at thiram concentrations of 100, 50, and 10
seen with respect to photosynthetic ability of plants g/ml, respectively (Figure 3a).
considering chlorophyll content, ascorbic acid Two sets of experiments performed by inoculating
concentration and leaf protein content (Figures 3a,b,c). fungicide treatment seeds in pot and synthetic media to
670 Afr. J. Biotechnol.
study the ascorbic acid content of G. max (Figure 3b) experiments. After 100 g/ml concentration of thiram a
showed significant negative correlation with increasing sharp decline in N2-ase activity occurred and no activity
concentration of thiram. Increase in ascorbic acid was seen beyond 500 g/ml concentration of thiram in
concentration up to 100 g/ml, and thereafter fell. both cases.
Ultimately, the pattern of increase and decrease in The pattern of chl b concentration followed similar
protein content of soybean leaf in both experimental trend as observed in case of for chl a content in thiram
conditions reflected similar trends as compared to other treated G. max. Ascorbic acid content of G. max showed
factors such as ascorbic acid content and total chl significant negative correlation with increasing
content and N2–ase activity (Figure 3c). Decrease in concentration of thiram. The increase and decrease
protein content was observed to be 14.0, 6.0, and 2.0%, pattern in protein content of the soybean plant also
and 12.0, 4.0, and 1.0% in pot and tube experiments, indicated negative significant correlation.
respectively, at 100, 50, and 10 g/ml thiram Nitrogen fixing contributes to fertility of soil resulting in
concentrations. increased production of subsequent crop. The observed
35% decrease in residual N2 at 750 g/ml thiram
concentration was due to the fact that the N2 available in
the soil in the absence of Rhizobium activity was by the
120 inoculated soybean for its initial growth and
100 development. Rhizobium failed completely to form
effective nodule with soybean in tube culture as well as
80 in pot experiment at 750 g/ml thiram concentration (Afifi
% Residual et al., 1969).
nitrogen The amount of chl, ascorbic acid and protein directly
40 indicates the growth of plant in favorable and unfavorable
conditions, which may be either due to fungicide or other
20 chemicals or toxins. The above findings consistent to
0 work done by Richards (1954), Zentmeyer (1995), Afifi,
0 10 50 100 150 250 500 750 et al. (1969), Szkolink (1978), Sullia and Anusuya (1989),
and Vyas et al. (1990). From the current study we
Thiram concentration (ug/ml)
conclude that soybean seeds either treated with thiram
before or after the sowing do not make any difference in
% Ressidual nitrogen nodule number and nodule dry weight (Bollon, 1961;
Domsch, 1964; Sullia and Anusuya, 1989; Vyas et al.,
1990). The N2–ase activity which was found to be
Figure 4. Effect of different concentrations of thiram on residual maximum at thiram concentration of 100 g/ml. This
nitrogen content in pot experiments after harvesting of G. max
nodulated by R. japonicum.
optimum concentration is effective with increase in
nodule number, nodule dry weight and growth estimation
factors such as chl, ascorbic acid and protein content of
Residual N2 G. max thereby enhancing the total production.
Presently, pre treatment of seeds with fungicide was
found to have positive role in amendment of biologically ACKNOWLEDGEMENTS
fixed N2 in the soil up to concentration of 100 g/ml
thiram (Figure 4). The authors are thankful to the Head Department of
Microbiology for providing the laboratory facility. The
financial support as JRF-NET, CSIR to Anupama Bikrol
DISCUSSION is highly acknowledged.
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pure synthetic media might be due to lowering of thiram
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