• African Journal of Biotechnology Vol. 4 (7), pp. 667-671, July 2005

Sponsored Links

Download the ebook

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
agricultural field.
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).
Tube Experiment
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).
Nitrogenase activity
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
120 20
% 80 0 10 50 100 150 250 500 750
Thiram concentration ( g/ml)
activity 60
Figure 3b.
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.
The increase in nodule dry weight in soil as compared to
pure synthetic media might be due to lowering of thiram
activity by soil parameters thereby making thiram less Afifi MN, Moharram AA, Hamdi YA (1969). Sensitivity of Rhizobium
effective in pot experiment as well as field conditions Spp. to certain Fungicides. Arch. Microbiol. 66: 121-128.
(Balasundaram and Subba Rao, 1977). Introduction of R. Anderson JR (1978). In: Pesticide Microbiology Academic Press,
London. pp. 313-533.
japonicum significantly increases the nodulation and Arnon DI (1949). Plant Physiology. In: S Sadashivam (eds) Biochemical
nodule dry weight (Balasundaram and Subba Rao, methods for Agricultural Sciences, Tamil Nadu Agri.Univ.
1977). Absence of nodules at the 750 g/ml of thiram Coumbator, India. pp. 241-257.
was seen and the N2–ase activity was found only upto Beijerick MW (1888b). Root nodule bacteria and leguminous plants.
Cited from Fred BE, Baldwin IL,Mc Coy(1932).University of
500 g/ml thiram concentration in both pot and tube Wisconsin Press Mandison. pp. 231-235.
Bikrol et al. 671
Burton JC (1979). New Development in inoculating legumes. In NS Rennie RJ (1986). Quantifying nitrogen fixation in field grown soybeans
Subba Rao (ed) Recent advances in Biological nitrogen Fixation. using 15 N isotopes dilution. In: Proceedings of Symposium,
Oxford and Publishing Co. Pvt. Ltd.,New Delhi, India, pp. 380-405. Soybean. In: Tropical and subtropical cropping system, held during
Ferrai A (1960). Nitrogen digestion by a continuous digestion and 26 September -1 October 1983, at Tsukuba, Japan. pp. 285-299.
analysis system. Ann. N.Y. Acad. Sci. pp. 87-89. Rivellin C, Leterme P, Catroux G (1993). Effect of some fungicide seed
Fred EB Beldwin IL, Mc Coy E (1932). Root nodule bacteria and treatment on the survival of Bradyrhizobium japonicum and on the
leguminous plants. Univ. Wisconsin, Madison, Winsconsin, USA. pp. nodulation and yield of soybean (Glycine max L. merr). Biol. Fertil.
142-154. Soils 16: 211-214.
Garrett SD (1963). In: Soil Fungi and Soil Fertility, Pergaman Press, Sadasivam S, Theymoli Balasubraminan (1987). In: Practical Manual
The Macmillan Company, New York, pp.102-106. in Biochemistry, Tamil Nadu Agricultural University, Coimbatore,
Gaur AC (1980). Effect of Pesticides on Symbiotic nitrogen fixation by India, p.14.
legumes. In. J. Microbiol. 20(40): 362-370. Saffald A (1888). Fine verwertung der Hellriegat Schan Versuche wit
Gibson AH (1963). Physical Environment and nitrogen fixation. The legumisoen in landwirtachaftlichen Bertriede, Dent. Landw. Press.
effect of root temperature on recently modulated Trifolium 15: 601-619.
subterraneum L. Plants. Austr. J. Biol. Sci. 16: 28-42. Subba Rao NS (1979). Chemically and biologically fixed nitrogen
Grahm PH (1964). An application of computer techniques to the potentials and prospects. In: Subba Rao NS (ed). Recent advances
taxonomy of the root nodule bacteria of legumes. J. Gen. Microbiol. in Biological Nitrogen fixation, Oxford and IBH publishing Co. Pvt.
35: 511-517. Ltd. New Delhi, India. pp. 1-7.
Guene NFD, Diouf A, Gueye M (2003). Nodulation and nitrogen fixation Subba Rao NS (1984a). Biofertilizer in Agriculture. Oxford and IBH
of field grown common bean (Phaseolus vulgaris) as influenced by Publishers, New Delhi, India. pp.16-76.
fungicide seed treatment. Afr. J. Biotech. 2(7): 198-201. Subba Rao NS (1984b). Soil microorganisms and plant growths.
Halverson LJ, Stacey G (1986). Signal exchange in plant microbe Oxford and IBH Publishers, New Delhi, India. pp. 278-289.
interactions. Microbiological Reviews. 50: 193-225. Thornton HG (1930). The early development of root nodules of Lucerne
Hardy RWF, Burns RC, Holsten RD (1973). Application of the (medicago sativa L.). Ann. Bot. 44: 385-392.
acetylene ethylene assay for measurement of nitrogen fixation. Soil Turner GL, Gibson AH (1980). Soybean growth and yield responses to
Biol. Biochem. 5: 17-27. starter fertilizers. Soil. Sci. Sco. Am. J. 50: 234-237.
Harris LJ, Ray SN (1935). In: Biochemical Methods for Agricultural Vincent JM (1970). A manual for the practical study of the Root nodule
Sciences. S Sadashivam (ed). Tamil Nadu Agric.Univ., Coimbatore, Bacterial. I.B.P. handbook No. 15, Black wells, Scientific
India. pp. 54-67. Publications, Oxford.
Kundu GG, Trimohan (1989). Effect of Rhizobium in association with Vyas SC, Vyas A, Mahajan KC, Shroff VN (1990). Effect of fungicides
granular insecticides on nodulation and yield in Soybean. Curr. Sci. on mycorrhizal and rhizobial development in soybean. Current trend
58(23): 1340-1342. in Mycorrhizal Research. Proc. On the Mycorrhizae at Haryana
Lowery OH, Rosebrough NJ, Farr AL, Randall RJ (1951). In: Practical Agricultural University, Hissar, India. pp. 188-189.
manual in Biochemistry. S. Sadashivam (ed). Tamil Nadu Agri. Univ. Witham FH Blaydes DF, Delvin RMl. (1971). Experiments in plant
Coumbatour, In. J. Biol. Chem. pp. 193-265. physiology van Nostrand, New York, pp. 232-245.

Use: 0.17