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Response of dual inoculation with VAM and Azospirillum on the yield and oil content of Palmarosa (Cymbopogon martinii var. motia)
Microbiol. Res. (1996) 151, 325 - 328
Microbiological Research © Gustav Fischer Verlag Jena
Response of dual inoculation with VAM and Azospirillum on the yield and oil content of Palmarosa (Cymhopogon martinii var. motia) Neelima Ratti, K. K. Janardhanan Division of Microbiology and Plant Pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India Accepted: February 11, 1996
Abstract Dual inoculation of a V AM fungus, Glomus aggregatum Schenck and Smith and Azospirillum brasilense increased the growth, yield and oil content of palmarosa (Cymbopogon martinii var. motia) significantly over uninoculated control as well as inoculations with G. aggregatum or A. brasilense alone. Azospirillum brasilense also stimulated the V AM colonization and increased V AM spore population in the rhizosphere soil of palmarosa. Nitrogen content of leaf tissue was higher in the plants inoculated with A. brasilense, while leaf P content was greater in G. aggregatum inoculated plants than in dual inoculation. Key words: Azospirillum brasilense - Glomus aggregatum - Palmarosa
Introduction Research in mycorrhizae until now has established that vesicular-arbuscular (VA) mycorrhizae can improve plant growth through increased uptake of phosphorus and other mineral nutrients especially in soils oflow fertility (Tinker 1975). Azospirillum spp. are ubiquitous soil organisms capable of colonizing the roots of tropical grasses and C3 plants. Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, increases dry weight yields of plants in both, green house and field experiments (Tien et al. 1979). The positive benefits from inoculation have been attributed to several mechanisms such as biological nitrogen fixation (Kapulnik et al. 1981) and secretion of plant growth Corresponding author: Neelima Ratti
hormones, which improve plant growth by their direct effects on metabolic processes (Tien et al. 1979; Okon 1985). Some free living N2 fixers occur closely associated with VAM fungi (Tilak and Subba Rao 1987). These reports suggest a direct interaction between the VAM fungi and Azospirillum. There are many reports of increased plant yields and/or increased N accumulation by plants from dual inoculation of VAM fungi and Azospirillum brasilense (Rai and Gaur 1982; Panwar 1993; Pacovsky 1988; Subba Rao et. al. 1979, 1985; Tilak and Singh 1988). In the present investigation dual inoculations were made using both, the VAM fungus Glomus aggregatum Schenck and Smith emend Koske and Azospirillum brasilense on the roots of palmarosa plants. The influence of these inoculations on plant growth, shoot biomass and essential oil content of palmarosa is reported.
Materials and methods Pot experiment. Sandy loam type soil having the following composition, organic carbon - 2.75%, total nitrogen - 0.09%, available P - 16.31 Ilg g-l, Na - 0.95mgg-l,K - 3.90mgg- 1 ,pH - 8.72,Ec - 0.817 dsm -1 determined by the standard method (Jackson 1973) was used in the experiment. The soil was filled in earthen pots (25 cm diameter) and autoc1aved at 121°C for 2 hrs on 3 consecutive days. Preparation of the cultures. VAM fungus: A soil based inoculum of a local isolate of Glomus aggregatum multiplied on palmarosa (Cymbopogon marMicrobiol. Res. 151 (1996) 3
325
tinii var. mOfia) in pot culture was used. The inoculum consisted of 500 spores/IOO g soil. Azospirillum brasilense: bacterial culture was obtained from the Department of Agricultural Microbiology, University of Agriculture and Technology, Bangalore. At the time of inoculation peat based inoculant had 2 x 10 8 bacterial cells g -1 of peat and were free from contamination.
were recorded after drying the samples at 60 DC for 48 hrs. For obtaining VAM spores 20.0 g soil from each replicate of a treatment were pooled and the spores were extracted from the soil by wet sieving and decanting (Gerdemann and Nicolson 1963). The spores were suspended in 50 ml water and counted in a well shaken 1 ml sample. The process was repeated Plant material. Palmarosa (Cymbopogon martinii var. ten times and the number of VAM spores present in motia) seeds were obtained from plants cultivated in each 100 g soil sample was determined. Nitrogen and phosphorus content of oven dried CIMAP farm. Seeds were surface sterilized with leaves and roots were determined by the micro0.5% sodium hypochlorite for 5 min, washed thoKjeldahl method and by means of an auto analyser roughly and used to raise the plants. (FIA), respectively. Results of the experiment were Plant inoculation method. Ten gram peat based cultu- analysed statistically by ANOVA. Statistical signifire of A. brasilense were spread over the soil surface. cant difference among the data was tested by critical For VAM inoculation treatments, a thin layer of difference (C.D.) test at 1 and 5 per cent level of 5.0 g soil based inoculant of G. aggregatum contain- probability. ing 500 spores/100 g inoculum was placed 2 cm below the surface of the soil. For VAM + Azospirillum treatments, 5.0 g VAM inoculum was placed 2 cm Results below the soil surface and 10 g A. brasilense inoculum was spread over the soil surface. Ten seeds were Total yield, oil content, phosphorus and nitrogen sown in each pot initially and 15 days after germina- content tion, 3 plants per pot were maintained to grow till 90 Inoculation with G. aggregatum and Azospirillum days at temperatures ranging from 30 to 35 DC in a brasilense singly or in combination increased plant glass house. The pots were irrigated with sterilized growth, oil content, nitrogen content, phosphorus water throughout the experiment. Seeds sown on content and yield of palmarosa significantly over steam sterilized soil in pots without any treatment uninoculated control. Although inoculation with G. served as controls. Each treatment was replicated five aggregatum or A. brasilense alone caused substantial times and the experiment was laid in a randomised increase in plant growth, total dry matter yield and block design. oil content, combined inoculation with both the
Measurements of VAM infection consequences. The percentage of mycorrhizal infection of roots was determined by the root slide technique (Daft and Nicolson 1966) after clearing the roots in 10% KOH and staining with trypan blue (Phillips and Hayman 1970). Dry weights of palmarosa roots and shoots
organisms further enhanced the growth, yield and oil content of palmarosa. The increase in total dry matter yield in all three treatments, VAM alone, VAM and A. brasilense together, A. brasilense alone, was 18.7%, 53.9% and 27.7% as compared with the noninoculated control (Table 1).
Table 1. Effect of VAM and Azospirillum inoculation on plant growth and oil content of palmarosa (Cymbopogon martiniij Treatments
Shoot fro wt. (g/pot)
Root fro wt. (g/pot)
Shoot dry wt. (g/pot)
Root dry wt. (g/pot)
Total dry wt. (g/pot)
Oil/toog fro wt. (ml)
Control G. aggregatum G. aggregatum + A. brasilense A. brasilense S. E. of means C. D. at 5% C. D. at 1%
120.8 142.0 183.8
42.2 55.6 80.8
44.4 52.0 65.6
9.0 11.4 16.6*'**
53.4 63.4 82.2**
0.45 0.67 0.90
156.6 2.29 7.07* 9.92**
60.4 1.79 5.50* 7.72**
56.2 2.67 8.23 11.53
12.0* 0.94 2.89 4.06
68.2** 2.57 7.91* 11.09
0.68 0.03 0.09* 0.13**
* Significantly different from the control at 5% level. ** Significantly different from the control at 1% level.
326
Microbiol. Res. 151 (1996) 3
Discussion
VAM root infection Azospirillum brasilense stimulated the VAM colonization of palmarosa roots. The plants inoculated with both, G. aggregatum and A. brasilense, showed 85.5% root colonization compared with 74.6% in the plants inoculated with G. aggregatum alone (Table 2). VAM spore population in rhizosphere soil An increase in VAM spore population in the rhizosphere soil of palmarosa inoculated with the combination of G. aggregatum and A. brasilense (574 spores/lOO g soil) as compared with G. aggregatum (472 spores/lOO g soil) was observed (Table 2).
Phosphorus and nitrogen content Nitrogen and phosphorus content of leaf and root tissue of palmarosa inoculated with G. aggregatum alone, G. aggregatum and A. brasilense together, and A. brasilense alone, was significantly higher than non inoculated control. Nitrogen content ofleafwas higher (3.54%) in A. brasilense inoculated plants, while phosphorus content of leaf and root was increased (2.23 mg g-t, 1.48 mg g-l) in plants inoculated with G. aggregatum alone (Table 3). Table 2. VAM infection in Palmarosa (Cymbopogon martinii) plants as influenced by Azospirillum brasilense
Treatments Control
G. aggregatum G. aggregatum + A. brasilense A. brasilense
Root colonization
Spores/l00
(%)
g soil
74.64 ± 1.56 85.50 ± 3.39
472 ± 16.43 574 ± 15.17
± = Standard deviation Table 3. Effect of VAM and Azospirillum inoculation on
nitrogen and phosphorus content of leaf and root of palmarosa plants Treatments
LeafN LeafP Root N Root P (%) (mgjg) (mg/g) (%)
Control
1.98 2.26 2.86
1.53 2.23 1.97
1.21 1.74 1.39
0.79 1.48*'** 0.85
3.54 0.03 0.08* 0.11 **
1.78 0.01 0.04* 0.06**
1.41 0.02 0.05* 0.07**
1.09*'** 0.02 0.07 0.09
G. aggregatum G. aggregatum + A. brasilense A. brasilense
S. E. of means C. D. at 5% C. D. at 1%
* Significantly different from the control at 5% level. ** Significantly different from the control at 1% level.
Azospirillum brasilense in combination with G. aggregatum enhanced the growth, yield and oil content of
palmarosa as compared to the effect produced by either organism alone. VA-mycorrhiza is known to increase biomass production of the plants (Manjunath and Bagyaraj 1981) and there are several reports that Azospirillum spp. are capable to improve the yield of graminaceous plants (Rai and Gaur 1982; Okon 1985; Panwar 1993; Henis et al. 1980). VAM colonization of palmarosa roots was increased in the presence of Azospirillum spp. Barea et al. (1983) also observed increase in VAM (G. mosseae) colonization of maize and ryegrass by the inoculation of
A. brasilense.
One of the factors for increase in plant growth by
Azospirillum spp. may be due to the fixation of nitrogen. Increase in yield of sorghum, maize and rye led to the gain of 21 kg N/ha when grass seeds were inoculated with Azospirillum in field trials (Tilak 1985). Earlier reports indicate higher yield and nutrient content (P, N) of plants inoculated with Azotobacter and VA mycorrhizal fungi than inoculated singly with either organism (Brown and Carr 1984; Mohandas 1987). In our study, palmarosa plants inoculated with both, Azospirillum and G. aggregatum showed less N content than A. brasilense alone presumably because of C competition between Azospirillum and Glomus. Presence of mycorrhizae was found to reduce N 2 fixation by A. brasilense by 60% and decreased net CO 2 fixation (Harris et al. 1985). The increase in growth of palmarosa plants might be due to the plant growth hormones produced by Azospirillum (Tien et al. 1979), as growth regulating substances increase the root biomass production (Dewan and Subba Rao 1979), but this needs confirmation. However, increase in yield and oil content in the palmarosa plants inoculated with both, G. aggregatum and A. bras ilense, compared with single inoculation shows synergistic beneficial activity of the two organisms on the growth of palmarosa. Acknowledgements
We are grateful to Dr. Sushil Kumar, Director, CIMAP, Lucknow for facilities and encouragement. We are also thankful to Dr. Bagyaraj, Department of Agricultural Microbiology, University of Agriculture and Technology, Bangalore, for providing us with an Azospirillum culture. We also wish to thank Dr. D. D. Patra and Dr. Chattopadhyay of the Department of Agronomy and Soil science, CIMAP for their help in determining P and N content of plants and to Shri Srikant Sharma for statistical analysis of Microbiol. Res. 151 (1996) 3
327
the data. One of us (Neelima Ratti) gratefully acknowledges CSIR, New Delhi for the award of Research Associateship.
References Barea, J. M., Bonis, A. F., Olivares, J. (1983): Interactions between Azospirillum and VA mycorrhiza and their effects on growth and nutrition of maize and ryegrass. Soil BioI. Biochem. 15, 705-709. Brown, M. E., Carr, G. R. (1984): Interactions between Azotobacter chroococcum and vesicular-arbuscular mycorrhiza and their effects on plant growth. J. Appl. Bacteriol. 56,429-437. Daft, M. J., Nicolson, T. H. (1966): Effect of endogenous mycorrhiza in plant growth II influence of soluble phosphate on endophyte and host in maize. New Phytology 65, 343-350. Dewan, G. K., Subba Rao, N. S. (1979): Seed inoculation with Azospirillum brasilense and Azotobacter chroococcum and the root biomass of rice (Oryza sativa L.). Plant and Soil 53, 295 - 302. Gerdemann, J. w., Nicolson, T. H. (1963): Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. British Mycol. Soc. 46, 235. Harris, D., Pacovsky, R. S., Paul, E. A. (1985): C-flow and N2 fixation in Sorghum-Azospirillum-Glomus associations. In: Proc. 6th North American Conference on mycorrhizae (Ed. R. Molina) Forest Res. Lab. Bend, Corvallis, OR., 25 - 29 June (1984) p. 374. Henis, Y., Kapulnik, Y., Sarig, S., Nur, I., Okon, Y. (1980): Yield increases on a commercial scale in grasses inoculated with Azospirillum in Israel. J. Appl. Bacteriol. 49, 7-8. Jackson, M. L. (1973): Soil chemical analysis. Prentice Hall. New Delhi, 498. Kapulnik, Y., Kigel, J., Okon, Y., Nur, I., Henis, Y. (1981): Effect of Azospirillum inoculation on some growth parameters and N content of wheat, sorghum and panicum. Plant and Soil 61, 65 - 70. Manjunath, A., Bagyaraj, D. J. (1981): Intensity of mycorrhiza infection and response of onion at different stages of growth. Plant and Soil 13, 295 - 298. Mohandas, S. (1987): Field response of tomato (Lycopersicon esculentum Mill 'pusa ruby') inoculation with a VA mycorrhizal fungus Glomus Jasciculatum and Azotobacter vinelandii. Plant and Soil 98, 295-297.
328
Microbiol. Res. 151 (1996) 3
Okon, Y. (1985): Azospirillum as a potential inoculant for agriculture. Trends Biotechnol. 3, 223 - 228. Pacovsky, R. S. (1988): Influence of inoculation with Azospirillum brasilense and Glomus Jasciculatum on sorghum nutrition. Plant and Soil 110, 283-287. Panwar, J. D. S. (1993): Response ofVAM and Azospirillum inoculation to water status and grain yield in wheat under water stress condition. Indian J. Plant Physiol. 36, 41-43. Phillips, J. M., Hayman, D. S. (1970): Improved procedures for clearing and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection. Trans. British Mycol. Society 55, 158 -161. Rai, S. N., Gaur, A. C. (1982): Nitrogen fixation by Azospirillum spp. and effect of Azospirillum /ipoJerum on the yield and N-uptake of wheat crop. Plant and Soil 69, 233-238. Subba Rao, N. S., Tilak, K. V. B. R., Singh, C. S., Lakshmi-Kumari, M. (1979): Response of a few economic species of graminaceous plants to inoculation with Azospirillum brasilense. Current Science 48, 133 -134. Subba Rao, N. S., Tilak, K. V. B. R., Singh, C. S. (1985): Synergistic effect of vesicular-arbuscular mycorrhizas and Azospirillum brasilense in the growth of barley in pot. Soil BioI. Biochem. 17, 119 -121. Tien, T. M., Gaskins, M. H., Hubbell, D. H. (1979): Plant growth substances produced by Azospirillum brasilense and their effect on the yield of pearl millet (Pennisetum americanum L.). Appl. Environment. Microbiol. 37, 1016-1024. Tilak, K. V. B. R. (1985): Recent developments in the field of Azospirillum and its interaction with vesicular-arbuscular mycorrhizal (VAM) fungi in increasing the crop yields. In: Crop Productivity (Eds: H. L. Srivastava, S. Bhaskaman, K. Menon), Oxford & IBH Book, Publ. Co., New Delhi, 287 - 305. Tilak, K. V. B. R., Subba Rao, N. S. (1987): Association of Azospirillum brasilense with pearl millet (Pennisetum americanum (L.) Leeke). Biology Fertil. Soils 4, 97 -102. Tilak, K. V. B. R., Singh, C. S. (1988): Response of pearl millet (Pennisetum americanum) to inoculation with vesicular-arbuscular mycorrhizae and Azospirillum brasilense with different sources of phosphorus. Current Science 57, 43 -44. Tinker, P. B. (1975): Effects of vesicular-arbuscular mycorrhizae on higher plants. In: 29th Symposium Soc. Expt. BioI., (Eds: D. G. Jennings, D. L. Lee), Cambridge Univ. Press, London, 325-349.
Microbiological Research © Gustav Fischer Verlag Jena
Response of dual inoculation with VAM and Azospirillum on the yield and oil content of Palmarosa (Cymhopogon martinii var. motia) Neelima Ratti, K. K. Janardhanan Division of Microbiology and Plant Pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India Accepted: February 11, 1996
Abstract Dual inoculation of a V AM fungus, Glomus aggregatum Schenck and Smith and Azospirillum brasilense increased the growth, yield and oil content of palmarosa (Cymbopogon martinii var. motia) significantly over uninoculated control as well as inoculations with G. aggregatum or A. brasilense alone. Azospirillum brasilense also stimulated the V AM colonization and increased V AM spore population in the rhizosphere soil of palmarosa. Nitrogen content of leaf tissue was higher in the plants inoculated with A. brasilense, while leaf P content was greater in G. aggregatum inoculated plants than in dual inoculation. Key words: Azospirillum brasilense - Glomus aggregatum - Palmarosa
Introduction Research in mycorrhizae until now has established that vesicular-arbuscular (VA) mycorrhizae can improve plant growth through increased uptake of phosphorus and other mineral nutrients especially in soils oflow fertility (Tinker 1975). Azospirillum spp. are ubiquitous soil organisms capable of colonizing the roots of tropical grasses and C3 plants. Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, increases dry weight yields of plants in both, green house and field experiments (Tien et al. 1979). The positive benefits from inoculation have been attributed to several mechanisms such as biological nitrogen fixation (Kapulnik et al. 1981) and secretion of plant growth Corresponding author: Neelima Ratti
hormones, which improve plant growth by their direct effects on metabolic processes (Tien et al. 1979; Okon 1985). Some free living N2 fixers occur closely associated with VAM fungi (Tilak and Subba Rao 1987). These reports suggest a direct interaction between the VAM fungi and Azospirillum. There are many reports of increased plant yields and/or increased N accumulation by plants from dual inoculation of VAM fungi and Azospirillum brasilense (Rai and Gaur 1982; Panwar 1993; Pacovsky 1988; Subba Rao et. al. 1979, 1985; Tilak and Singh 1988). In the present investigation dual inoculations were made using both, the VAM fungus Glomus aggregatum Schenck and Smith emend Koske and Azospirillum brasilense on the roots of palmarosa plants. The influence of these inoculations on plant growth, shoot biomass and essential oil content of palmarosa is reported.
Materials and methods Pot experiment. Sandy loam type soil having the following composition, organic carbon - 2.75%, total nitrogen - 0.09%, available P - 16.31 Ilg g-l, Na - 0.95mgg-l,K - 3.90mgg- 1 ,pH - 8.72,Ec - 0.817 dsm -1 determined by the standard method (Jackson 1973) was used in the experiment. The soil was filled in earthen pots (25 cm diameter) and autoc1aved at 121°C for 2 hrs on 3 consecutive days. Preparation of the cultures. VAM fungus: A soil based inoculum of a local isolate of Glomus aggregatum multiplied on palmarosa (Cymbopogon marMicrobiol. Res. 151 (1996) 3
325
tinii var. mOfia) in pot culture was used. The inoculum consisted of 500 spores/IOO g soil. Azospirillum brasilense: bacterial culture was obtained from the Department of Agricultural Microbiology, University of Agriculture and Technology, Bangalore. At the time of inoculation peat based inoculant had 2 x 10 8 bacterial cells g -1 of peat and were free from contamination.
were recorded after drying the samples at 60 DC for 48 hrs. For obtaining VAM spores 20.0 g soil from each replicate of a treatment were pooled and the spores were extracted from the soil by wet sieving and decanting (Gerdemann and Nicolson 1963). The spores were suspended in 50 ml water and counted in a well shaken 1 ml sample. The process was repeated Plant material. Palmarosa (Cymbopogon martinii var. ten times and the number of VAM spores present in motia) seeds were obtained from plants cultivated in each 100 g soil sample was determined. Nitrogen and phosphorus content of oven dried CIMAP farm. Seeds were surface sterilized with leaves and roots were determined by the micro0.5% sodium hypochlorite for 5 min, washed thoKjeldahl method and by means of an auto analyser roughly and used to raise the plants. (FIA), respectively. Results of the experiment were Plant inoculation method. Ten gram peat based cultu- analysed statistically by ANOVA. Statistical signifire of A. brasilense were spread over the soil surface. cant difference among the data was tested by critical For VAM inoculation treatments, a thin layer of difference (C.D.) test at 1 and 5 per cent level of 5.0 g soil based inoculant of G. aggregatum contain- probability. ing 500 spores/100 g inoculum was placed 2 cm below the surface of the soil. For VAM + Azospirillum treatments, 5.0 g VAM inoculum was placed 2 cm Results below the soil surface and 10 g A. brasilense inoculum was spread over the soil surface. Ten seeds were Total yield, oil content, phosphorus and nitrogen sown in each pot initially and 15 days after germina- content tion, 3 plants per pot were maintained to grow till 90 Inoculation with G. aggregatum and Azospirillum days at temperatures ranging from 30 to 35 DC in a brasilense singly or in combination increased plant glass house. The pots were irrigated with sterilized growth, oil content, nitrogen content, phosphorus water throughout the experiment. Seeds sown on content and yield of palmarosa significantly over steam sterilized soil in pots without any treatment uninoculated control. Although inoculation with G. served as controls. Each treatment was replicated five aggregatum or A. brasilense alone caused substantial times and the experiment was laid in a randomised increase in plant growth, total dry matter yield and block design. oil content, combined inoculation with both the
Measurements of VAM infection consequences. The percentage of mycorrhizal infection of roots was determined by the root slide technique (Daft and Nicolson 1966) after clearing the roots in 10% KOH and staining with trypan blue (Phillips and Hayman 1970). Dry weights of palmarosa roots and shoots
organisms further enhanced the growth, yield and oil content of palmarosa. The increase in total dry matter yield in all three treatments, VAM alone, VAM and A. brasilense together, A. brasilense alone, was 18.7%, 53.9% and 27.7% as compared with the noninoculated control (Table 1).
Table 1. Effect of VAM and Azospirillum inoculation on plant growth and oil content of palmarosa (Cymbopogon martiniij Treatments
Shoot fro wt. (g/pot)
Root fro wt. (g/pot)
Shoot dry wt. (g/pot)
Root dry wt. (g/pot)
Total dry wt. (g/pot)
Oil/toog fro wt. (ml)
Control G. aggregatum G. aggregatum + A. brasilense A. brasilense S. E. of means C. D. at 5% C. D. at 1%
120.8 142.0 183.8
42.2 55.6 80.8
44.4 52.0 65.6
9.0 11.4 16.6*'**
53.4 63.4 82.2**
0.45 0.67 0.90
156.6 2.29 7.07* 9.92**
60.4 1.79 5.50* 7.72**
56.2 2.67 8.23 11.53
12.0* 0.94 2.89 4.06
68.2** 2.57 7.91* 11.09
0.68 0.03 0.09* 0.13**
* Significantly different from the control at 5% level. ** Significantly different from the control at 1% level.
326
Microbiol. Res. 151 (1996) 3
Discussion
VAM root infection Azospirillum brasilense stimulated the VAM colonization of palmarosa roots. The plants inoculated with both, G. aggregatum and A. brasilense, showed 85.5% root colonization compared with 74.6% in the plants inoculated with G. aggregatum alone (Table 2). VAM spore population in rhizosphere soil An increase in VAM spore population in the rhizosphere soil of palmarosa inoculated with the combination of G. aggregatum and A. brasilense (574 spores/lOO g soil) as compared with G. aggregatum (472 spores/lOO g soil) was observed (Table 2).
Phosphorus and nitrogen content Nitrogen and phosphorus content of leaf and root tissue of palmarosa inoculated with G. aggregatum alone, G. aggregatum and A. brasilense together, and A. brasilense alone, was significantly higher than non inoculated control. Nitrogen content ofleafwas higher (3.54%) in A. brasilense inoculated plants, while phosphorus content of leaf and root was increased (2.23 mg g-t, 1.48 mg g-l) in plants inoculated with G. aggregatum alone (Table 3). Table 2. VAM infection in Palmarosa (Cymbopogon martinii) plants as influenced by Azospirillum brasilense
Treatments Control
G. aggregatum G. aggregatum + A. brasilense A. brasilense
Root colonization
Spores/l00
(%)
g soil
74.64 ± 1.56 85.50 ± 3.39
472 ± 16.43 574 ± 15.17
± = Standard deviation Table 3. Effect of VAM and Azospirillum inoculation on
nitrogen and phosphorus content of leaf and root of palmarosa plants Treatments
LeafN LeafP Root N Root P (%) (mgjg) (mg/g) (%)
Control
1.98 2.26 2.86
1.53 2.23 1.97
1.21 1.74 1.39
0.79 1.48*'** 0.85
3.54 0.03 0.08* 0.11 **
1.78 0.01 0.04* 0.06**
1.41 0.02 0.05* 0.07**
1.09*'** 0.02 0.07 0.09
G. aggregatum G. aggregatum + A. brasilense A. brasilense
S. E. of means C. D. at 5% C. D. at 1%
* Significantly different from the control at 5% level. ** Significantly different from the control at 1% level.
Azospirillum brasilense in combination with G. aggregatum enhanced the growth, yield and oil content of
palmarosa as compared to the effect produced by either organism alone. VA-mycorrhiza is known to increase biomass production of the plants (Manjunath and Bagyaraj 1981) and there are several reports that Azospirillum spp. are capable to improve the yield of graminaceous plants (Rai and Gaur 1982; Okon 1985; Panwar 1993; Henis et al. 1980). VAM colonization of palmarosa roots was increased in the presence of Azospirillum spp. Barea et al. (1983) also observed increase in VAM (G. mosseae) colonization of maize and ryegrass by the inoculation of
A. brasilense.
One of the factors for increase in plant growth by
Azospirillum spp. may be due to the fixation of nitrogen. Increase in yield of sorghum, maize and rye led to the gain of 21 kg N/ha when grass seeds were inoculated with Azospirillum in field trials (Tilak 1985). Earlier reports indicate higher yield and nutrient content (P, N) of plants inoculated with Azotobacter and VA mycorrhizal fungi than inoculated singly with either organism (Brown and Carr 1984; Mohandas 1987). In our study, palmarosa plants inoculated with both, Azospirillum and G. aggregatum showed less N content than A. brasilense alone presumably because of C competition between Azospirillum and Glomus. Presence of mycorrhizae was found to reduce N 2 fixation by A. brasilense by 60% and decreased net CO 2 fixation (Harris et al. 1985). The increase in growth of palmarosa plants might be due to the plant growth hormones produced by Azospirillum (Tien et al. 1979), as growth regulating substances increase the root biomass production (Dewan and Subba Rao 1979), but this needs confirmation. However, increase in yield and oil content in the palmarosa plants inoculated with both, G. aggregatum and A. bras ilense, compared with single inoculation shows synergistic beneficial activity of the two organisms on the growth of palmarosa. Acknowledgements
We are grateful to Dr. Sushil Kumar, Director, CIMAP, Lucknow for facilities and encouragement. We are also thankful to Dr. Bagyaraj, Department of Agricultural Microbiology, University of Agriculture and Technology, Bangalore, for providing us with an Azospirillum culture. We also wish to thank Dr. D. D. Patra and Dr. Chattopadhyay of the Department of Agronomy and Soil science, CIMAP for their help in determining P and N content of plants and to Shri Srikant Sharma for statistical analysis of Microbiol. Res. 151 (1996) 3
327
the data. One of us (Neelima Ratti) gratefully acknowledges CSIR, New Delhi for the award of Research Associateship.
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