WFL Publisher

Science and Technology Food, Agriculture & Environment Vol.2 (1) : 238-242. 2004

www.world-food.net

Effects of inoculation of Azospirillum sp. in maize seeds under field conditions

Osmar R. Dalla Santa 1, Carlos R. Soccol 2*, Pedro Ronzelli Junior 3, Ramona Fernndez Hernndez4, Georgina L. Michelena Alvarez4 , Herta S. Dalla Santa1 and Ashok Pandey 5
Laboratrio de Microbiologia, Universidade Estadual do Centro-Oeste, Paran, Brazil. 2Laboratrio de Processos Biotecnolgicos, Universidade Federal do Paran,. CEP 81531-970, Curitiba-PR, Brazil. 3Laboratrio de Fitotecnia, Universidade Federal do Paran, Brazil. 4Instituto Cubano de Investigaciones de los Derivados de la Caa de Azcar, Cuba. 5 Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum 695019, India. *email: soccol@ufpr.br
1

Received 22 November 2003, accepted 15 January 2004.

Abstract
Two field experiments were performed to examine the effects of inoculation of Azospirillum sp. RAM-7 and RAM-5 strains on maize Cargil909. The treatments involved inoculation with or without different levels of nitrogen (N) and control. Simple seed inoculation with Azospirillum sp. RAM-7 strain increased yields as compared to control, but the yield difference was not significant (p<0.05). The inoculation along with high level of N resulted significantly higher yields and increase in total N and total lipids contents of the seed in comparison to the control. Increase in the yield of maize with the application of inoculant over control was recorded at N 150 kg ha-1 The inoculation of Azospirillum sp. RAM-5 in maize Cargill-909 also increased yield as compared to the control, but the difference was not significant (p<0.05). The inoculation using Azospirillum sp. RAM-7 or RAM-5 strains reduced 40% of the amount of N fertilizer recommended for each experiment (p<0.05). Key words: Plant growth promotion, Nitrogen fixation, Inoculants, Azospirillum, Zea mays

Introduction The genus Azospirillum was first studied by J. Dbereiner for nitrogen fixation capacity and occurrence in the rhizosphere, forming different kinds of association with non-leguminous plants. Azospirillum sp are nitrogen-fixing rhizobacteria with the potential to increase the yield of economically important cereals and grasses in different climatic regions. Members of the genus Azospirillum are pleomorphic, Gram-negative, aerobic, chemoorganotrophs, and under free-living conditions, fix nitrogen at low oxygen tension in a medium devoid of ammonia1-8. The beneficial effects on plant growth are not only nitrogen fixed in the rhizosphere, but are also related to the ability of these bacteria to reduce nitrate, solubilize phosphates, synthesize antibiotics and growth-promoting substances, including phytohormones and siderophores. The positive effects of bacterial inoculation are mainly attributed to improved root development and subsequent increase in the rate of water and mineral uptake9-13. Success in crop yield enhancement by microorganisms depends on efficient plant root colonization. Several bacterial properties are believed to contribute to this capacity, such as chemotaxis toward root exudates, metabolism of root exudates components, suppression of competing microorganisms, and the ability to bind to the plant root surface7, 14, 15. There are many reports indicating positive plant responses to the inoculation of Azospirillum sp. The effects depend on the bacterial strain, soil structure, plant cultivar and environmental conditions. In the field, deleterious agents such as pesticides, toxic metals, saline stress, etc. may reduce or eliminate the beneficial effects of Azospirillum on plant growth10, 4, 16. There are reports describing increase in the yields in field experiments 238

with different maize cultivars inoculated with different bacterial strains17-19. In this work, two experiments were carried out in field conditions with the aim to evaluate the effects of the inoculation of Azospirillum sp RAM-7 and RAM-5 strains in maize Cargill-909. Material and Methods Bacterial strain and growth conditions: Azospirillum sp. RAM7 and RAM-5 strains were obtained from the Instituto Cubao de Investigaciones de los Derivados de la Can de Azcar (ICIDCA). These were grown in a medium containing (g L-1) fructose 12, yeast extract 1, Na2PO4 4.2, (NH4)2HPO4 3.5, MgSO4.7H2O 0.2, KCl 0.02, CaCl2 0.01, NaMoO4.2H2O 0.01, FeCl3 0.015. The initial pH was adjusted to 7.3. Both the strains were cultivated in a 8-L fermenter (MDL Marubishi) with 5-L working volume at 370C and 250 rpm for 18 h with 0.9 vvm (volume of air/volume of media by minute) of aeration20. Inoculants formulation: For the formulation of the inoculants, 40 mL of the bacterial suspension was mixed with 200 g of powder peat (pre-sterilized with -irradiation) and placed in a sterile plastic packet and stored at room temperature. The viable cell in the mixture was about 10 9 UFC g -1 . Final cell concentration in the fermenter was 1010 CFU mL-1. Experimental designs: The treatments included inoculation with or without different levels of nitrogen (N) as follows: (1) control (uninoculated without N), (2) Azospirillum inoculation (IN) without N, (3) IN plus N 30 kg ha-1, (4) IN plus N 60 kg N.ha-1, (5) IN plus N 90 kg ha-1, (6) IN plus N 120 kg ha-1, (7)
Food, Agriculture & Environment, Vol.2 (1), January 2004

IN plus N 150 kg ha-1, and (8) N 150 kg ha-1. The blocks were distributed in a completely randomized design with four replicate per treatment. The individual replicated plot size was the 3.2 m per 8 m with four plants rows and the density of 55,000 plants ha-1. For the evaluation, external rows and 1.5 m in each extremity was considered borders. Field experiments: The experiment was conducted during 1998-99 on the Fundao Agrria de Pesquisa Agropecuria FAPA in Guarapuava, Brazil. The soil is Latossolo Bruno Alumnico Tpico. The inorganic nitrogen was applied during sowing and as cover fertilizer. Phosphorus (P 105 kg ha-1) and potassium (K 70 kg ha-1) were applied during sowing and were similar for all the treatments. The inoculation in maize seeds was carried out by the seed coating method with powder peat inoculants. The rate of inoculum was 106 CFU g-1 of seeds. The commercial maize seeds Cargill 909 were used. Analysis: The effect of the both Azospirillum sp. strains on maize was analyzed on grain yield. Total N accumulation was estimated by the Kjeldahl method21 and total lipids were estimated by the Bligh Dyer method22, and thousand kernel weight and rate of grains contaminated with fungi were also determined. Statistical analysis. The yield in each plot for each treatment was transformed to kg ha-1. The data were analyzed using Tukeys multiple test at significance p = 0.05. Results and Discussion Azospirillum sp. RAM-7 inoculation. Table 1 shows the effects of Azospirillum RAM-7 inoculation on maize Cargill-909. In general, the higher the nitrogen level was applied to the plots, the higher was the yield. Increase in the total N and total lipid contents were also observed. These results were statistically significant (p<0.05). Increase in the yield and growth-related measurements due to inoculation with Azospirillum have been previously also reported9, 12, 17. The treatment having only inoculation showed an increase of

9.4% maize grain yield, but yield was not significantly different from the control. In some studies consistent increase in crop yields derived from Azospirillum inoculation in field conditions has been found at low levels of nitrogen fertilizer or without any nitrogen addition 17, 23. In the present study, statistically significant positive responses on grain yield were obtained due to inoculation even at higher levels of nitrogen. At N fertilization 150 kg ha-1 grain yield of inoculated treatment was 13.5% higher than without inoculation (p<0.05). Earlier studies have also described consistent effects of Azospirillum inoculation on maize yields, associated with nitrogen levels24. The yield of the treatments having inoculum with N 90 and 120 kg ha-1 was equivalent to that obtained N 150 kg ha-1 but no inoculum (p<0.05). These results showed that the inoculation of Azospirillum could save 40% of the amount of nitrogen fertilizer recommended for this experiment, without any negative effects on yield. Figure 1 shows the effects of the treatments on a thousand kernel weight and the rate of grains contaminated with fungi. Significant effects were observed in the thousand kernel weight, and the rate of the grains contaminated when nitrogen fertilizer was increased. However, no apparent inoculation effects was observed on these quality components. Azospirillum sp. RAM-5 inoculation: In this trial, significant increases in yield were obtained again. Both, the inoculation and the nitrogen level had significant effects on grain yield, but the inoculation effects depended on the nitrogen level. Table 2 shows the effects of Azospirillum sp. RAM-5 inoculation in maize Cargill-909. The simple seed inoculation with Azospirillum sp. RAM-5 without any nitrogen additions increased grain yield by 17% over the control. At N 150 kg ha-1 the inoculation increased grain yield only 6% over the corresponding uninoculated control. Although these increases were not statistically significant (p<0.05), the inoculation showed a trend to increase the grain yield. The significant positive responses in grain yield due to inoculation were highest at N fertilization levels 90 and 120 kg ha-1. The yields in these treatment were approximately equivalent to that obtained with N 150 kg ha-1 without inoculation (p<0.05). Thus, also inoculation with Azospirillum

Table 1. Effect of Azospirillum sp. RAM-7 inoculation on maize Cargill-909 grain yield, total nitrogen and total lipids in grain at different nitrogen levels. Treatment Control Inoculated (IN) IN + N 30 kg ha-1 IN + N 60 kg ha-1 IN + N 90 kg ha-1 IN + N 120 kg ha-1 IN + N 150 kg ha-1 150 kg ha-1
1

Grain yield(kgha-1) 2.686 d1 2.939 d 5.826 bc 5.497 c 6.518 b 6.702 b 7.633 a 6.720 b

Grain total nitrogen (%) 0.89 b 0.92 ab nd nd nd nd 0.99 a 0.94 ab

Grain total lipids (%) 3.04 b 3.22 b nd nd nd nd 3.92 a 3.91 a

Means followed by the different letters in the same column are statistically different (P<0.05) according Tukeys multiple range test. nd not determined

Food, Agriculture & Environment, Vol.2 (1), January 2004

239

Table 2. Effect of Azospirillum sp. RAM-5 inoculation on maize Cargill-909 grain yield, total nitrogen and total lipids accumulation in grain at different nitrogen levels. Treatment Control Inoculated (IN) IN + N 30 kg ha-1 IN + N 60 kg ha-1 IN + N 90 kg ha-1 IN + N 120 kg ha-1 IN + N 150 kg ha-1 N 150 kg ha-1
1

Grain yield(kg ha-1) 2.851 d 3.341 d 5.201 c 5.508 c 6.615 b 6.957 b 7.765 a 7.311 ab

Grain total nitrogen (%) 0.88 a 0.90 a nd nd nd nd 1.00 a 0.96 a

Grain total lipids (%) 2.90 b 3.04 b nd nd nd nd 3.34 a 3.35 a

Means followed by the different letters in the same column are statistically different (P<0.05) according Tukeys multiple range test. nd not determined

A
300

290

280

270

260

250 1 2 3 4 5 6 7 8

Treatments

B
2,5

1,5

0,5

0 1 2 3 4 5 6 7 8

Treatments

Figure 1. Effect of the treatments in a field experiment with Azospirillum sp. RAM-7 on maize Cargill-909 thousand kernel weight (A), and rate of grains contaminated with fungi (B): (1) Control, (2) Inoculated (IN), (3) IN plus N 30 kg ha-1, (4) IN plus N 60 kg ha-1, (5) IN plus N 90 kg ha-1, (6) IN plus N 120 kg ha-1, (7) IN plus N 150 kg ha-1, and (8) N 150 kg ha-1 alone.

sp. RAM-5 showed the efficiency to reduce 40% of the amount of nitrogen fertilizer recommended for this experiment. It has been reported that Azospirillum sp. inoculation can increase nitrogen contents in grains17, 25, 26. In our experiment, inoculum with Azospirillum sp. RAM-5 had no apparent effects on nitrogen content of the grains. The lipid content in the treatments with N 150 kg ha-1 was significantly higher (p<0.05) than without N fertilization, but inoculation did not have any significant effect on lipid content (Table 2). The positive effects of bacterial inoculation are mainly attributed to the production of phytohormones that improve root development. These developments within the root system are important because they increase absorptive area and volume of soil substrate available to the plant, with subsequent increase in the rate of water and mineral uptake27-29. The influence of the treatments on the thousand kernel weight and the rate of the grain contaminated with fungi was measured (Figure 2). Higher thousand kernel weight on the control were obtained when the nitrogen fertilizer was increased. The increase of the N level in the treatments showed a significant reduction for the grains contaminated. The increase in the thousand kernel weight and the rate of grain contaminated are dependent on the nitrogen nutrition of the plant30. The results of the experiments on Azospirillum sp. RAM-7 and RAM-5 inoculated in maize Cargill-909 showed increased grain yield. Increase in grain yield was obtained even at high levels of nitrogen mineral fertilization, but the bacterial inoculation does not replace total nitrogen fertilizer recommended for this crop. Different response was observed among the inoculation with Azospirillum sp. RAM-7 and Azospirillum sp. RAM-5 strains, indicating some specificity between the bacteria genotype and the culture genotype. This specificity has been reported previously 12, 31. Yield improvement by inoculation with bacteria requires an efficient screening system aiming to select constantly the most successful combinations between the plant genotype and a particular bacterial strain9, 17, 24, 27, 31-32.

240

Grains fungi (%)

Thousand kernel weight (g)

Food, Agriculture & Environment, Vol.2 (1), January 2004

A
2,5

1,5

0,5

0 1 2 3 4 5 6 7 8

Treataments

B
300

290

280

270

260

250 1 2 3 4 5 6 7 8

Treatments
Figure 2. Effect of the treatments in a field experiment with Azospirillum sp. RAM-5 on maize Cargill-909 rate of grains contaminated with fungi (A), and thousand kernel weight (B): (1) control, (2) Inoculated (IN), (3) IN plus N 30 kg ha-1, (4) IN plus N 60 kg ha-1, (5) IN plus N 90 kg ha-1, (6) IN plus N 120 kg ha-1, (7) IN plus N 150 kg ha-1, and (8) N 150 kg ha-1.

References
Tarrand, J. J., Krieg, N. R. and Dbereiner, J. 1978. A taxonomic study of the Spirillum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can. J. Microbiol. 24:967980. 2 Dbereiner, J. and Pedrosa, F. O. 1987. Nitrogenfixing bacteria in nonleguminous group plants. EUA, Springer-Verlag. 120 p. 3 Arsne, F., Katupitiya, S., Kennedy, I. R. and Elmerich, C. 1994. Use of lacZ fusions to study the expression of nif genes of Azospirillum brasilense in association with plants. Mol. Plant-Microbe Interact. 7:748-757. 4 Bashan, Y. and Holguin, G. 1997. Azospirillum-plant relationships: environmental and physiological advances (19901996). Can. J. Microbiol. 43:103-121. 5 Gillis, M. and Reinhold-Hurek, B. 1994. Taxonomy of Azospirillum. In: Okon, Y. (ed.). Azospirillum plant associations. USA, CRC Press. p. 1-14. 6 Fani, R., Bandi, C., Bazzicalupo, M., Damiani, G., Di Cello, F., Francelli, S., Gallori, E., Gerace, L., Grifoni, A. Li, P. and Mori, E. 1995. Phylogenetic studies of the genus Azospirillum. In: Fendrik, I. et al. (eds.). Azospirillum and related microorganisms. Germany,
1

Springer Verlag. p. 59-75. Alexandre, G., Ren, R. and Ren, B. 1999. A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagela. Appl. Environ. Microbiol. 65:4701-4704. 8 Fischer, S. E., Miguel, M. J. and Mori, G. B. 2003. Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress. FEMS Microbiol. Lett. 219:53-62. 9 Pandey, A., Sharma, E. and Palni, L. M. S. 1998. Influence of bacterial inoculation on maize in upland farming systems of the Sikkim Himalaya. Soil Biol. Biochem. 30: 379384. 10 Jofr, E., Fischer, S., Rivarola, V., Balengo, H. and Mori, G. 1998. Saline stress affects the attachment of Azospirillum brasilense Cd to maize and wheat roots. Can. J. Microbiol. 44:416-422. 11 Olubayi, I., Caudales, R., Atkinson, A. and Neyra, C. A. 1998. Differences in chemical composition between nonflocculant and flocculated Azospirillum brasilense Cd. Can. J. Microbiol. 44:386-390. 12 Fallik, E. and Okon, Y. 1996. The response of maize (Zea mays) to Azospirillum inoculation in various types of soils in the field. World J. Microb. Biot. 12: 511-515. 13 Steenhoudt, O. and Vanderleyden, J. 2000. Azospirillum, a free-living nitrogen fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol. Rev. 24:487-506. 14 Pereg-gerk, L., Paquelin, P., Kennedy, I. R. and Elmerich, C. 1998. A transcriptional regulator of the luxR-UhpA family, FIcA, controls flocculation and wheat root surface colonization by Azospirillum brasilense Sp7. Mol. Plant-Microbe Interact. 11:177-187. 15 Hauwaerts, D., Alexandre, G., Das, S. K., Vanderleyden, J. and Zhulin, I. B. 2002. A major chemotaxis gene cluster in Azospirillum brasilense and relationships between chemotaxis operons in alfaproteobacteria. FEMS Microbiol. Lett. 208:61-67. 16 Chotte, J., Schwartzmann, A., Bally, R. and Monrozier, L. J. 2002. Changes in bacterial communities and Azospirillum diversity in soil fractions of a tropical soil under 3 or 19 years of natural fallow. Soil Biol. Biochem. 34:1083-1092. 17 Sumner, M. E. 1990: Crop responses to Azospirillum inoculation. In: Stewart, B. A (ed.). Advances in Soil Science. New York, Springer Verlag. 120 p. 18 Hecht-Buchholz, C. 1998. The apoplast-habitat of endophytic dinitrogen-fixation bacteria and their significance for the nitrogen nutrition of nonleguminous plants. Z. Pflanzenernhr. Bodenk. 161:509-520. 19 Fulchieri, M. and Frioni, L. 1994. Azospirillum inoculation on maize (Zea mays): Effects on yield in field experiment in central Argentina. Soil Biol. Biochem. 26: 921-923. 20 Fernndez, R., Dalla Santa, O. R., Soccol, C. R. and Michelena, G. 1999. Obtencin de un inoculante comercial a partir de Azospirillum sp. en condiciones brasileas. Revista sobre los derivados de la caa de azcar - ICIDICA. 33:66-72. 21 AOAC 1996. Official Method of Analysis. Association of Official Analytical Chemists, 17th ed. Arlington, Virginia, U.S.A. 22 Bligh, E. G. and Dayer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 23 Rao, N. S. S. 1981. Response of crops to Azospirillum inoculation in ndia. In: Vose, P. B. and Ruschel, A. P. (eds.). Associative N2-Fixation. Florida: CRC Press. p. 137144. 24 Fages, J. 1994. Azospirillum inoculants and field experiments. In: Okon, Y. (ed.). Azospirillum Plant Associations. USA, CRC Press. p. 88-105. 25 Boddey, R. M. and Dbereiner, J. 1988. Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research. Plant Soil. 108:53-65. 26 Nur, I., Okon, Y. and Henis, Y. 1980. An increase in nitrogen content of Setaria italica and Zea mays inoculaited with Azospirillum. Can.
7

Food, Agriculture & Environment, Vol.2 (1), January 2004

Thousand kernel weight (g)

Grain fungi (%)

241

J. Microbiol. 26:482-485. Okon, Y., and Vanderleyden, J. 1998. Root-associated Azospirillum species can stimulate plants. Features. 63:366-370. 28 Kucey, R. M. N. 1988. Alteration of size of wheat root systems and nitrogen fixation by associative nitrogen-fixing bacteria measured under field conditions. Can. J. Microbiol. 34:735-739. 29 Lin, W., Okon, Y. and Hardy, R. W .F. 1983. Enhanced mineral uptake by Zea mays and Sorghum bicolor roots inoculated with Azospirillum brasilense. Appl. Environ. Microb. 45:1775-1779. 30 Molin, R. 2000. Ocorrncia e preveno de gros ardidos e micotoxinas na cultura do milho. In: Sandini, I. E. and Fancelli, A. L. (eds). Milho, Estratgias de manejo para a regio sul. Guarapuava: Fundao Agrria de Pesquisa Agropecuria. p.117-129. 31 Millet, E., Avivi, Y. and Feldman, M. 1984. Yield response of various wheat genotypes to inoculation with Azospirillum brasilense. Plant Soil. 80:261-266. 32 Reis, V. M., Baldani, J. I., Baldani, V.L.D. and Dobereiner, J. 2000. Biological dinitrogen fixation in Gramineae and palm trees. Plant Sci. 19:227-247.
27

242

Food, Agriculture & Environment, Vol.2 (1), January 2004