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Nitrogen Fixation by Azospirillum spp. in Certain Saline and Saline-Alkali Soils of India
zu. Bakt. II. Abt. 136
(1981), 641-643
[Division of Microbiology, Indian Agricultural Research Institute, New Delhi· 110012, India]
Short Communication
Nitrogen Fixation by Azospirillum spp. in Certain Saline and Saline-Alkali Soils of India K. V. B. R. TILAK and G. S. R. KRISHNA MURTI
Summary Azospirillum survives in saline and saline-alkali soils and seed inoculation with the organism in these soils helps in increasing yields of a salt-tolerant crop like barley.
Zusammenfassung Azospirillum uberlebt in salzhaltigen und alkalischen salzhaltigen Boden. Durch Saatgutbeimpfung mit diesem Organismus lal3t sich der Ertrag einer salztoleranten Pflanze wie Gerste erhohen,
Survival of Azospirillum in air-dried and stored, neutral and mildly alkaline (pH 6 to 8) soils and in carriers have been reported (LAKSHMI et al. 1977, TILAK et al. 1979). The influence of various soil factors such as redox potential, pH and organic matter on nitrogen fixation by Azospirillum in rice soils has also been reported (CHARYULU and RAO 1980). The significant effects of Azospirillum inoculation on the grain yield of rice, wheat, oats, barley and sorghum in normal soils have also been documented (SUBBA RAO et al. 1979a, b). However, the occurrence and response of Azospirillum in saline and saline-alkali soils are littl eknown. Therefore, the present communication deals with the distribution of Azospirillum and its performance on a salt-tolerant crop like barley (Hordeum vulgare) in sa.line and saline-alkali soils.
Materials and Methods The sail samples from Karnal (Haryana), Bilaspur and Rampur (U.P.), India, were collected from 0-15 em depth (a few characteristics of these soils are presented in Table 1) and were stored in polyethylene bags till plating. The ability of Azospirillum to survive in these soils under natural conditions was tested by plating a loopful of soil to a flask containing 20 ml of sterile nitrogen' free semi-solid sodium malate medium (DOBEREINER et al. 1976). After 24 hr at 30°C a typical white, dense pellicle developed a few mm below the surface of the medium. The microscopic examination of a 48 hr old culture revealed characteristic curved rods with fat droplets and spiral movements. The population of Azospirillum was enumerated quantitatively by following the most-probable number method by using the probability tables (ALEXANDER 1965). Semi-solid sodium malate tubes were inoculated with different dilutions of soil samples. The tubes in which characteristic pellicle formed a few mm below the surface of the medium were scored as positive for Azospirillum. Six replicates were maintained for each dilution. The nitrogen fixing ability of the organism was estimated by incubating the soil(s) for 5 days by the micro-Kjeldahl method (JACKSON 1967). Results were corrected for the nitrogen content of the medium and soil. The nitrogen fixing ability was expressed as /1g N 2 fixed per flask.
642
K. V. B. R. TILAK and G. S. R. KRISHNA MURTI
Two strains of Azospirillum isolated from the roots of salt-tolerant varieties of barley (Ratna and Jyoti) identified as Azospirillum braeilense (nir r] (TARRAND et al. 1978) were grown separately on Okon's medium (Okon et al. 1977) for 72 hr at 37 DC and were subsequently mixed together. The seeds of barley var. Ratna were soaked in the mixed broth culture for overnight and were sown in 20 cm (dia) pots containing 6 kg unsterilised saline and saline-alkali soils of Kamal Plants raised from uninoculated seeds and seeds soaked in medium alone (without organism) were served as controls. Treatment receiving urea (40 kg N/ha) with and without inoculation were also maintained. Table 1. Characteristics of saline and saline-alkali soils Nature of soil
Saline Saline Saline Saline Saline-alkali Saline-alkali Saline-alkali
Place of collection
Bilaspur Bilaspur Karnal Rampur Rampur Karnal Karnal
pH
8.2 8.0 8.0 7.8 9.2 8.8 10.5
Exchangeable sodium content
Electrical conductivity (mmhos/cm)
(%)
4.2 3.5 5.2 4.5 10.8 8.5 14.8
12.8 13.2 14.8 13.5 25.5 22.8 34.7
'I'able 2. Quantitative enumeration and nitrogen fixation by Azospirillum in saline and saline-alkali soils (mean of six replicates) Soil Saline Saline Saline Saline Saline-alkali Saline-alkali Saline-alkali
Most- pro bable-number (105 • g-l air-dried soil)
(flg N 2 fixed/flask)
125 150 98 105 1.5 8.5 0.9
2,250 1,875 1,050 1,125 625 350 125
N 2 fixation
Table 3. Response of seed inoculation with Azospirillum on yield (g/pot) of barley var. Ratna in saline and saline-alkali soils of Kamal (mean of 5 pots'j) Treatment
Uninoculated control Medium alone (control) Azospirillum inoculation 40 kg N/ha (urea) 40 kg N/ha + Azospirillurn.
Saline-alkali soil
Saline soil Straw yield
Grain yield
Straw yield
Grain yield
20.5 22.7 30.5 2 ) 33.4 41.6
15.2 15.9 20.2 2 ) 28.7 33.2 2 )
14.2 15.0 18.5 21.7 28.5
10.1 10.5 12.5 13.5 14.5
1) Each pot had 4 plants. 2) Significant increase over corresponding control.
Nitrogen Fixation by Azospirillum spp.
643
Results and Discussion The occurrence of Azospirillum was notioed in both saline and saline-alkali soils (Table 2). The population of Azospirillum was more in saline soils than in saline-alkali soils. Further, the nitrogen fixing ability of the organism was higher in saline soils than in saline-alkali soils. A strong negative correlation (r - -0.7032) existed between the nitrogen fixing ability of the organism and exchangeable sodium content of the soils indicating thereby that the exchangeable sodium content suppresses the nitrogen fixation. The seed inooulation brought a marked increase in grain and straw yields with Azospirillum brasilense over uninoculated control in both the soils, although the increase in yields was significant in saline soil only (Table 3). The increase in grain yield due to inoculation over uninoculated oontrol was 33.3 and 23.8 per cent in saline and saline-alkali soils, respectively. The results in Table 3 also clarify the possible influence of malate medium alone on the performance of the cereal. Generally speaking, the medium had very little beneficial influenoe on the straw and grain yields of the oereal tested. Acknowledgement Our sincere thanks are due to Dr. N. S. SUBBA RAo, Head, Division of Microbiology, Indian Agric, Res. Institute, New Delhi, India, for providing facilities and constant encouragement during the course of these investigations.
References ALEXANDER, M.: Most-probable-number method for microbial populations. In: Methods of Soil Analysis (C. A. BLAOK, ed.], Part II, pp. 1467 -1472. Amer. Soc. Agron., Madison 1965. CHARYULU, P. B. B. N., and RAo, V. R.: Influence of various soil factors on nitrogen fixation by Azospirillum spp. Soil BioI. Biochem. lZ (1980), 343-346. DOBEREINER, J., MARRIELL, I. E., and NERY, M.: Ecological distribution of Spirillum lipoJerum Beijerinck. Can. J. Microbiol. 22 (1976), 1464-1473. JAOKSON, M. L.: Soil Chemical Analysis, pp. 95-110. Prentice Hall of India Pvt. Ltd., New Delhi, India. 1967. LAKSHMI, V., RAo, A. S. N., VIJAYA LAKSHMI K., LAKSHMI-KuMARI, M., TILAK, K. V. B. R., and SUBBA RAo, N. S.: Survival and establishment of Spirillum lipoJerum. Proc. Indian Acad. Sci. 86 (1971),397-404. OKON, Y., ALBREOHT, S. L., and BURRIS, R. H.: Methods for growing Spirillum lipoferum and for counting it in pure culture and in association with plants. Appl, Environ. Microbiol. 33 (1977), 85-88. SUBBA RAo, N. S., TILAK, K. V. B. R., LAKSHMI-KuMARI, M., and SINGH, C. C. S.: Azospirillum a new bacterial fertilizer for tropical crops. Sci. Reporter 10 (1979), 690-692. - - SINGH, C. S., and LAKSHMI-KuMARI, M.: Response of a few economic species of graminaceous plants to inoculation with Azospirillum brasilense. Curro Sci. 3 (1979), 133-134. TARRAND, J. J., KRIEG, N. R., and DOBEREINER, J.: A taxonomic study of the Spirillum lipo[erum group with descriptions of a new genus Azospirillum gen. nov. and Azospirillum brasilense sp. nov. Can. J. Microbiol. 24 (1978), 967 - 980. TILAK, K. V. B. R., LAKSHMI-KuMARI, M., and NAUTIYAL, C. S.: Survival of Azospirillum brasilense in different carriers. Curro Sci. 9 (1979), 412 - 413. Authors' address: Prof. Dr. K. V. B. R. TILAK, Div. of Microbiology, and Dr. G. S. R. KRISHNA MURTI, Div. of Agric. Physics, Indian Agricultural Research Institute, New Delhi - 110012, India.
44
Zbl. Bakt. II. Abt., Bd. 136
(1981), 641-643
[Division of Microbiology, Indian Agricultural Research Institute, New Delhi· 110012, India]
Short Communication
Nitrogen Fixation by Azospirillum spp. in Certain Saline and Saline-Alkali Soils of India K. V. B. R. TILAK and G. S. R. KRISHNA MURTI
Summary Azospirillum survives in saline and saline-alkali soils and seed inoculation with the organism in these soils helps in increasing yields of a salt-tolerant crop like barley.
Zusammenfassung Azospirillum uberlebt in salzhaltigen und alkalischen salzhaltigen Boden. Durch Saatgutbeimpfung mit diesem Organismus lal3t sich der Ertrag einer salztoleranten Pflanze wie Gerste erhohen,
Survival of Azospirillum in air-dried and stored, neutral and mildly alkaline (pH 6 to 8) soils and in carriers have been reported (LAKSHMI et al. 1977, TILAK et al. 1979). The influence of various soil factors such as redox potential, pH and organic matter on nitrogen fixation by Azospirillum in rice soils has also been reported (CHARYULU and RAO 1980). The significant effects of Azospirillum inoculation on the grain yield of rice, wheat, oats, barley and sorghum in normal soils have also been documented (SUBBA RAO et al. 1979a, b). However, the occurrence and response of Azospirillum in saline and saline-alkali soils are littl eknown. Therefore, the present communication deals with the distribution of Azospirillum and its performance on a salt-tolerant crop like barley (Hordeum vulgare) in sa.line and saline-alkali soils.
Materials and Methods The sail samples from Karnal (Haryana), Bilaspur and Rampur (U.P.), India, were collected from 0-15 em depth (a few characteristics of these soils are presented in Table 1) and were stored in polyethylene bags till plating. The ability of Azospirillum to survive in these soils under natural conditions was tested by plating a loopful of soil to a flask containing 20 ml of sterile nitrogen' free semi-solid sodium malate medium (DOBEREINER et al. 1976). After 24 hr at 30°C a typical white, dense pellicle developed a few mm below the surface of the medium. The microscopic examination of a 48 hr old culture revealed characteristic curved rods with fat droplets and spiral movements. The population of Azospirillum was enumerated quantitatively by following the most-probable number method by using the probability tables (ALEXANDER 1965). Semi-solid sodium malate tubes were inoculated with different dilutions of soil samples. The tubes in which characteristic pellicle formed a few mm below the surface of the medium were scored as positive for Azospirillum. Six replicates were maintained for each dilution. The nitrogen fixing ability of the organism was estimated by incubating the soil(s) for 5 days by the micro-Kjeldahl method (JACKSON 1967). Results were corrected for the nitrogen content of the medium and soil. The nitrogen fixing ability was expressed as /1g N 2 fixed per flask.
642
K. V. B. R. TILAK and G. S. R. KRISHNA MURTI
Two strains of Azospirillum isolated from the roots of salt-tolerant varieties of barley (Ratna and Jyoti) identified as Azospirillum braeilense (nir r] (TARRAND et al. 1978) were grown separately on Okon's medium (Okon et al. 1977) for 72 hr at 37 DC and were subsequently mixed together. The seeds of barley var. Ratna were soaked in the mixed broth culture for overnight and were sown in 20 cm (dia) pots containing 6 kg unsterilised saline and saline-alkali soils of Kamal Plants raised from uninoculated seeds and seeds soaked in medium alone (without organism) were served as controls. Treatment receiving urea (40 kg N/ha) with and without inoculation were also maintained. Table 1. Characteristics of saline and saline-alkali soils Nature of soil
Saline Saline Saline Saline Saline-alkali Saline-alkali Saline-alkali
Place of collection
Bilaspur Bilaspur Karnal Rampur Rampur Karnal Karnal
pH
8.2 8.0 8.0 7.8 9.2 8.8 10.5
Exchangeable sodium content
Electrical conductivity (mmhos/cm)
(%)
4.2 3.5 5.2 4.5 10.8 8.5 14.8
12.8 13.2 14.8 13.5 25.5 22.8 34.7
'I'able 2. Quantitative enumeration and nitrogen fixation by Azospirillum in saline and saline-alkali soils (mean of six replicates) Soil Saline Saline Saline Saline Saline-alkali Saline-alkali Saline-alkali
Most- pro bable-number (105 • g-l air-dried soil)
(flg N 2 fixed/flask)
125 150 98 105 1.5 8.5 0.9
2,250 1,875 1,050 1,125 625 350 125
N 2 fixation
Table 3. Response of seed inoculation with Azospirillum on yield (g/pot) of barley var. Ratna in saline and saline-alkali soils of Kamal (mean of 5 pots'j) Treatment
Uninoculated control Medium alone (control) Azospirillum inoculation 40 kg N/ha (urea) 40 kg N/ha + Azospirillurn.
Saline-alkali soil
Saline soil Straw yield
Grain yield
Straw yield
Grain yield
20.5 22.7 30.5 2 ) 33.4 41.6
15.2 15.9 20.2 2 ) 28.7 33.2 2 )
14.2 15.0 18.5 21.7 28.5
10.1 10.5 12.5 13.5 14.5
1) Each pot had 4 plants. 2) Significant increase over corresponding control.
Nitrogen Fixation by Azospirillum spp.
643
Results and Discussion The occurrence of Azospirillum was notioed in both saline and saline-alkali soils (Table 2). The population of Azospirillum was more in saline soils than in saline-alkali soils. Further, the nitrogen fixing ability of the organism was higher in saline soils than in saline-alkali soils. A strong negative correlation (r - -0.7032) existed between the nitrogen fixing ability of the organism and exchangeable sodium content of the soils indicating thereby that the exchangeable sodium content suppresses the nitrogen fixation. The seed inooulation brought a marked increase in grain and straw yields with Azospirillum brasilense over uninoculated control in both the soils, although the increase in yields was significant in saline soil only (Table 3). The increase in grain yield due to inoculation over uninoculated oontrol was 33.3 and 23.8 per cent in saline and saline-alkali soils, respectively. The results in Table 3 also clarify the possible influence of malate medium alone on the performance of the cereal. Generally speaking, the medium had very little beneficial influenoe on the straw and grain yields of the oereal tested. Acknowledgement Our sincere thanks are due to Dr. N. S. SUBBA RAo, Head, Division of Microbiology, Indian Agric, Res. Institute, New Delhi, India, for providing facilities and constant encouragement during the course of these investigations.
References ALEXANDER, M.: Most-probable-number method for microbial populations. In: Methods of Soil Analysis (C. A. BLAOK, ed.], Part II, pp. 1467 -1472. Amer. Soc. Agron., Madison 1965. CHARYULU, P. B. B. N., and RAo, V. R.: Influence of various soil factors on nitrogen fixation by Azospirillum spp. Soil BioI. Biochem. lZ (1980), 343-346. DOBEREINER, J., MARRIELL, I. E., and NERY, M.: Ecological distribution of Spirillum lipoJerum Beijerinck. Can. J. Microbiol. 22 (1976), 1464-1473. JAOKSON, M. L.: Soil Chemical Analysis, pp. 95-110. Prentice Hall of India Pvt. Ltd., New Delhi, India. 1967. LAKSHMI, V., RAo, A. S. N., VIJAYA LAKSHMI K., LAKSHMI-KuMARI, M., TILAK, K. V. B. R., and SUBBA RAo, N. S.: Survival and establishment of Spirillum lipoJerum. Proc. Indian Acad. Sci. 86 (1971),397-404. OKON, Y., ALBREOHT, S. L., and BURRIS, R. H.: Methods for growing Spirillum lipoferum and for counting it in pure culture and in association with plants. Appl, Environ. Microbiol. 33 (1977), 85-88. SUBBA RAo, N. S., TILAK, K. V. B. R., LAKSHMI-KuMARI, M., and SINGH, C. C. S.: Azospirillum a new bacterial fertilizer for tropical crops. Sci. Reporter 10 (1979), 690-692. - - SINGH, C. S., and LAKSHMI-KuMARI, M.: Response of a few economic species of graminaceous plants to inoculation with Azospirillum brasilense. Curro Sci. 3 (1979), 133-134. TARRAND, J. J., KRIEG, N. R., and DOBEREINER, J.: A taxonomic study of the Spirillum lipo[erum group with descriptions of a new genus Azospirillum gen. nov. and Azospirillum brasilense sp. nov. Can. J. Microbiol. 24 (1978), 967 - 980. TILAK, K. V. B. R., LAKSHMI-KuMARI, M., and NAUTIYAL, C. S.: Survival of Azospirillum brasilense in different carriers. Curro Sci. 9 (1979), 412 - 413. Authors' address: Prof. Dr. K. V. B. R. TILAK, Div. of Microbiology, and Dr. G. S. R. KRISHNA MURTI, Div. of Agric. Physics, Indian Agricultural Research Institute, New Delhi - 110012, India.
44
Zbl. Bakt. II. Abt., Bd. 136