Effect of ammonia-excreting strain of Azotobacter chroococcum on cereals and legumes

Zentralbl. ::Ilikrobiol. 142 (1987), 363-368 VEB Gustav Fischer Verlag Jena [Department of Microbiology, Panjab University, Chandigarh, Inelia]

Effect of Ammonia-Excreting Strain of Azotobacter chroococcum on Cereals and Legumes NEERU NARULA and K. G. GUPTA

Summary The effect of an ammonia-excreting strain of A. chroococcum AC2 was stuclied on cereals (wheat, maize, and barley) and legumes (mungbean and chickpea in association with crop-specific rhizobia). Most of the crops studied with regard to percent nitrogen, dry weight and acetylene reduction acti\-ity, were beneficially influenced by the inoculation of the ammonia-excreting strain of A. chroococcum AC2.

Zusammenfassung Die "-irkung des Ammoniak ausscheidenc1en Azotobacter chmococcunl-Slammes AC2 wurde an Getreide- (Weizen, ::Vlais und Gerste) und Gemtisepflanzen (slung-Bohnen und Kichererbse) in Verbinclung mit wirtsspezifischen Rhizobien studiert. Die meisten cler untersuchten Pflanzen zeigten in bezug auf prozentualen Stickstoffgellalt, Trockengewicht und Azetylenrecluktion eine giinstige Beeinflussung durch die Inokulation von A_ chroococcUnl AC2.

Azotobacter in the root zone of plants is known to improve the nitrogen-fixation of plants in soil, especially when its propagation is stimulated. Since 1930, 'Azobakterin', containing A. chroococcl1m, has been used in USSR and in many other countries as a soil improver purely as nitmgenous fertilizer, suitable for improving various kinds of crops (ALLISON 1947; KOSTYCHEV 1926; BEREZOVA 1938; BROWN 1964). The beneficial effect of its application was largely associated with the formation of physiologically active substances of various types, protection against root pathogens, stimulation of rhizospheric microorganisms, modification of nutrient uptake, and enhancement of nitrogen fixation (BROWN 1975). Marked increase in nitrogen fixation and yield was found when seeds (MONIB 1979a, 1979b) and seedlings of various crops, like wheat (JAIN 1973; SHARMA 1979; POI 1979; CHAHAL 1979; DHILLON 1980), maize (ApTE 1981), barley (HARPER and LYNCH 1979), were treated with Azotobacter cultures. There is also a significant increase in plant weight and total nitrogen by foliar spray of Azotobacter (SUHAS 1980; KANNAIYAN 1980)_ Therefore, the present studies were undertaken to study the effect of an ammonia-excreting strain of Azotobacter chroococcum on cereals and legumes (in association with Rhizobium) under both sterilized and unsterilized conditions.

Materials and Methods Organisms The ammonia-excreting strain AC2 and non-ammonia excreting strain AC45 of A_ chroococcum was iRolated from local soils by standard method (BROWN 19(;4). Rhizobium sp. for mung bean and chickpea were obtained from the Department of :Microbiology, Haryana Agricultural University, Risar, India.

364

N. N ARULA and K. G. GUPTA

Media for cultivation

A. chroococcum strain was grown III ammonia-excreting Jensen's medium, containing 2 % sucrose as described by NARuLA et al. (1981). Rhizobium sp. for chickpea and mungbean were maintained on yeast mannitol agar medium. Seeds Seeds of mungbean (Vigna radiata variety K-851), chickpea (Gicer a,rietinum variety H-208), wheat (Triticum vulgare, local variety) were obtained from the Department of Plant Breeding, Haryana Agricultural University, Hisar. :Vlaize (Zea mays local variety) and barley (Hordeum vulgare local variety) were obtained from National Seed Corporation of India, Challdigarh. Preparation of soil Garden soil and sand were mixed in 2: 1 ratio, sieved to ensure proper mixing and amended with 0.5 % K 2 HP0 4 and 0.1 % calcium carbonate. The experiments were done both under sterilized and unsterilized conditions. The soil was sterilized at 15 Ib/sq. inch pressure for 1 h on three consecutive days and filled in pots. Plant inoculation Seeds of wheat, maize, barley, mungbean, and chickpea, used in these studies, were surfacesterilized by the method described by VnwENT (1970) and germinated on 1 % agar. Prior to sowing, the seeds were treated with different strains of rhizobia and Azotobacter according to the treatments, then they were planted in sterilized and unsterilized soil. 6 seedlings each were kept in a pot. The plants were watered twice a day in summer and once a day in winter. After 35 d of growth the plants were harvested. Nitrogen fixing (C 2 H 2 reduction) activity Nitrogenase activity was measured in all the crops studied according to the method by HARDY (1973). The assay was conducted in 100 llll serum bottles with a hole on top, but protected by the inner rubber sept,um. The plants were dug out carefully and adhering soil was gently removed (HARDY et al. 1973). The plants were decapitated and two or four nodulated root systems were introduced in a 100 ml serum bottle. 10 % volume of air was removed from the serum bottle with the help of a disposable syringe and an equal volume of pure acetylene gas was injected. The bottles were buried in a shady place and incubated for 1 h. All samplings and inoculations were done between 10.00 a.m. to 11.30 a.m. The samples were removed and ethylene determined by gas chromatography, using a portable gas chromatograph. Nitrogenase activity was expressed as nanomoles of C2 H 4 h-1 plant- 1 per g fresh weight of nodules. Determination of nodule number and fresh weight of nodules Following the assay, the nodules were detached, washed, blotted, dry and fresh weight of nodular tissue and their number was determined. Determination of dry weight of plants The shoots were dried to constant weight at 80°C and the dry weight was determined. All values in each crop are mean values of at least six replicates. Total nitrogen Total nitrogen was determined by conventional micro-Kjeldahl's method (KJELDAHL 1883).

Results and Discussion The present work represents the effect of Azotobacter chroococcum strain AC2 (ammonia excretor) and AC45 (non-excretor) on cereal crops like wheat, maize, and barley and leguminous crops, like mungbean and gram. In case of legumes the study was made in presence of rhizobia. The parameters studied were the dry weight and per cent nitrogen in case of cereals and nodule number, fresh weight of nodules, nitrogenase activity, dry weight, and per cent nitrogen in case of legumes.

Effect of Al1lmonia-Excretmg Strain of A. chrooooccum

365

Effect of strain AC2 on cereals (wheat, maize, and barley) under sterilized and unsterilized conditions The effect of AC2 on wheat, maize, and barley under both sterilized and unsteriiized conditions was studied (Table 1). The results show that in presence of ammonia excretor both the dry weight and per cent nitrogen was increased in wheat, maize, barley. In case of wheat, the dry weight increased from 0.126 (control) to 0.147 g/plant (inoculated) under sterilized conditions and 0.204-0.:31 g/plant under unsterilized soil conditions. Similarly, the per cent nitrogen was increased from 1.911 (control) to 2.5 % (inoculated) in sterilized and from UJ:3 (control) to 2.168 per cent under nnsterilized conditions. Similar results were observed in maize and barley. Effect of AC2 on leguminous sterilized conditions

CI'OpS

(mungbean and chickpea) under

The effect of A. chroococcum AC2 was studied on leguminous crops under sterilized conditions in association with their respective Rhizobium (Table 2). From the results, it can be observed that there was a decrease in nodule number in presence of strain AC2 (5.96/plant), compared to the inoculation of lllungbean with Rhizobium sp. alone (6.92/plant). Increased nitrogenase activity (13602,8 nmoles of C2 H 4 ), dry weight (0.288 g/plant), and per cent nitrogen fixed (2.688) were observed in the treatment with Azotobacter AC2 along with Rhizobium sp. mungbean. The effect of ammonia excretor strain Azotobacter AC2 on chickpea showed an increase in nodule number (19.4/plant), decrease in wet weight of nodules (0.133 g/plant), dry weight of plants (0.31 g/plant), and significant increase in nitrogenase activity (46,600 nmoles of C2 H 1, whereas an increased wet weight (0.244 g/plant) and dry weight (0.373 g/plant) was observed in treatment with Rhizobinm sp. chickpea alone.

Table 1. Effect of A. chroococcum .\('2 on cereal crops (wheat, maize, anti barley) under sterilized and unsterilized soil conditions Treatments

Sterilized

U nsterilized

Dry wt. (gjplant)

% nitrogen (in total shoot)

Dry wt. (gjplant)

%Nitrogen (in total shoot)

0.125 0.147

1.911 2.500

0.204 0.310

1.930 2.lfiS

O.5S0 1.490

1.120 1.420

li.tHiO

1.5S4

1.232 !.fiGS

0.435 0.833

1.100 1.300

0.442 1.SS2

1.202 1.420

Wheat Control A. chroococcum

+

:Maize Control + A. chroococcum Barley Control + A. chroococcum

Results are the avarage of six pots. Sowing was done in H1S1 for wheat and maize respectively. Not much difference was observed between non-excretors al1(i the control plants. Level of significance at (Ull.

366

N. NARuLA and

K.

G. GUPTA

Table 2. Effect of Azotob(lcter chroococcum AC2 on leguminous crops in association with Rhizobium under sterilized conditions Treatment

Dry wt. (g/plant)

% Nitrogen (in total) shoot)

nil

0.192

1.904

nil

0.200

1.940

5,674.0

0.269

2.179

13,H02.8

0.288

2.688

No. of nodules/ plant

Fresh wt. of nodules (g)

Nitrogenase activity nmoles/h/g fresh wt. of nodules

nil

nil

nil 6.92

nil 0.047

5,96

0.022

Mungbean Control

+ A. chroococcum (AC2) + Rhizobium + Rhizobium + AC2

Chickpea Control

+ A. chroococcum (AC2) + Rhizobium + Rhizobium + AC2

1.0

OJJ3

nil

0,129

1.400

nil

nil

nil

0.132

1.532

9.07

0.224

23,320.0

0.373

1.631

19.40

0.133

46,GOO.0

0.310

1.701

Results are the average of six plants. Sowing for mungbean was done in 1981 and Sept. 1982 for chickpea. Level of significance at 0.01.

Effect of AC2 on leguminous crops (mungbean and chickpea) under unsterilized conditions The effect of AC2 was also studied on mungbean and chickpea in association with rhizobia under unsterilized conditions (Table 3). From the results it is evident that there was increase in nodule number when inoculated with Rhizobium sp. and Azotobacter AC2 strains together (l0.5/plant), followed by inoculation with Rhizobium sp. alone (16.37) and control (3.71). Maximum fresh weight of nodules (0.066 plant), nitrogenase activity (9,182.4 nmoles of C2 H 4 ), and dry weight (0.298 g/plant) were obtained in mungbean inoculation with Rhizobium sp. mungbean and Azotobacter AC2 together. The per cent nitrogen was highest in treatment with Rhizobium sp. alone (2.86 %), followed by inoculation with Rhizobium and Azotobacter strains together (2.34 %). In case of chickpea, highest nodule number per plant (28.6 g/plant), fresh weight of nodules (0.473 g/plant), and dry weight of plants (0.536 g/plant) was obtained when inoculated with Rhi?obiu.m sp. and Azotobacter strains together. Highest nitrogenase activity was observed in treatment with Rhizobium sp. (21,180 nmoles of C2 H 4 ), followed by treatment with Rhizobium sp. and Azotobacter together (10,600 nmoles). In legumes, the effect of Azotobacter chroococcum strain AC2 and crop-specific rhizobia together indicate that there might be the production of growth-promoting substances, ammonia or any other suitable conditions, brought about by both the organisms and acting well in association. The observation is in line with the work by SANORIA (1980). The role of A7otobacter in increasing crop yield has been found to be variable (MORRIS et al. 1978; MONIB 1979a, b). The results (Table 1 and 3) show that there is increase in per cent nitrogen fixed and dry weight in all the crops, studied under sterilized and unsterilized conditions. The work was found to be in line with findings by ALLISON (1947), MEHROTRA (1970, 1971), SUHAS (1980), MONJB (1979a, b) and

Effect of Ammonia-Excreting Strain of A. chroococcum

367

Table 3. Effect of A. chroococcum AC2 on legnminons crops in association with Rhizobium under unsterilized conditions Treatments

No. of nodules/ plant

Fresh wt. of nodules (g)

Dry wt. (gjplant)

% Nitrogen (in total) shoot)

240.0

0.181

2.184 2.400

Nitrogenase activity mnolesjhjg fresh wt. of nodules

Mungbean Control

+ AC2 + Rhizobium + Rhizobium + AC2

3.71

0.007

5.00

0.018

225.0

0.200

16.37

0.065

6,246.9

2.241

2.860

19.54

0.066

9,182.4

0.248

2.340

3.84

0.060

380.0

Gram 0.176

1.700

nil

nil

nil

0.180

1,842

14.4

0.162

21,180.0

0.185

1.882

28.6

0.473

10,600.0

0.536

2.001

Control

+ AC2 + Rhizobium + Rhizobium + AC2

Level of significance at 0.01.

APTE (1981), but the A. chroococcum strains seem to be crop-specific (POI et al. 1979), as observed in the previous studies. Considering the above-mentioned results, it can be concluded that the stimulatory effect of ammonia-excreting strains of A. chroococcum AC2 was observed. The increase in per cent nitrogen fixed in crops might be due to the capacity of particular crops to utilize ammonia. One point is clear, i.e., crops like maize and barley are favourably influenced by AC2, but in that case the right strain as well as crop has to he selected.

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KASNAIYAN, S., GOVINDARAYAN, K., and LEWIS, H. D.: Effect of foliar spray of Azotobacter chroococcum on rice crops. Plant and Soil 56 (1980), 487-490. KJELDAHL: Neue Methoden zur Bestimmung des Stickstoffs in organischen K6rpern. J. Anal. Chem. 22 (IHH3), 3!i6-382. KOSTYCHEV, S. P., SHELOUlIlOVA, A. ]\1., and SHULGINA, O. G.: Investigations of the biodynamics of soils. Trudy Od3S,kogCl nauchno-issled. Inst. Epidemiologii Mikrobiologiya 1 (1926),5-46 MEHROTRA, C. L., and LEHRI, L. K.: Studies on Azotobacter inoculation under tropical conditions. Proc. Symp. Recent Advances in Crop Production. Kanpur, India (1970), 170-179. MEHROTRA, C. L., and LEHRJ, L. K.: Effects of Azotobacter inoculation on crop yields. J. Indian Soc. Soil Sci. 19 (1971), 242-248. MONTE, M., ABD-EL-}IALEK, E. Y., HOS:NY, 1., and FAYEZ, ]\1.: Associative symbiosis of Azotobacter chroococcum and higher plants. Zbl. Bakt. II 134 (1979a), 140-148. - Seed inoculation with Azotobacter chj'oococcum in sand culture and effect of nitrogen balance. Zbl. Bakt. II 134 (1979b), 243-24H. MORRIS, H. D., GIDDENS, J., and BROWN, A. R.: Effect of Azotobacter inoculation on growth of crops. Agric. Res. 19 (1978),1-9. NARULA, N., LAKSHlIHNARAYANA, K., and TAuRo, P.: Ammonia excretion by Azotobc!cter chroococcum. Biotech. Bioeng. XXIII (1981), 467-470. POI, S. C., and KABI, M. C.: Effect of Azotobacter inoculation on the growth and yield of jute and wheat. Indian J. Agl'ic. Sci. 49 (1979), 478-480. SANORIA, C. L., and RANA, G.: Single and combined culture effect of Rhizobium and Azotobacter on yield and quantity of pea in natural and alkali soils. Indian J. of Exptl. BioI. 18 (1980), 666-66S. SHARlIIA, R. L., and RAo, A. V.: Response to biofertilizers of legumes and wheat under temperate climate. Indian J. Microbiol. 9 (1979), H9-90. SUHAS, P. \V., and RAI, P. V.: Response of sorghum to foliar sprays and seed inoculation with Azotobacter. Indian J. }Iicrobiol. 20 (1980), 319-320. VINCENT, J. }f.: A manual for the practical study of the root nodule bacteria. Oxford 1970. Author's address: Dr. NEERU NARULA, 128 \V, Model Town, Hissar - 125004 (Haryana), India.