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Interactions between Azotohacter spp. and Rhizobium sesbani into the rhizosphere of Sesbania sesban (L.) Merrill plants and its efficiency on growth and symbiontic nitrogen fixation
Zentralbl. Mikrobiol. 147 (1992), 112- 118 Gustav Fischer Verlag Jena (Botany Department , Faculty of Science , Al-Azhar University, Cairo , Egypt)
Interactions between Azotobacter spp. and Rhizobium sesbani into the Rhizosphere of Sesbania sesban (L.) MERRILL Plants and its Efficiency on Growth and Symbiontic Nitrogen Fixation M. S. EL-GAMAL Key words : Azotobacter , Rhizobium sesba ni, rhizosphere, symbiontic nitrogen fixation
Summary The present work was made to study the effect of adding Azotobacter spp .; A. chroococcum and/or A. vinelandii into the rhizosphere of Sesbania sesban seedlings which inoculated with R. sesbani as well as the addition of urea into rhizosphere of Sesbania plants (inoculated with R. sesbani); on growth , nodulation and dinitrogen fixation in Sesbania plants in comparison with the same plant inoculated only with R. sesbani (controlled plants). Results indicated that Sesbania plants fortified with urea or inoculated with A. chroococc um and/or A. vinelandii into the rhizosphere gave significant increases in both chlorophyll contents, total soluble carbohydrates, total soluble proteins, root, shoot lengths, dry weight of roots and shoots. Moreover, stimulation was also observed in nitrogenase activity in Sesbania plants treated with A. chroococcum and/or A. vinelandii into the rhizosphere, while no change appear in nitrogenase activity in Sesbania plants suplemented with urea as organic fertilizer. Therefore , it can be suggested that the association between Azoto bacter spp . and Rhizobium sesbani into the rhizosphere of Sesba nia plants enhanced the development of Sesbania growth in the above traits.
Zusammenfassung Die vorliegende Arbeit wurde durchgefuhrt, urn die Wirkung einer Zusatzbeimpfung mit Azotobacte r spp . , A. chroococcum und/oder A. vinelandii in die Rhizosphare von Sesbania sesban- Sam lingen , welche schon mit Rhizobium sesb ani beimpft waren als auch bei Zugabe von Hamstoff in die Rhizosphare zu ermitteln. Wachstum , Nodulation und Dinitrogen-Fixierung in Sesbania-Pflanzen wurden ermittelt. Als Kontrolle dienten die nur mit R. sesbani beimpften Pflanzen. Die Ergebnisse zeigen, daBdie Sesbania-Pflanzen , die mit Hamstoff oder mit A . chroococcum und/oder mit A. vinelandii zusatzlich beimpft waren, einen signifikanten Anstieg im Chlorophyllgehalt, von total gelosten Kohlenhydraten und Proteinen sowie Wurzel- und Schotilange und -trockengewicht aufwiesen. Bei Zusatzbeimpfung mit A. chroococcum und/oder A . vinelandii wurde weiterhin eine Stimulation der NitrogenaseAktivitat beobachtet, wahrend dies bei Harnstoff- oder organischer Dlingung nicht eintrat. Daher kann festgestellt werden, daf die Assoziation zwischen Azotobacter spp. und Rhizobium sesbani in der Rhizosphare das Wachstum von Sesban ia-P flanzen erhoht.
Introduction
Recently much work has been done on the significance of Azotobacter as a biological nitrogen fertilizer for improving the growth of different species of plant crops (FEDOROV1940 ; FEDOROV and NEPOMlLOVA 1954 ; GALIMOVA 1960; SMIRNOVA 1960; POCHON and CHALVIGNAC 1964). Concerning the importance of diazotrophs bacteria in the rhizosphere, several workers showed that association of Rhizobium sp. with a suitable strain of Azotobacter sp.
Interactions between Azotobacter spp. and Rhizobium sesbani
113
(A. chroococcum or A. vinelandii) increased root nodules of legumes e.g. Glycine max, Vigna radiata, Cicer arietinum, Pisum sativum and Trifolium repens (RAVAT and SANORIA 1976; JAUHRI et al. 1979; BURNS et al. 1981; KOTHARI et al. 1986). In addition, EL-HoSEINY (1979 a, b) indicated that introducing of Azotobacter chroococcum into the rhizosphere of both Zeay mays and Hordeum vulgare plants stimulated growth of these plants. However, APTE et al. (1971); SINGH et al. (1979); KOTHARI et al. (1986); and PATIL et al. (1987) reported that other microorganisms such as Beijerinckia, Bacillus, Spirillospora, Azospirillum brasilense enhanced nodulation and dry yield of some leguminous plants. On the contrary, JENSEN (1942) and RAO et a!. (1976) mentionedthat associationbetween Azotobacterand Rhizobiahad no effect on
nodulation of some leguminous plants. On the other hand, many investigators found that mineral nitrogen fertilizers stimulated nodulation and nitrogen fixation in leguminous plants at low doses of nitrogen (10-100 p.p.m.), above this concentration nodules formation was suppressed while plants developed well (RICHARDSON et a!., 1957; KOLOsovA, 1965; VINCENT, 1970; EL-GAMAL, 1985), SINGH and SUBBA RAo (1979) noticedthat additionof urea to leguminousplants at levels (0-80 kg N/ha) stimulated nodules formation and dry yields of its. The present paper has been carried out to study the effect of introducing viable cells of Azotobacter spp. ,. (i.e., A. chroococcum and A. vinelandii, either adding singly or together)into the rhizosphereof Sesbania sesban seedlings which inoculated with R. sesbani with the aim of studying their efficiency on growth, nodulation and dinitrogen fixation of the treated plants as well as the same plants fortified with urea as organic fertilizer and inoculated with R. sesbani in comparison to Sesbania plants which inoculated only with R. sesbani (controlled plants).
Material and Methods Microorganisms and Treatments A pure culture of R. sesbani was isolated from root nodules of Sesbania sesban and identified by ELWAN et al. (1983). Sesbania sesban seeds were grown in pots (2 plants/Pot) containing sterile sandy soil. The moisture content of each pot was kept at 30 % water holding capacity. The pots were inoculated with Rhizobium sesbani. Each pot was supplied with equal amounts of nitrogen free nutrient solution (modified from THORNTON, 1930 according to VINCENT, 1970), it has the following constituents; 2 g Ca3 (P04 h ; 0.5 g K 2HP04 ; 0.2 g MgS04 ' 7H 20; O.I g NaCI in I litre of distilled water. Irrigation was supplied with equal amounts of tap water per pot at intervals of five days. For setting up the experiment, five sets of pots were used; the first set (4 pots) represents control group tSesbania plants inoculated with R. sesbani only). The second set (4 pots) represents Sesbania plants inoculated with R. sesbani and supplemented with urea into the rhizosphere (at concentration 0.2 giL = 93 p.p.m. N) which allowed nitrogenase activity of R. sesbani. The third set (4 pots) represents sesbania plants inoculated with R. sesbani and injected with standard inoculum of Azotobacter chroococcum. The fourth set (4 pots) represents Sesbania plants inoculated with R. sesbani and injected with standard inoculum of Azotobacter vinelandii. The fifth set (4 pots) represents Sesbania plants inoculated with R. sesbani and injected with standard inocula of both A. chroococcum and A. vinelandii together. Rhizobium inoculum: A standard inoculum of R. sesbani was prepared by growing the organism on yeast extract mannitol agar medium for 3 days at 30°C. The microorganisms were harvested and suspended in sterile tap water to prepare a standard bacterial suspension containing about I x 106 cells per ml. It was inoculated in sterile sand cultures at five days after sowing Sesbania seeds (in all treatments including control) at a dose 5 ml/pot. Azotobacter inoculum: A. chroococcum and A. vinelandii were kindly provided from Agriculture Research center, Giza. Egypt. Standard bacterial inocula were prepared by growing each species singly on nitrogen-free agar medium (PHILIPP et aI., 1981) at 30°C for 3 days. Bacterial growth was harvested and suspended in sterile tap water to obtain suspension containing about I X 106 cell per ml for every species. Sesbania sesban seedlings of fifteen days old were inoculated into the rhizosphere with a standard viable inoculum of each Azotobacter sp. or both of them together. The plants were harvested after 45 days from sowing seeds, then readings & measurements were carried out.
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Growth criteria a) Preparation of sample for Chlorophyll estimation Determination of chlorophyll "a", "b" and total chlorophyll contents were carried out according to VERNON et al. (1966). One gram of fresh leaves was cut into small pieces with a scissor, chlorophylls were extracted by grinding the tissues in an electrical blender for 5 minutes in 100 ml of 80 % acetone. The homogenate was filtered through a Whatman NO I filter paper and the filterate was completed into 100 ml with 80% acetone. The optical density of the extract was measured using spectroco!ourimeter at 649 and 665!lm for chlorophyll a & b respectively. The chlorophyll concentration was calculated using the following equations. Chlorophyll (a) mg/g tissue = 11.63 (A665) - 2.39 (A649). Chlorophyll (b) mg/g tissue = 20.11 (A649) - 5.18 (A665). Total chlorophyll (a + b) mg/g tissue = 6.45 (A665) + 17.72 (A649).
b) Preparation of sample for determination of Total water soluble Carbohydrates (mg/g oven dry weight) Total soluble carbohydrates of roots, shoots and nodules of Sesbania sesban plants were carried out using the anthrone technique according to UMBREIT et al. (1959). The plant tissues (roots, shoots or nodules) were dried at 65°C till a constant dry weights were obtained, then ground to a fine powder. One g of the powder was transferred into a 100 ml capacity conical flask, 5 m! of 2 % phenol water and 10 ml of 30 % trichloroacetic acid were added. The mixture was shaken and Kept over night before being filtered, the filterate was made up to 50 ml with distilled water. Ten ml of the filtrate of each sample were mixed with 2 gm of activated charcoal and shaken well for 15 minutes and then filterated. The clear filtrate was diluted quantitatively with dist water. 3 ml of the diluted filterate were transferred to a pyrex test tube of 60 ml capacity, to which 6 ml of freshly prepared anthrone reagent (2 g of anthrone/L of 95 % sulphuric acid) were added. The contents of the tube were thoroughly mixed by swiriling. The tube was then placed in a boiling water bath for 3 minutes & then left to cool. The developed colour was measured using colourimeter at 620 urn filter. A blank mixture containing dist. water and reagent was also measured under the same condition to set up the apparatus at zero optical density. A standard curve was constructed (using different concentrations of sucrose) from which amount of total soluble carbohydrates were determined.
c) Preparation of sample for determination of "Total soluble proteins" (mg/g oven dry weight) Plant tissues (roots, shoots or nodules) of Sesbania sesban plants were dried well at 65°C till a constant dry weights, then ground to a fine powder. One gram of the powder was transferred into 100 m! capacity conical 5 ml of 2 % phenol water and 10 ml dist. water were added. The mixture was shaken and kept overnight before being filtered. The filterate was completed to 50 ml by dist. water. Total water soluble proteins were determined by using the method of LOWRY et al. (1951), using bovine serum albumen as a standard protein.
d) Preparation of Sample for evaluating nitrogenase activity in Sesban sesban nodules The acetylene reduction technique was applied for estimating amount of nitrogen fixed (ug/g dry nodules/h) according to the method of DlLOWORTH (1970). The fresh roots of Sesbania sesban with attached nodules were washed thoroughly and transfered to 420 ml serum bottles, then sealed with tide rupper stoppers. The appropriate volume of acetylene was injected into the bottles using plastic syring to give acetylene concentration of 10% of the atmosphere. The bottles were incubated at 30°C for I h. Then, gas samples were withdrawn and assayed for ethylene concentration using Fishere gas liquid chromatograph. Standard curve for pure ethylene was carried out. Thus !l moles CzRJgm dry nodules/hr X 0.42 = ug nitrogen fixed/g dry nodules/h were estimated. After determination of Nitrogenase activity in nodules, then Number of nodules/plant were counted in a fresh state and nodules were dried in electric oven at 60°C for several times till a constant dry weight of nodules were determined (mg/plant).
e) Preparation of Samples for measuring Roots & shoots lengths (em) and determination of Dry weight of Roots & shoots (mg/plant): Separation of roots and soils The whole content of pots were gently removed, placed a sieve net work and feeble current of water was applied on the plant to remove sand particles without affecting nodules of the root system, counting number of nodules per plant, separation of the root system from the shoot system and then measuring the length for roots & shoots, drying of both roots & shoots in oven at 105°C for 2 days, until a constant dry weight was obtained. The T-test was used in statistical analysis to determine the significancy between different treatments and control.
Interactions between Azotobacter spp . and Rhi zobium sesbani
115
Results and Discussion Results in Table 1 indicated that Sesbania plants supplemented with 0 .2 g/L of Urea or injected with A. chrooco ccum and/or A. vinelandii had more or less, the same amount of both chlorophyll "a" , "b" and total chlorophyll of Sesbania leaves, whereas all treatments caused significant increa ses in the above traits in comparison to Sesbania plants inoculated only with R. sesbania (control). Table I. Chlorophylls "a". "b" and total chlorophyll (mglg) of the leaves of Sesbania sesban plants as affected by presence of urea ; Azotobacter chroococcum, andlor Azotobacter vinelandii in its rhizosphere . Treatment
Chlorophyll contents (mglg fresh weight) Chlorophyll "a "
Chlorophyll "b"
Total chlorophyll
Plants inoculated with R. sesbani only (Control)
7.42 ± 0.12
3.13 ± 0.19
10.55 ± 0.19
Plants inoculated with R. sesbani and fortified with urea.
7.92 (S)
± 0.04
3.97 ± 0.06 (S)
11.89 (HS)
± 0.09
Plants inoculated with R. sesbani and injected with Azotobacter chroococc um.
7.84 ± 0.04 (S)
3.90 ± 0. 12 (S)
11.74 (S)
± 0.20
Plants inoculated with R. sesbani and injected with Azotobacter vinelandii.
7.84 (S)
± 0.08
11.67 (S)
± 0.18
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
7.87 ± 0.04 (S)
± 0.05
3.83 (S)
3.89 ± 0.12
11.76 ± 0.21
(S)
(S)
S Significant at 5 % level of probability, HS Significant at I % level of probability.
It is possible to suggest that addition of urea as an organic fertilizer in a small doses (0 .02 % WIV) allowed Sesbania plants to developed well without inhibition of nodulation and nitrogen fixation of Sesbania plants as compared to control plants . In addition, data in Table 2 showed that Sesbania plants fortified with urea or injected with A. chroococcum andlor A. vinelandii into the rhizosphere gave significant increases in both total carbohydrates and total proteins of their roots , shoots and nodules as compared to Sesbania plants which inoculated only with R. sesbania. These results are in accord ance with MAZE (1898) , EL-HoSEINY et al. (1979 a) and SINGH et al. (1979) who found that introducing of Azotobacter spp . or addition of urea into the rhizosphere of most plants enhanced growth of these plants. It can be suggested that occuraence of synergism between Azotobacter spp . and Rhizobium sesbani into the rhizos phere of Sesbania seedlings, this stimulation of R. sesbani by Azo tobacter spp. can be interpreted to the ability of Azotobacter spp. to fix atmo spheric nitrogen in free conditions which utilized by Rhizobia. Results in Table 3 man ifested that Sesbania plants injected with A. chroococcum and/or A. vinelandii into the rhizosphere exhibited significant increa se in nitrogena se activit y in Sesbania nodule s parallel with both number and dry weight of nodules, whereas add ition of urea as a fertilizer for Sesbania plants into the rhizosphere showed non significant effect on its nitrogenase activity in nodule s. This result proved that mech anism of nitrogen fixation in Sesbania plants controlled by some biological active substance which secretes by Azotoba cter spp . (either added singly or together). Similar results were reported by SMALL! et al. (1957), VOZNYAKOVSKAYA (1963) and SOBIESZCZANSKI (1965) who mentioned that Azotobacter can able to synthesize biologic ally active subs tances which influence seedlings growth. In addition , data in Table 4 indicated that Sesbania plants fortified with urea or treated with Azotobacter spp. into the rhizosphere gave significant increases in both root , shoot lengths, dry weight of roots
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M. S.
EL-GAMAL
Table 2. Total water soluble carbohydrates (TWSC) and total water soluble proteins (TWSP) (mg/g oven dry weight) of Sesbania sesban plants as affected by treatments with A. chroococcum, and/or A. vinelandii "as compared to fortification with urea as fertilizer" into their rhizospheres. Treatment
TWSC
TWSP
Roots
Shoots
Nodules
Roots
Shoots
Nodules
Plants inoculated with R. sesbani only (control)
32.66 ± 0.67
63.66 ± 0.33
53.00 ± 0.58
28.70 ± 0.33
45.66 ± 0.88
46.30 ± 0.67
Plants inoculated with R. sesbani and fortified with urea.
39.66 ± 0.88 (S)
79.30 ± 0.67 (HS)
62.75 ± 0.38 (HS)
35.50 ± 0.28 (HS)
62.50 ± 0.50 (HS)
54.30 ± 0.88 (S)
Plants inoculated with R. sesbani and injected with Azotobacter chroococcum.
37.30 ± 0.33 (S)
70.50 ± 0.64 (S)
56.50 ± 0.28 (S)
32.25 ± 0.63 (S)
52.00 ± 0.58 (S)
60.75 ± 0.63 (HS)
Plants inoculated with R. sesbani and injected with Azotobacter vinelandii
36.30 ± 0.33 (S)
66.66 ± 0.67 (S)
56.00 ± 0.40 (S)
31.66 ± 0.33 (S)
52.30 ± 0.63 (S)
59.00 ± 0.58 (HS)
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
38.00 ± 0.58 (S)
71.30 ± 0.33 (HS)
58.70 ± 0.67 (S)
33.30 ± 0.72 (S)
54.30 ± 0.33 (S)
63.66 ± 0.33 (HS)
S Significant at 5 % level of probability, HS Significant at I % level of probability.
Table 3. Effect of adding A. chroococcum and/or A. vinelandii into the rhizosphere of Sesbania sesban plants "in comparison to fortification with urea as fertilizer" on nodulation and dinitrogen fixation in Sesbania sesban plants. Treatment
Number of nodules (per plant)
Dry weight of nodules (mg/plant)
Nitrogenase Activity ([1 moles CZH 4/g (ug Nz fixed/g dry nodules/h) dry nodules/h)
Plants inoculated with R. sesbani only (control).
11.30 ± 0.67
18.66 ± 0.88
243.00 ± 1.52
102.06
Plants inoculated with R. sesbani and fortified with urea.
14.66 ± 0.33 (S)
21.30 ± 0.67 (NS)
251.30 ± 1.34 (NS)
105.54
Plants inoculated with R. sesbani and injected with A. chroococcum.
17.00 ± 0.58 (S)
23.66 ± 0.67 (S)
277.30 ± 2.73 (HS)
116.47
Plants inoculated with R. sesbani and injected with A. vinelandii.
16.30 ± 0.88 (S)
23.30 ± 0.33 (S)
260.00 ± 2.65 (S)
109.20
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
18.00 ± 0.71 (S)
24.00 ± 0.41 (S)
289.30 ± 2.34 (HS)
121.50
S Significant at 5 % level of probability, HS Significant at I % level of probability, NS Non Significant.
and shoots. Such results showed that Azotobacter spp. produce some growth substances which increased cell elongation & cell division such as gibberellins and indo1es (VANCURA and MACURA 1960; VANCURA 1961 and ELWAN et a1. 1972). Therefore, it cannot be neglected the
117
Interactions between Azotoba cter spp . and Rhizobium sesbani
Table 4. Growth of roots and shoots in Sesbania sesban plants as affected by introdu cing A. chroococcum and/ or A. vinelandii into the rhizosphere of Sesbania sesban plants " in comparison to fortification with urea as ferti lizer" . Dry weight of roo ts (rng/plant)
Shoot length (ern)
Dry weight of shoots mg/plan t)
Treatment
Root length (cm)
Plants inoculated with R. sesbani only (control).
13.50
± 0.64
68 .25
± 0.85
18.75
± 0.48
165.00
± 0.91
Plants inoculated with R. sesbani and fortified with urea.
17.75 (S)
± 0 .75
75 .00 (S)
± 0.91
22 .30 (S)
± 0.75
175.30 (HS)
± 0.63
Plants inoculated with R. sesbani and injected with A. chroo coccum .
16.50 (S)
± 0.29
72 .30 (S)
± 0 .48
22 .00 (S)
± 0 .71
169.50 (S)
± 0 .67
Plants inoculated with R. sesbani and injecte d with A. vinelandii.
16.00 (S)
± 0.4 1
72. 50 (S)
± 0 .65
21 .50 (S)
± 0.65
170.30 (S)
± 0.48
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandi i.
16.50 (S)
± 0.65
73.00 (S)
± 0.41
22.25 (S)
± 0 .69
170.50 (S)
± 0.65
S Signifi cant at 5 % level of probabi lity, HS Signi ficant at I % level of probability.
physiological role of Azotobacter spp. "as a multipurpose function" into the rhizosphere of leguminous plants which acts as a biofertilizer as well as its ability to synthesize plant hormones which stimulate plant growth and development.
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(1963), 168. Authors address: M. S. EL-GAMAL, Botany Department, Faculty of Science, Al-Azhar University, Cairo, Egypt.
Interactions between Azotobacter spp. and Rhizobium sesbani into the Rhizosphere of Sesbania sesban (L.) MERRILL Plants and its Efficiency on Growth and Symbiontic Nitrogen Fixation M. S. EL-GAMAL Key words : Azotobacter , Rhizobium sesba ni, rhizosphere, symbiontic nitrogen fixation
Summary The present work was made to study the effect of adding Azotobacter spp .; A. chroococcum and/or A. vinelandii into the rhizosphere of Sesbania sesban seedlings which inoculated with R. sesbani as well as the addition of urea into rhizosphere of Sesbania plants (inoculated with R. sesbani); on growth , nodulation and dinitrogen fixation in Sesbania plants in comparison with the same plant inoculated only with R. sesbani (controlled plants). Results indicated that Sesbania plants fortified with urea or inoculated with A. chroococc um and/or A. vinelandii into the rhizosphere gave significant increases in both chlorophyll contents, total soluble carbohydrates, total soluble proteins, root, shoot lengths, dry weight of roots and shoots. Moreover, stimulation was also observed in nitrogenase activity in Sesbania plants treated with A. chroococcum and/or A. vinelandii into the rhizosphere, while no change appear in nitrogenase activity in Sesbania plants suplemented with urea as organic fertilizer. Therefore , it can be suggested that the association between Azoto bacter spp . and Rhizobium sesbani into the rhizosphere of Sesba nia plants enhanced the development of Sesbania growth in the above traits.
Zusammenfassung Die vorliegende Arbeit wurde durchgefuhrt, urn die Wirkung einer Zusatzbeimpfung mit Azotobacte r spp . , A. chroococcum und/oder A. vinelandii in die Rhizosphare von Sesbania sesban- Sam lingen , welche schon mit Rhizobium sesb ani beimpft waren als auch bei Zugabe von Hamstoff in die Rhizosphare zu ermitteln. Wachstum , Nodulation und Dinitrogen-Fixierung in Sesbania-Pflanzen wurden ermittelt. Als Kontrolle dienten die nur mit R. sesbani beimpften Pflanzen. Die Ergebnisse zeigen, daBdie Sesbania-Pflanzen , die mit Hamstoff oder mit A . chroococcum und/oder mit A. vinelandii zusatzlich beimpft waren, einen signifikanten Anstieg im Chlorophyllgehalt, von total gelosten Kohlenhydraten und Proteinen sowie Wurzel- und Schotilange und -trockengewicht aufwiesen. Bei Zusatzbeimpfung mit A. chroococcum und/oder A . vinelandii wurde weiterhin eine Stimulation der NitrogenaseAktivitat beobachtet, wahrend dies bei Harnstoff- oder organischer Dlingung nicht eintrat. Daher kann festgestellt werden, daf die Assoziation zwischen Azotobacter spp. und Rhizobium sesbani in der Rhizosphare das Wachstum von Sesban ia-P flanzen erhoht.
Introduction
Recently much work has been done on the significance of Azotobacter as a biological nitrogen fertilizer for improving the growth of different species of plant crops (FEDOROV1940 ; FEDOROV and NEPOMlLOVA 1954 ; GALIMOVA 1960; SMIRNOVA 1960; POCHON and CHALVIGNAC 1964). Concerning the importance of diazotrophs bacteria in the rhizosphere, several workers showed that association of Rhizobium sp. with a suitable strain of Azotobacter sp.
Interactions between Azotobacter spp. and Rhizobium sesbani
113
(A. chroococcum or A. vinelandii) increased root nodules of legumes e.g. Glycine max, Vigna radiata, Cicer arietinum, Pisum sativum and Trifolium repens (RAVAT and SANORIA 1976; JAUHRI et al. 1979; BURNS et al. 1981; KOTHARI et al. 1986). In addition, EL-HoSEINY (1979 a, b) indicated that introducing of Azotobacter chroococcum into the rhizosphere of both Zeay mays and Hordeum vulgare plants stimulated growth of these plants. However, APTE et al. (1971); SINGH et al. (1979); KOTHARI et al. (1986); and PATIL et al. (1987) reported that other microorganisms such as Beijerinckia, Bacillus, Spirillospora, Azospirillum brasilense enhanced nodulation and dry yield of some leguminous plants. On the contrary, JENSEN (1942) and RAO et a!. (1976) mentionedthat associationbetween Azotobacterand Rhizobiahad no effect on
nodulation of some leguminous plants. On the other hand, many investigators found that mineral nitrogen fertilizers stimulated nodulation and nitrogen fixation in leguminous plants at low doses of nitrogen (10-100 p.p.m.), above this concentration nodules formation was suppressed while plants developed well (RICHARDSON et a!., 1957; KOLOsovA, 1965; VINCENT, 1970; EL-GAMAL, 1985), SINGH and SUBBA RAo (1979) noticedthat additionof urea to leguminousplants at levels (0-80 kg N/ha) stimulated nodules formation and dry yields of its. The present paper has been carried out to study the effect of introducing viable cells of Azotobacter spp. ,. (i.e., A. chroococcum and A. vinelandii, either adding singly or together)into the rhizosphereof Sesbania sesban seedlings which inoculated with R. sesbani with the aim of studying their efficiency on growth, nodulation and dinitrogen fixation of the treated plants as well as the same plants fortified with urea as organic fertilizer and inoculated with R. sesbani in comparison to Sesbania plants which inoculated only with R. sesbani (controlled plants).
Material and Methods Microorganisms and Treatments A pure culture of R. sesbani was isolated from root nodules of Sesbania sesban and identified by ELWAN et al. (1983). Sesbania sesban seeds were grown in pots (2 plants/Pot) containing sterile sandy soil. The moisture content of each pot was kept at 30 % water holding capacity. The pots were inoculated with Rhizobium sesbani. Each pot was supplied with equal amounts of nitrogen free nutrient solution (modified from THORNTON, 1930 according to VINCENT, 1970), it has the following constituents; 2 g Ca3 (P04 h ; 0.5 g K 2HP04 ; 0.2 g MgS04 ' 7H 20; O.I g NaCI in I litre of distilled water. Irrigation was supplied with equal amounts of tap water per pot at intervals of five days. For setting up the experiment, five sets of pots were used; the first set (4 pots) represents control group tSesbania plants inoculated with R. sesbani only). The second set (4 pots) represents Sesbania plants inoculated with R. sesbani and supplemented with urea into the rhizosphere (at concentration 0.2 giL = 93 p.p.m. N) which allowed nitrogenase activity of R. sesbani. The third set (4 pots) represents sesbania plants inoculated with R. sesbani and injected with standard inoculum of Azotobacter chroococcum. The fourth set (4 pots) represents Sesbania plants inoculated with R. sesbani and injected with standard inoculum of Azotobacter vinelandii. The fifth set (4 pots) represents Sesbania plants inoculated with R. sesbani and injected with standard inocula of both A. chroococcum and A. vinelandii together. Rhizobium inoculum: A standard inoculum of R. sesbani was prepared by growing the organism on yeast extract mannitol agar medium for 3 days at 30°C. The microorganisms were harvested and suspended in sterile tap water to prepare a standard bacterial suspension containing about I x 106 cells per ml. It was inoculated in sterile sand cultures at five days after sowing Sesbania seeds (in all treatments including control) at a dose 5 ml/pot. Azotobacter inoculum: A. chroococcum and A. vinelandii were kindly provided from Agriculture Research center, Giza. Egypt. Standard bacterial inocula were prepared by growing each species singly on nitrogen-free agar medium (PHILIPP et aI., 1981) at 30°C for 3 days. Bacterial growth was harvested and suspended in sterile tap water to obtain suspension containing about I X 106 cell per ml for every species. Sesbania sesban seedlings of fifteen days old were inoculated into the rhizosphere with a standard viable inoculum of each Azotobacter sp. or both of them together. The plants were harvested after 45 days from sowing seeds, then readings & measurements were carried out.
114
M. S. EL-GAMAL
Growth criteria a) Preparation of sample for Chlorophyll estimation Determination of chlorophyll "a", "b" and total chlorophyll contents were carried out according to VERNON et al. (1966). One gram of fresh leaves was cut into small pieces with a scissor, chlorophylls were extracted by grinding the tissues in an electrical blender for 5 minutes in 100 ml of 80 % acetone. The homogenate was filtered through a Whatman NO I filter paper and the filterate was completed into 100 ml with 80% acetone. The optical density of the extract was measured using spectroco!ourimeter at 649 and 665!lm for chlorophyll a & b respectively. The chlorophyll concentration was calculated using the following equations. Chlorophyll (a) mg/g tissue = 11.63 (A665) - 2.39 (A649). Chlorophyll (b) mg/g tissue = 20.11 (A649) - 5.18 (A665). Total chlorophyll (a + b) mg/g tissue = 6.45 (A665) + 17.72 (A649).
b) Preparation of sample for determination of Total water soluble Carbohydrates (mg/g oven dry weight) Total soluble carbohydrates of roots, shoots and nodules of Sesbania sesban plants were carried out using the anthrone technique according to UMBREIT et al. (1959). The plant tissues (roots, shoots or nodules) were dried at 65°C till a constant dry weights were obtained, then ground to a fine powder. One g of the powder was transferred into a 100 ml capacity conical flask, 5 m! of 2 % phenol water and 10 ml of 30 % trichloroacetic acid were added. The mixture was shaken and Kept over night before being filtered, the filterate was made up to 50 ml with distilled water. Ten ml of the filtrate of each sample were mixed with 2 gm of activated charcoal and shaken well for 15 minutes and then filterated. The clear filtrate was diluted quantitatively with dist water. 3 ml of the diluted filterate were transferred to a pyrex test tube of 60 ml capacity, to which 6 ml of freshly prepared anthrone reagent (2 g of anthrone/L of 95 % sulphuric acid) were added. The contents of the tube were thoroughly mixed by swiriling. The tube was then placed in a boiling water bath for 3 minutes & then left to cool. The developed colour was measured using colourimeter at 620 urn filter. A blank mixture containing dist. water and reagent was also measured under the same condition to set up the apparatus at zero optical density. A standard curve was constructed (using different concentrations of sucrose) from which amount of total soluble carbohydrates were determined.
c) Preparation of sample for determination of "Total soluble proteins" (mg/g oven dry weight) Plant tissues (roots, shoots or nodules) of Sesbania sesban plants were dried well at 65°C till a constant dry weights, then ground to a fine powder. One gram of the powder was transferred into 100 m! capacity conical 5 ml of 2 % phenol water and 10 ml dist. water were added. The mixture was shaken and kept overnight before being filtered. The filterate was completed to 50 ml by dist. water. Total water soluble proteins were determined by using the method of LOWRY et al. (1951), using bovine serum albumen as a standard protein.
d) Preparation of Sample for evaluating nitrogenase activity in Sesban sesban nodules The acetylene reduction technique was applied for estimating amount of nitrogen fixed (ug/g dry nodules/h) according to the method of DlLOWORTH (1970). The fresh roots of Sesbania sesban with attached nodules were washed thoroughly and transfered to 420 ml serum bottles, then sealed with tide rupper stoppers. The appropriate volume of acetylene was injected into the bottles using plastic syring to give acetylene concentration of 10% of the atmosphere. The bottles were incubated at 30°C for I h. Then, gas samples were withdrawn and assayed for ethylene concentration using Fishere gas liquid chromatograph. Standard curve for pure ethylene was carried out. Thus !l moles CzRJgm dry nodules/hr X 0.42 = ug nitrogen fixed/g dry nodules/h were estimated. After determination of Nitrogenase activity in nodules, then Number of nodules/plant were counted in a fresh state and nodules were dried in electric oven at 60°C for several times till a constant dry weight of nodules were determined (mg/plant).
e) Preparation of Samples for measuring Roots & shoots lengths (em) and determination of Dry weight of Roots & shoots (mg/plant): Separation of roots and soils The whole content of pots were gently removed, placed a sieve net work and feeble current of water was applied on the plant to remove sand particles without affecting nodules of the root system, counting number of nodules per plant, separation of the root system from the shoot system and then measuring the length for roots & shoots, drying of both roots & shoots in oven at 105°C for 2 days, until a constant dry weight was obtained. The T-test was used in statistical analysis to determine the significancy between different treatments and control.
Interactions between Azotobacter spp . and Rhi zobium sesbani
115
Results and Discussion Results in Table 1 indicated that Sesbania plants supplemented with 0 .2 g/L of Urea or injected with A. chrooco ccum and/or A. vinelandii had more or less, the same amount of both chlorophyll "a" , "b" and total chlorophyll of Sesbania leaves, whereas all treatments caused significant increa ses in the above traits in comparison to Sesbania plants inoculated only with R. sesbania (control). Table I. Chlorophylls "a". "b" and total chlorophyll (mglg) of the leaves of Sesbania sesban plants as affected by presence of urea ; Azotobacter chroococcum, andlor Azotobacter vinelandii in its rhizosphere . Treatment
Chlorophyll contents (mglg fresh weight) Chlorophyll "a "
Chlorophyll "b"
Total chlorophyll
Plants inoculated with R. sesbani only (Control)
7.42 ± 0.12
3.13 ± 0.19
10.55 ± 0.19
Plants inoculated with R. sesbani and fortified with urea.
7.92 (S)
± 0.04
3.97 ± 0.06 (S)
11.89 (HS)
± 0.09
Plants inoculated with R. sesbani and injected with Azotobacter chroococc um.
7.84 ± 0.04 (S)
3.90 ± 0. 12 (S)
11.74 (S)
± 0.20
Plants inoculated with R. sesbani and injected with Azotobacter vinelandii.
7.84 (S)
± 0.08
11.67 (S)
± 0.18
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
7.87 ± 0.04 (S)
± 0.05
3.83 (S)
3.89 ± 0.12
11.76 ± 0.21
(S)
(S)
S Significant at 5 % level of probability, HS Significant at I % level of probability.
It is possible to suggest that addition of urea as an organic fertilizer in a small doses (0 .02 % WIV) allowed Sesbania plants to developed well without inhibition of nodulation and nitrogen fixation of Sesbania plants as compared to control plants . In addition, data in Table 2 showed that Sesbania plants fortified with urea or injected with A. chroococcum andlor A. vinelandii into the rhizosphere gave significant increases in both total carbohydrates and total proteins of their roots , shoots and nodules as compared to Sesbania plants which inoculated only with R. sesbania. These results are in accord ance with MAZE (1898) , EL-HoSEINY et al. (1979 a) and SINGH et al. (1979) who found that introducing of Azotobacter spp . or addition of urea into the rhizosphere of most plants enhanced growth of these plants. It can be suggested that occuraence of synergism between Azotobacter spp . and Rhizobium sesbani into the rhizos phere of Sesbania seedlings, this stimulation of R. sesbani by Azo tobacter spp. can be interpreted to the ability of Azotobacter spp. to fix atmo spheric nitrogen in free conditions which utilized by Rhizobia. Results in Table 3 man ifested that Sesbania plants injected with A. chroococcum and/or A. vinelandii into the rhizosphere exhibited significant increa se in nitrogena se activit y in Sesbania nodule s parallel with both number and dry weight of nodules, whereas add ition of urea as a fertilizer for Sesbania plants into the rhizosphere showed non significant effect on its nitrogenase activity in nodule s. This result proved that mech anism of nitrogen fixation in Sesbania plants controlled by some biological active substance which secretes by Azotoba cter spp . (either added singly or together). Similar results were reported by SMALL! et al. (1957), VOZNYAKOVSKAYA (1963) and SOBIESZCZANSKI (1965) who mentioned that Azotobacter can able to synthesize biologic ally active subs tances which influence seedlings growth. In addition , data in Table 4 indicated that Sesbania plants fortified with urea or treated with Azotobacter spp. into the rhizosphere gave significant increases in both root , shoot lengths, dry weight of roots
116
M. S.
EL-GAMAL
Table 2. Total water soluble carbohydrates (TWSC) and total water soluble proteins (TWSP) (mg/g oven dry weight) of Sesbania sesban plants as affected by treatments with A. chroococcum, and/or A. vinelandii "as compared to fortification with urea as fertilizer" into their rhizospheres. Treatment
TWSC
TWSP
Roots
Shoots
Nodules
Roots
Shoots
Nodules
Plants inoculated with R. sesbani only (control)
32.66 ± 0.67
63.66 ± 0.33
53.00 ± 0.58
28.70 ± 0.33
45.66 ± 0.88
46.30 ± 0.67
Plants inoculated with R. sesbani and fortified with urea.
39.66 ± 0.88 (S)
79.30 ± 0.67 (HS)
62.75 ± 0.38 (HS)
35.50 ± 0.28 (HS)
62.50 ± 0.50 (HS)
54.30 ± 0.88 (S)
Plants inoculated with R. sesbani and injected with Azotobacter chroococcum.
37.30 ± 0.33 (S)
70.50 ± 0.64 (S)
56.50 ± 0.28 (S)
32.25 ± 0.63 (S)
52.00 ± 0.58 (S)
60.75 ± 0.63 (HS)
Plants inoculated with R. sesbani and injected with Azotobacter vinelandii
36.30 ± 0.33 (S)
66.66 ± 0.67 (S)
56.00 ± 0.40 (S)
31.66 ± 0.33 (S)
52.30 ± 0.63 (S)
59.00 ± 0.58 (HS)
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
38.00 ± 0.58 (S)
71.30 ± 0.33 (HS)
58.70 ± 0.67 (S)
33.30 ± 0.72 (S)
54.30 ± 0.33 (S)
63.66 ± 0.33 (HS)
S Significant at 5 % level of probability, HS Significant at I % level of probability.
Table 3. Effect of adding A. chroococcum and/or A. vinelandii into the rhizosphere of Sesbania sesban plants "in comparison to fortification with urea as fertilizer" on nodulation and dinitrogen fixation in Sesbania sesban plants. Treatment
Number of nodules (per plant)
Dry weight of nodules (mg/plant)
Nitrogenase Activity ([1 moles CZH 4/g (ug Nz fixed/g dry nodules/h) dry nodules/h)
Plants inoculated with R. sesbani only (control).
11.30 ± 0.67
18.66 ± 0.88
243.00 ± 1.52
102.06
Plants inoculated with R. sesbani and fortified with urea.
14.66 ± 0.33 (S)
21.30 ± 0.67 (NS)
251.30 ± 1.34 (NS)
105.54
Plants inoculated with R. sesbani and injected with A. chroococcum.
17.00 ± 0.58 (S)
23.66 ± 0.67 (S)
277.30 ± 2.73 (HS)
116.47
Plants inoculated with R. sesbani and injected with A. vinelandii.
16.30 ± 0.88 (S)
23.30 ± 0.33 (S)
260.00 ± 2.65 (S)
109.20
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandii.
18.00 ± 0.71 (S)
24.00 ± 0.41 (S)
289.30 ± 2.34 (HS)
121.50
S Significant at 5 % level of probability, HS Significant at I % level of probability, NS Non Significant.
and shoots. Such results showed that Azotobacter spp. produce some growth substances which increased cell elongation & cell division such as gibberellins and indo1es (VANCURA and MACURA 1960; VANCURA 1961 and ELWAN et a1. 1972). Therefore, it cannot be neglected the
117
Interactions between Azotoba cter spp . and Rhizobium sesbani
Table 4. Growth of roots and shoots in Sesbania sesban plants as affected by introdu cing A. chroococcum and/ or A. vinelandii into the rhizosphere of Sesbania sesban plants " in comparison to fortification with urea as ferti lizer" . Dry weight of roo ts (rng/plant)
Shoot length (ern)
Dry weight of shoots mg/plan t)
Treatment
Root length (cm)
Plants inoculated with R. sesbani only (control).
13.50
± 0.64
68 .25
± 0.85
18.75
± 0.48
165.00
± 0.91
Plants inoculated with R. sesbani and fortified with urea.
17.75 (S)
± 0 .75
75 .00 (S)
± 0.91
22 .30 (S)
± 0.75
175.30 (HS)
± 0.63
Plants inoculated with R. sesbani and injected with A. chroo coccum .
16.50 (S)
± 0.29
72 .30 (S)
± 0 .48
22 .00 (S)
± 0 .71
169.50 (S)
± 0 .67
Plants inoculated with R. sesbani and injecte d with A. vinelandii.
16.00 (S)
± 0.4 1
72. 50 (S)
± 0 .65
21 .50 (S)
± 0.65
170.30 (S)
± 0.48
Plants inoculated with R. sesbani and injected with both A. chroococcum and A. vinelandi i.
16.50 (S)
± 0.65
73.00 (S)
± 0.41
22.25 (S)
± 0 .69
170.50 (S)
± 0.65
S Signifi cant at 5 % level of probabi lity, HS Signi ficant at I % level of probability.
physiological role of Azotobacter spp. "as a multipurpose function" into the rhizosphere of leguminous plants which acts as a biofertilizer as well as its ability to synthesize plant hormones which stimulate plant growth and development.
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(1963), 168. Authors address: M. S. EL-GAMAL, Botany Department, Faculty of Science, Al-Azhar University, Cairo, Egypt.