Biol Fertil Soils (1997) 24:123-128 © Springer-Verlag 1997

T.K. Ghosh • K.C. Saha

Effects of inoculation of cyanobacteria on nitrogen status and nutrition of rice (Oryza satire L.) in an Entisol amended with chemical and organic sources of nitrogen

Received: 8 June 1994

Abstract A field experiment was conducted with wetland rice (Oryza sativa cv. 1R-36) in a sandy clay loam soil (Entisol) to study the effect of inoculation with a soil- based mixed culture of four diazotrophic cyanobacteria, Aulosira fertilissima, Nostoc muscorum, N. commune and Anabaena spp., on the N-flux in inorganic (NH2+NO3+ NO[), easily oxidizable, hydrolysable and non-hydrolys- able forms of N in soil during vegetative growth periods of the crop. Effects on grain and straw yield and N uptake by the crop were estimated. The effects of applying urea N and N as organic sources, viz. Sesbania aculeata, Neem (Azardirachta indica) cake and FYM, each at the rate of 40 kg N ha -1, to the soil were also evaluated. Inoculation significantly increased the release of inorganic N, evi- denced by its increased concentrations either in soil or in soil solution. However, such increases rarely exceeded even 4% of total N gained in different froms in the soil system by inoculation during the vegetative growth stages of the rice plant, when the nutritional requirement of the plants is at a maximum. Most of the N2 fixed by cyano- bacteria remained in the soil as the hydrolysable form (about 85%) during this period. Inoculation caused an in- significant increase in grain (8%) and straw (11%) yield, which was, however, accompanied by a significant in- crease in N uptake by the grain (30%) and an increase in total uptake of 15.3 kg N h a 1. Such beneficial effects of inoculation varied in magnitude with the application of or- ganic sources, with farmyard manure (FYM) being the most effective. Application of urea N, on the other hand, markedly reduced such an effect.

Key words Cyanobacteria • Soil inoculation • N-transformation. Inorganic N - Easily oxidizable N • Hydrolysable N - Non-hydrolysable N • Wetland rice • Farmyard manure. Oryza sativa

T.K. Ghosh • K.C. Saha ( 9 ) Department of Agricultural Chemistry and Soil Science, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-74t252, West Bengal, India

Introduction

Much interest has been generated over many years in the tropics on the improvement of the fertility status of rice soils to sustain rice yields by utilizing diazotrophic cyano- bacteria as a biological input (De 1939; De and Sulaiman 1950; Venkataraman 1972; Singh and Bisoyi 1989). Cya- nobacteria give a considerable build-up of N fertility in rice soils (Roger and Kulasooriya 1980; Saha and Mandal 1980; Roger and Reynaud 1982), but their inoculation has failed to increase rice yields consistently (Watanabe 1986; Roger et al. 1993). The reasons of this are not well under- stood. Nitrogen fixed by cyanobacteria may become avail- able to rice plants only after its release into the surround- ings, either as extracellular products and/or on mineraliza- tion of their intracellular contents. Release of N from rapid decomposition of fresh or dry cyanobacterial mass incor- porated into the soil has also been reported (Saha et al. 1982; Tirol et al. 1982; Mian and Stewart 1985); but N2 fixation by cyanobacteria vis-a-vis its release in the soil- water system may be more useful for crop production dur- ing the vegetative growth stages of rice than at later stages (Ghosh and Saha 1993; Roger et al. 1993). Quantitative information regarding transfer of cyanobacterial nitrogen to a different N-pool of soil during the vegetative growth period of rice is lacking.

In the present investigation, we aimed to study the ef- fects of cyanobacterial inoculation in association with chemical and organic N sources on the N-flux in different forms of soil N during the vegetative growth periods of rice and on the nutrition and yield of the crop.

Materials and methods

Inoculum

The open-air soil-based dry inocula containing a mixture of Aulosira fertilissima, Nostoc muscorum, Nostoc commune and Anabaena spp. were prepared as described by Roger and Kulasooriya (1980). These strains were selected because of their high N2-fixing ability (Ghosh and Saha 1993). The inocula contained about 11 xl 0 ° colony-forming

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units (CFUs) of N2-fixing cyanobacteria (agarized N-free medium) per gram dry weight, of which inoculated strains contributed about 82% (A. fertilissima 23%; N. muscorum 30%; N. commune 10% and Anabaena spp. 19%), the rest being indigenous in the soil. The inocu- la were applied at the rate of 10 kg ha -1 in the experimental plots.

Yield and N uptake

The crop was harvested at maturity and the grain and straw yields were recorded from each plot. Samples of grain and straw were ana- lysed for total N (Kjeldahl) content.

Soil of experimental site

The soil was a sandy claly loam (Entisol) and had 40×102 CFUs indi- genous cyanobacteria g- soil, containing predominantly species of the genera Nostoc and Cylindrospermum, identified according to Desi- kachary (1959). The other relevant characteristics of the soil were a pH of 8.3 (soil:H20 1:2.5); organic C 0.98% (Wet digestion); NH~-N 4.5 mg kg-1; NO~-N 0.8 ms kg-1; hydrolysable N 856 mg kg-1; non- hydrolysable N 385 nag kg-~; total N 0.12% (Kjeldahl); 1N ammonium acetate extractable Ca 2+ 20.7 cmol (p+) kg -1 and Mg 2+ 2.4 cmol (p+) kg 1; and Olsen available P 3.1 mg kg -1. All the physicochemical char- acteristics of the soil were analysed according to Jackson (1973) and Bremner (1965).

Experimental design

A field experiment was laid out with wetland rice (cv. IR-36) and with a plot size of 5×5 m following a randomized block design at the Viswavidyalaya Farm (23030 ' N, 89°E and 9.75 m altitude). The treatments used were: (1) control (no N) 40 kg N h a I as (2) urea; (3) Sesbania acuIeata L. twigs; (4) Neem (Azardirachta indica) cake; or (5) farmyard manure (FYM). Each of the treatments was divided into: (a) inoculated (cyanobacterial flakes at 10 kg h a 1) and (b) uninocu- lated (sterilized flasks at 10 kg ha -1) series and replicated 4 times.

All the organic inputs, Sesbania aculeata (3.6% N), Neem cake (3.1% N) and FYM (0.6%N), were incorporated into the soil 5 days prior to transplanting of rice seedlings. All the plots received 30 kg P and 30 kg K ha -1 from single superphosphate and mufiate of potash as basal dressings. Three rice seedlings (3 weeks old) were trans- planted into each hill, maintaining row-to-row and hill-to-hill dis- tances of 20 cm and 15 cm, respectively. Cyanobacterial inoculation was performed at the rate of 10 kg flakes ha -1 after 7 days of trans- planting in the form of a top dressing. The plots were kept sub- merged by maintaining a water depth of approximately 3_+0.5 cm on the soiI surface up to the maturity of the crop.

Collection and analysis of soil samples

Representative soil samples (0-15 cm) from each of the plots were ta- ken at 30, 60 and 90 days after transplanting (DAT) with the help of a soil sampler and were analysed for (1) inorganic N (NH~+NO~+ NO~) fraction by extraction with 2 N KC1 (Bremner and Keeney 1966); (2) alkaline KMnO4 oxidizable organic N fraction (Sahrawat and Burford 1982) from inorganic N extracted soils; (3) hydrolysable N fraction by extraction with 6 N HC1 (soil:HC1 1:5) from inorganic N extracted soils, and (4) non-hydrolysable N by digesting the resid- ual soil with conc. H2SO4, following the procedure outlined by Brem- her (1965).

Collection and analysis of soil solution

Samples of soil solution were collected, under anaerobic conditions, at 30 and 60 DAT from each plot with the help of peizometers (PateI et al. 1990) installed in the soil at a depth of 20 cm. Previous to the sampling date, a vacuum was created inside the peizometer tubes by a hand-operated pump and samples of soil solution were drawn into 500-ml stoppered bottles by means of suction. A layer of liquid paraf- fin was introduced in the stoppered bottles to protect the samples from direct air contact. The samples were evaporated after acidifica- tion with dilute H2SO4 to reduce the volume and were analysed for N content by alkaline distillation.

Results and discussion

Inorganic N in soil and soil solution

The results (Fig. 1A) show that inorganic N (NH~+NO3+ NO2) content in soil increased due to submergence and at-

tained a m a x i m u m at 90 DAT. Appl ica t ion o f urea and

22

20

18

A ¢ 16

14

12

10

6 -

4

n

m

2 [ ] A ¸

0 30 6'0 9'0

~ 5 B

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4-

5 3'0 6'0 9'0

TL i MTL i PI i

Days after transplatation LS.D. (UI & I) Treatment means Stage means

5 % 2.66 1.46 1% 3.65 1.95

Fig. 1A, B Influence of cyanobacterial inoculation on inorganic N (NH~+NO3+NO2) content in soil. A No inoculation (U/), B inocula- tion (/). • no N, © urea-N at 40 kg ha -1, • Sesbania aculeata-N at 4 0 k g h a -1, [] Neem cake-N at 4 0 k g h a -1, • FYM-N at 40kg ha -1, }--1 SD of the mean of four replicates, bars indicate average inorganic N values, LSD least significant difference, TL tillering, MTL maximum tillering, PI panicle initiation

Table 1 Influence of cyanobac- terial inoculation on inorganic N content in soil solution (N in mg V a +SD of the means of four re- plicates). Figures in parentheses indicate percentage increase (No No N, N4o 40 kg N ha 1, UI un- inoculated, I inoculated, I-UI in- oculation effect, LSD least signif- icant difference, TL tillering, MTL maximum filleting)

N source Inoculation Days after transplantation Mean

30 TL 60MTL

No UI 0.08+0.00 0.19_+0.03 0.14 I 1.02+0.17 0.28_+0.14 0.65 I-UI 0.94 (1175.0) 0.09 (47.4) 0.51 (364.3)

Urea (N4o) UI 0.17_+0.08 0.15_+0.00 0.16 I 0.79-+0.03 0.28-+0.24 0.54 I-UI 0.62 (364.7) 0.13 (86.7) 0.38 (237.5)

S. aculeata (N4o) UI 0.35+0.7 0.15_+0.00 0.25 I 0.50_+0.19 0.15_+0.00 0.33 I-UI 0.15 (42.9) 0.00 0.08 (32.0)

Neem cake (N4o) UI 0.20_+0.09 0.68+0.22 0.44 I 0.35+0.07 0.71-+0.07 0.53 I-UI 0.15 (75.0) 0.03 (4.4) 0.09 (20.5)

FYM (N4o) UI 0.08+0.00 0.30+0.00 0.19 I 0.08_+0.00 0.40-+0.07 0.24 I-UI 0.00 0.10 (33.3) 0.05 (26.3)

Treatment means Stage means

LSD 5% 0.17 0.14 1% 0.23 0.20

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also organic sources (Sesbania aculeata, Neem cake and FYM) significantly increased this form of N in the soil, with urea giving a more prominent increase than the or- ganic sources at the early stages of crop growth. This clearly depicts the more rapid release of inorganic N from urea to soil than from organic sources.

Inoculation of cyanobacteria (Fig. 1B) increased the in- organic N content in soil marginally (4.8%) at 30 DAT, sig- nificantly (55.8%) at 60 DAT and with a decline thereafter, resulting in an average increase of 2.14 nag N kg -I soil over its uninoculated counterpart. Of the organic inputs, in- oculation in the presence of FYM also significantly in- creased the soil content of this form of N at both 30 (82%) and 60 (35%) DAT, with an average increase of 2.35 mg N kg -1 soil, and performed better than the other two sources, viz. S. aculeata and Neem cake. On the other hand, inoculation in urea-N applied plots significantly re- duced the inorganic N content in soil, which was most pro- nounced up to 60 DAT. However, at 90 DAT there was a trend towards an improvement in the content of this form of N by inoculation in this treatment.

Inorganic N content in soil solution was also increased by inoculation in most of the treatments (Table 1), but the proportion of increase differed from that of the soils in re- spect of DAT. Inoculation of cyanobacteria alone signifi- cantly increased inorganic N content in soil solution at 30 DAT (1175%), followed by a relatively small increase at 60 DAT (47%), which cumulatively produced an aver- age N gain of 0.51 mg N 1-1 soil solution compared with uninoculated controls. A similar trend was observed with inoculation in urea-N-treated plots, where the correspond- ing figures were 365% and 87%, depicting a completely opposite trend to the depression of inorganic N content in soil under the same treatment (Fig. 1B). Inoculation in the presence of all the organic sources failed to increase sig-

nificantly this form of N in soil solution at both 30 DAT and 60 DAT. However, with the incorporation of Neem cake and FYM, there was a considerable increase in soil solution N content at 60 DAT as compared to 30 DAT in both the uninoculated and inoculated series.

The increase in inorganic N in soil and soil solution due to inoculation indicated a release of fixed N of cyano- bacterial cells. However, the variation in the magnitude of the increase at different periods might be the result of many interacting processes including mineralization-immo- bilization and losses through various means. Immobiliza- tion of the rapid hydrolysis product of urea by growing cyanobacterial cells and hence suppression of their Nz-fix- ing activity might be the cause of the reduction in inorgan- ic N content in soil due to inoculation in urea-N-treated plots. However, such a negative effect of inoculation be- came of less importance later with the depletion of hydro- lysis products of urea in the system. Vlek and Craswell (1979) reported a net immobilization of 18-30% of N from urea fertilizer by growing algal cells within 3 weeks after application. Possibly prevention of loss due to immo- bilization by inoculated organism and the release of immo- bilized N in turn led to the reverse trends in inorganic N accumulation in soil and soil solution under the urea-N-ap- plied conditions. The wide C:N ratio of the organic sources added and variations in the mineralization rates and release of products may possibly be related to the dif- ferential contribution of cyanobacterial inoculation in their presence in increasing the inorganic N contents in soil and soil solution at different stages. However, the changes in different organic N fraction contents in the soil due to in- oculation may also explain the variation in inorganic N ac- cumulation.

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Table 2 Influence of cyanobac- terial inocuiation on loosely bound or easily oxidizable organ- ic N fraction in soil (N in mg kgq). For explanation of ab- breviations see Table 1

N source Inoculation Days after transplantation Mean

30 TL 60MTL

N o UI 96.6-+6.2 97.3_+14.7 96.9 I 102.4_+ 11.8 105.8+3.4 104.1 I-UI 5.8 (6.0) 8.6 (8.8) 7.2 (7.4)

Urea (N4o) UI 119.6+13.8 114.2_+10.5 116.9 I 110.8_+15.1 116.2_+18.2 113.5 I-UI -8.8 (-7.3) 2.0 (1.7) -3.4 (-2.9)

S. aculeata (N4o) UI 98.0-+8.1 113.2_+6.3 105.6 I 114.6-+21.0 119.6_+1.0 117.1 I-UI 16.6 (16.9) 6.4 (5.7) 11.5 (10.9)

Neem cake (N4o) UI 96.1_+5.9 107.7_+6.8 101.9 I 111.3_+15.3 114.6_+9.1 113.0 I-UI 15.2 (15.8) 6.9 (6.4) 11.1 (10.8)

FYM (N4o) UI 101.1_+7.5 119.9_+7.3 110.5 I 103.9_+15.2 121.3_+9.5 112.6 I-UI 2.7 (2.7) 1.4 (1.2) 2.1 (1.9)

Treatment means Stage means

LSD 5% NS NS

Easily oxidizable organic N fraction in soil

The results (Table 2) show that inoculation of cyanobacter- ia, in general, increased the easily oxidizable organic N fraction in the soil except in urea-N-treated plots at 30 DAT, where the content was reduced from its uninocu- lated control value, resembling a similar negative effect of inoculation on inorganic N content in soil under the same treatments (Fig. 1B). However, the magnitude of the in- crease or decrease of this fraction of N in soil due to in- oculation was insignificant in all the treatments. Compara- tively, the effect of inoculation on increasing the easily oxidizable N fraction in soil, on average, was prominent with S. aculeata (10.9%) and Neem cake (10.8%) and negligible with FYM (1.9%), and was slightly differently associated with the trend toward increasing inorganic N content in the soil under the same three treatments (Fig. 1B). ¢The influence of the organic sources or their de- composition products on cyanobacterial N2 fixation and its release in soil, in addition to the transitional nature of the easily oxidizable N fraction in the N transformation pro- cess in soil, might be responsible for this phenomenon.

Hydrolysable and non-hydrolysable N in soil

The hydrolysable N content of uninoculated soils (Table 3) was significantly higher in all the treatments at 60 DAT as compared to 30 DAT, indicating a positive influx from other N fractions, including contributions from the indi- genous cyanobacteria. On average, urea-N supported the highest concentration of this form of N in soil, closely fol- lowed by FYM.

Inoculation of cyanobacteria increased hydrolysable-N content in the soil by about 5% and 14% at 30 and 60 DAT, respectively, with an average value of about

74 mg N kg q soil. A positive N flux in this form of N was also observed in the presence of all the three organic sources, with varied efficiency of inoculation. This may be explained by the increase in undecomposed intracellular N of cyanobacteria resulting from enhanced N2-fixation by the inoculated organisms. In contrast, inoculation in the presence of urea-N resulted in a decrease of this form of N content in soil at an earlier stage followed by a margin- al increase later compared with uninoculated controls, again indicating the suppressive effect of urea-N on cyano- bacterial N2-fixation depends upon concentration.

Non-hydrolysable N content in soil (Table 3) showed a significant decrease at 60 DAT from the values at 30 DAT in all the treatments, indicating transformation of this form of N to other forms. The inoculation effect of cyanobacter- ia was marginal or inconsistent in terms of improvement of this form of N in the soil.

Yields and N uptake

The results (Table 4) show that application of urea-N as well as each of the organic sources, without inoculation, increased more or less significantly (P=0.05) the rice yields and N uptake by the crop, the highest values being obtained with urea. Inoculation of cyanobacteria alone, or in the presence of all other treatments, caused a positive grain yield response compared with the uninoculated con- U'ols, but none of them were significant. A higher effi- ciency of inoculation, however, was obtained with inocula- tion None followed by inoculation in the presence of FYM, where the increase in grain yield was 8% for inocu- lation alone and 7% for inoculation in the presence of FYM. The effects of inoculation on grain yield with S. aculeata, Neem cake and urea-N were comparatively small.

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Table 3 Hydrolysable (HL) and non-hydrolysable (NHL) N content in soil as influenced by the inoculation of cyanobacteria (N in mg kg-l). For explanation of abbreviations see Table 1

N source Inoculation HL-N Mean NHL-N Mean

Days after transplantation Days after transplantation

30 TL 60 MTL 30 TL 60 MTL

No UI 713_+55 815-+28 764 198_+6 154_+32 176 I 750_+ 14 925+42 838 210+20 162_+ 10 186 I-UI 37 (5) 111 (14) 74 (10) 12 (6) 8 (5) 10 (6)

Urea (N4o) UI 758_+137 985_+56 872 212_+35 194_+0 203 I 747_+ 138 1007_+89 877 214_+6 190_+21 202 I-UI 12 (-2) 23 (2) 6 (1) 2 (1) -4 (-2) -1 (-0.4)

S. aculeata (N4o) UI 764+69 934_+14 849 202+56 176+5 189 I 815+73 962+16 888 214_+30 170_+10 192 I-UI 51 (7) 28 (3) 39 (5) 12 (6) -6 (-4) 3 (2)

Neem cake (N4o) UI 747_+111 905_+16 826 224+15 174_+25 199 I 781_+139 1019_+35 900 224+5 178-+15 201 I-UI 34 (5) 113 (13) 74 (9) 0 4 (2) 2 (1)

FYM (N4o) UI 781_+73 95l-+0 866 206-+10 166_+t2 186 I 790_+42 1053_+73 921 209_+ 11 162_+ 12 185 I-UI 9 (1) 102 (11) 56 (6) 3 (1) -4 (-2) -1 (-0.4)

Treatment means Stage means Treatment means Stage means

LSD 5% NS 58 NS 12 1% 79 17

Table 4 Influence of cyanobacterial inoculation on yields and N uptake by grain and straw of rice (cv. IR-36). For explanation of abbrevia- tions see Table 1

N source Inoculation Yield (q ha -l) N uptake (N in kg ha -j)

Grain Straw Grain Straw Total

No UI 35.1-+0.5 45.9-+7.7 35.2+0.5 27.0-+5.6 62.2 I 37.9-+1.9 50.8+1.6 45.7+1.7 31.8+0.4 77.5 I-UI 2.8 (8.0) 4.9 (10.7) 10.5 (30.0) 4.8 (17.6) 15.3 (24.6)

Urea (N4o) gl 39.5-+0.1 58.1+4.9 53.9-+1.6 48.2-+5.9 102.1 I 41.l_+2.1 61.5_+1.4 53.6_+1.9 41.5_+3.4 95.1 I-UI 1.6 (4.1) 3.4 (5.9) -0.3 (-0.6) -6.7 (-14.0) -7.0 (-6.9)

S. aculeata (N4o) UI 39.4_+1.9 57.3-+1.2 48.6_+2.0 42.3-+1.0 90.9 I 41.0_+1.3 59.8_+2.1 53.0-+1.6 49.5_+3.6 102.5 I-UI 1.6 (4.1) 2.5 (4.4) 4.4 (9.1) 7.2 (17.1) 11.6 (12.8)

Neem cake (N4o) U1 38.8-+0.7 57.8_+2.8 46.9_+1.4 39.6-+3.0 86.5 I 39.2_+2.4 59.7_+2.4 48.4_+2.2 40.8+3.8 89.2 I-UI 0.4 (1.0) 1.9 (3.3) 1.5 (3.2) 1.2 (3.0) 2.7 (3.2)

FYM (N40) UI 37.9_+1.7 55.0_+7.3 47.5_+2.8 34.6_+4.8 82.1 I 40.6_+1.5 61.2_+3.5 52.2_+2.3 41.6_+3.1 93.8 I-UI 2.7 (7.1) 6.2 (11.3) 4.7 (10.0) 7.0 (20.2) 11.7 (14.3)

LSD 5% 3.5 8.3 3.8 7.9 1% 4.7 11.6 5.2 10.8

The increase in straw yield due to inoculation showed a similar trend to that of the grain yield.

Cyanobacterial inoculation alone or in the presence of FYM and S. aculeata significantly (P=0.05) increased N uptake by grain, the increases being about 30%, 10% and 9%, accompanied by increases in total N uptake (grain+straw) of about 15.3 kg, 11.8 kg and 11.7 kg ha q ,

respectively, compared with the uninoculated controls. In- cidentally, these three treatments also produced a higher inoculation effect on soil inorganic N content (Fig. 1B). As for grain yield, the effect of inoculation in this respect was much lower (about 2.8 kg N uptake ha -1) with Neem cake. On the other hand, inoculation reduced, although in- significantly, the N uptake by rice in urea-N-treated plots.

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The marginal concurrent increases in grain and straw yield caused by inoculation under the same treatment indicate that cyanobacteria possess other properties in addition to N2 fixation which are partially responsible for increasing rice yields.

The results of this investigation have clearly demon- strated that inoculation of cyanobacteria improves the N fertility of the soil, rice yield and N uptake by the crop. However, in terms of N supply, inoculation benefited the N status of soil nearly 10 times more than that utilized by the crop, indicating transformation of a very small fraction of cyanobacterial fixed N to the plant-available form in the soil system during the vegetative growth period of the crop. Roger and Reynaud (1982) also found that only one- third of field cyanobacteria were decomposed and ab- sorbed by the rice plants in the 1 st year, the rest remaining as residual soil N. Saha and Mandal (1980), however, ob- served that most of the residual N from cyanobacteria re- maining in wet soil after the crop harvest did not persist with air drying of the soil.

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