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Organic Manures in Relation to Rhizosphere Effect III. Effect of Organic Manures on Population of Ammonifying Bacteria and Mineralization of Nitrogen in Rice and Succeeding Wheat Rhizosphere Soils
Zentralbl. Mikrobiol. 141 (1986), 267-277 VEB Gustav Fischer Verlag Jena [From University College of Agriculture, Calcutta University, Calcutta- 700-019, India, and Department of Agricultural Chemistry and Soil Science, Bidhan Chandra Krishi Viswavidyalaya, Kalyani-741-235, India]
Organic Manures in Relation to Rhizosphere Effect III. Effect of Organic Manures on Population of Ammonifying Bacteria and Mineralization of Nitrogen in Rice and Succeeding Wheat Rhizosphere Soils") P.
BHATTACHARYYA, B.
K.
DEY,
S.
NATH,
and S.
BANIK
With 1 Figure
Summary Ammonifying bacteria were stimulated in the rhizosphere of rice with green manuring (control), while depressed with town compost (TC) at early tillering stage. The growth of cellulose decomposers was greatest with ammonium sulphate (AS). Except under control and farm yard manure (FYM), the number increased with time and with the age of the crop at final harvest, the highest was with rice straw (RS) + rock phosphate (RP). At this stage, the abundance of cellulose decomposers was greatest with RS + AS. AS induced the best residual effect on the proliferation of ammonifying bacteria and RS + RP on cellulose decomposers in the rhizosphere of wheat. Green manuring (control) promoted the growth of ammonifying Bacillus, which was further intensified with TC in the rice rhizosphere. Residually Pseudomonas was stimulated by the manures in the wheat rhizosphere. At early tillering stage of rice, mineralization of nitrogen in the rhizosphere decreased with the manures, excepting AS and AS + RP. Barring under RS + RP, the transformation declined with the age of the crop. AS and RP, either alone or together, also provided a favourable condition for nitrogen mineralizat.ion, residually in the rhizosphere of wheat. In this respect, the rhizosphere effect of wheat under the residual influence was superior to that of rice under the direct influence of the manures. The process was positively correlated with the number of ammonifiers, being significantly in the wheat rhizosphere at final harvest.
Zusammenfassung Tm fruhen Entwicklungsstadium des Reises wurden in der Reis-Rhizosphare die Ammonifikanten durch alleinige Grundungung (Kontrolle) stimuliert, durch Stadtkompost (TC) jedoch herabgesetzt. Die Vermehrung der Zellulosezersetzer war am hochsten bei Verabreichung von Ammonsulfat (AS). Au/3er bei Griindiingung und Stalldung (FYM) stiegen die Keimzahlen im Laufe der Zeit und mit dem Alter der Pflanzen an, die hochsten wurden in der Variante Reisstroh (RS) Rohphosphat (RP) nachgewiesen. Zum Erntetermin war die Zahl der Zelhrlosezerset.zer in der Variante RS -I-- AS am hochsten, In der Rhizosphure des nachfolgenden Weizens stimulierte AS die Ammonifikanten und RS + RP die Zellulosezersetzcr am stiLrksten. Unter Reis wurde der Ammonifikant Bacillus durch Grundungung und TC stimuliert. Unter Weizen wurde Pseudomonas durch die Dunger stimuliert. Au/3er bei AS und AS + RP wurde irn fruhen Entwicklungsstadium des Reises die N-Mineralisation in der Rhizosphare durch die Dungungen herabgesetzt. Sie ging auch zur Ernte hin zuruek. Unt.er Wei zen sehufen AS und RP, allein o der kombiniert, gunstige Bedingungen fur die N-Mineralisation, wie uberhaupt die Nachwirkung der Dunger dem direkten Einflu/3 uberlegen war. Die N-Mineralisation korrelierte mit der AmmonifikantenKeimzahl.
+
1) Part of Ph.D. Thesis, submitted by the senior author to the University of Calcutta. 18"
268
P. BHATTACHARYYA et a!'
Mineral nitrogen is produced in soils as a by-product of microbial metabolism. A stimulation in the growth and activity of microorganisms carrying out mineralization of nitrogen and decomposition of cellulosic materials in the rhizosphere of ri ce, am end ed with different manures, followed by a subsequent increase in the proliferation of the ammonifiers, and a decline of the cellulose decomposers in the rhizosphere of the succeeding wheat, grown without further am endment have been found (N ATH 1974 ; DEY et al. 1976). The present work was undertaken to gather information regarding the dir ect effect of organic manures, applied after green manuring, on the numerical preponderan ce of ammonifying ba ct eria and cellulose-decomposing organisms, generic distribution of t he ammonifiers and th e mineralization of nitrogen in the rhizosphere soils of rice (Oryza 8ativa L. cv. IR-8) and their residual effect on the same in the rhizosphere of wheat (Tritic7lm aestioum L. cv, Sonalika). For better understanding, the data have been compared and correlated.
Materials and Methods The ex p er im en t al field conta in ed a g angetic alluvial (Fluv aquent, DOLUI 1977) silty cla y so il, h a ving pH 7.5, EC 0.2 m m hoe/em, org a n ic carbon 0.75 %' t ot al nitrogen 0.057 %' C : N r atio 13 : I, available P 0.0012 %' a v a ilab le K 0.0048 %' a n d CEC 22.0 5 m eq/l00 g soil. The la y-out w as in a randomised bl ock des ign with 5.5 X 5.3 m pl ots a fter green manuring with 6- wk old S esbania aculeata P el's. pl ant s , g rown with a seed rate of 45 kgjha. The following tre atments were d on e at t he time of puddl ing for ri ce wi th four replicati ons : (a) (b) (c) (d) (e) (f) (g)
n o m anure - con trol farm y ard manure (FY"l) with 0.93 % N - p er 40 kg N /h a ; t own co m p ost (TC) wi th 0.64 % N - p er 40 kg N /ha; ammo n ium sulp hate (A S) wi th 20 % N - p er 40 kg N /ha; ri ce str aw (R S) wi th 0.4 % N - p er 40 kg N /ha plus rock p hosph ate (R P ) wi th 25 % P 20S pe r 30 kg P 20 5/ha; AS - p er 40 kg N /ha plus RP - p er 30 kg P 205 /ha; R S - p er 20 kg N /ha plus A S - p er 20 kg N /h a.
-
\ Vith a hill to row spa cing of 15 c m b y 20 em , 30-day-old ri ce seed ling s were transplant ed, two pl an t s p et· hill. After h arvesting of rice, wheat seeds were s own by drilling with a spacing of 15 cm b etween t he rows without further a p plicat ion of m anures. Usual ag ro no m ic pra ctices were follow ed for cultiv at ion of the cr op s. Rhizosphere so ils were coll ected as p er s tandard m ethods (KATZNELSON et a l. 194 8), for rice at early t ille ring stag e, 24 days after transpl anti ng, and at final harvest, 105 d ays after transplanting, and for wheat a t ear emergence stage after 58 d ays of growth, and at f in al h arvest after 102 days of grow th and those from all the replicated plots of the same treatment wer e p ool ed together. The initial so il sam p le was a composite one an d collected from all the pl ots after green manuring b efore ap plication of manures, prior to the cu lt ivat ion of rice crop. The soils were immediately u sed wi thou t drying to attain a n effect a t field condition, keeping s id e b y si de sets for estimati on of m oisture con t en t . T ot al number of ammonify ing b a ct eri a w a s estimate d in REMY' S nutrient agar m edium, a do p t in g pl ate count m ethod foll owing seria l d ilution technique, and t hat of cell ulose-d ec o m p osi n g m icroorg anisms in D UBOS' m ediu m , a dop t ing mo st prob able nu mber t ech n iq ue (ALLEN 1957). T he isol a t ed a m m on ify ing b a ct eri a were id entified up to gen eric lev el accor d ing to the k ey pro vided in B ER G EY 'S Manu al o f D et ermin a ti v e B a cteriol og y (8th ed , 1974). The amo unt of ni trog en min er ali zed w a s estim at e d b y d et ermining t h e am mon ia ni trogen (NHa-N) a n d nitrate-nitrogen (NOa-N ) content of t he initi al an d rh izosp here so ils after in cubating 100 g lot s of soil s in 100 m l b e ak er s at 30 °C, m aintaining the mo is t u re lev el at field co ndition . After 7 an d 14 d ay s of incub a ti on, 10 g of soi l w as extracted twice in a 25 0 ml conical fl ask, each t ime wit h 15 0 ml of 10 % K Cl so lution b y shak in g in a mech anical s haker for 30 mi n . The con ten ts w ere fil t er ed t h ro ugh What .man No. 42 filt er p aper. Fro m t he fil t r ate, 100 m l of t h e K CI e xtract w as taken t o a di still ation fl a sk wi th 400 ml of distilled w at er a n d d istilled wi th 3 g of pow d er ed
269
Organic Manures in Relation to Rhizosphere Effect. III.
ignited magnesium oxide, collecting 250 ml distillate for ammonia-nitrogen. The residue, after cooling and adding 250 ml of distilled water, was distilled with 0.5 g of DEVARDA'S alloy for nitrate nitrogen.
Results Enumeration of ammonifying bacteria and cellulose-decomposing organisms As compared to initial soil, the number of the ammonifying bacteria increased in the rhizosphere soil of rice, except under TC, AS alone, or with RS or RP at early tillering stage and FYM at final harvest (Table 1). Only RS + RP could increase the Table 1. Total number of ammonifying bacteria present in the rhizosphere of rice, treated with different manures, and their number in the rhizosphere of wheat due to residual manurial effect*) Treatment
Total number of ammonifying bacteria (X 105 ) per g dry soil ++ R I **
Control> FYM TC AS RS + RP AS + RP RS + AS Mean S.Em. ±
161 62 19 35 80 54 35 63.7 17.91
R2
WI
79
112 93 51 130 94 57 86 89.0 10.60
5:~
57
n
83 62 72 69.8 3.85
W2 85 43 45 121
n
52 48 66.7 10.82
Mean 109.2 62.7 45.5 89.7 82.5 56.2 60.2 72.3 8.43
S.Em.
±
23.29 8.88 9.98 22.12 4.37 3.35 11.50 5.70
* Initial number: Ammonifying bacteria = 54 X 105 ; ** R I = Rhizosphere soils of rice, collected at early tillering stage after 24 days of transplanting; R 2 = Rhizosphere soils of rice, collected at final harvest after 105 days of transplanting; WI = Rhizosphere soils of wheat, collected at ear emergence after 58 days of growth; W 2 = Rhizosphere soils of wheat, collected at final harvest after 102 days of growth; + Control = Green manure alone; FYM = Farm yard manure; TC = Town Compost; AS Ammonium sulphate; RS = Rice straw; RP = Rock phosphate; ++ Average of duplicate plates.
number of control series in the rhizosphere of rice and that, too, at final harvest, while AS could do so residually throughout in the wheat rhizosphere. With the age of the crop the number decreased uniformly in the wheat rhizosphere, while in the rice rhizosphere an increase was observed under TC, AS alone, or with RS or RP and RS + RP (Fig. 1). The overall difference in the number in rice and wheat rhizosphere was not significant (Table 5). The number of cellulose decomposers in the rhizosphere of rice was in general more than the initial number, except under AS and FYM at final harvest (Table 2). AS, AS + RP, RS + RP, TC, FYM, and RS + AS at early tillering stage and RS + AS, TC, RS + RP, AS + RP at final harvest could increase the number of control series in the rice rhizosphere, while RS + RP and AS + RP had that effect as residue in the wheat rhizosphere at ear emergence stage and AS at final harvest. With the age of the crop the number of cellulose decomposers increased in the rhizosphere of rice, getting RS + AS and TC and in the rhizosphere of wheat getting AS. Despite a gradual variation, the overall difference in the number in rice and wheat rhizosphere was not significant (Table 5).
270
P.
BHATTACH AR Y Y A
et al,
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N u m ber of ammonifie rs a nd tot al mineralized n itrogen in ric e a nd su cceeding wheat rhi zosphere so ils .
Gene ric distribution o f ammo n if y i n g bacteri a A positive rhizosphere effec t towards Bacillus, the most pr evalent ammonifying bacteria in t he rhizosphere was observed in the series under TC and control at early t illering stage and under F YM and TC at final harvest of rice (Ta ble 3). With the age of the crop , B acill us was enha nced un der FYM, while AS with RS or RP or alone dep ressed t he nu mber at both t he stages. Sarcina was stimulated wit h RS + AS and AS + RP at early t illering stage and AS alone at final har vest. P seud omonas was elimi nated from t he rice rhizosp here, except unde r RS + RP at final har vest , while it was incited in the wheat rhizosphere u nd er TC and RS + RP at ear emergence stage and wit h FYM, TC, and control at final har vest. P roteus was stimulated wit h AS + RP, RS + AS, an d AS alone in t he rhizosph ere of both ri ce and wheat an d with RS + RP in t he lat t er. FYM at early t illeri ng stage an d TC and RS + RP at final harvest incit ed Serratia in t he rhizosphere of ri ce. Micrococcus was stimulated only in t he rhiz osphere of wheat ex clusively under t he residual influence of F YM.
271
Organic Manures in Relation to Rhizosphere Effect. III.
Table 2. Most probable number of cellulose-decomposing organisms in rice rhizosphere, as affected by different organic manures, and in wheat rhizosphere, due to their residual effect* Most probable number ( X 103) per g dry soil
Treatment
R 1 ** MPN Control + FYM TC AS RS + RP AS + RP RS + AS Mean S.Em. ±
R2 MPN
p**
116 8.98 212 0.02 270 0.11 3,460 2.25 1,090 2.73 1,500 10.83 188 0.12 976.6 460.35
155 94 792 45
esr
P 0.08 0.07 10.73 14.62 0.01 0.14 0.14
210 2,780 672.4 367.35
W2
W1
MPN P
Mean
MPN
0.11 18.14 14.99 0.14 0.01 0.11 0.01
308 38 56 237 2,023 1,453 283 628.3 295.85
S.Em.
±
P
27 0.11 21 0.14 45 0.99 278 0.14 17 0.04 23 0.01 17 0.04 61.1 :36.32
151.5 91.2 290.7 1005.0 940.2 796.5 817.0 584.6 82.38
58.64 29.53 174.92 619.80 422.57 :394.56 656.64 190.90
* Initial most probable number of cellulose-decomposing organisms = 115 X 103 pcr g dry soil, P value = 0.84; ** See foot note Table 1; + See foot note Table 1; ++ P = Percent of time identical results would be obtained with similar experiments.
Table 3. Distribution of the predominant genera of ammonifying bacteria in the rhizosphere of rice, treated with different manures, and their distribution in the rhizosphere of wheat due to residual manurial effect* Genus
Bacillus
Rhizosphere
Number of strains ++ Control r
FYM
TC
AS
RS
R 1 ** R2
5 4 4 :3
4 5 3 :3
6 5 4 3
2 2 2 2
4 3 :3 2
RI R2
2 2 1
1 1 2 1
1 2 1
WI W2
Micrococcus
WI W2 Proteus
Pseudomonas
RI R2
WI W2 RI R2
Sarcina
2
1 1 1 1
2 1 1 2 3 :3 3
2 2 2 :3
2
:3 3 3 3
2 :3
2 4
:3 4
2 1
1 3 2
2
2 2
RI R2
1 1
2 1
1
4 5 4 3
3 1 1 1
5 4 4 2
6 4 3 3
2
:3 2 3 2
2 3 2 3
1 1
2 3 3 3
RI
R2 WI
W2
*
1 2 1 1
WI W2 WI W2
Serratia
+ RP AS + RP RS + AS
2 :3 3
Initial soil contained: Bacillus (4), Sarcina (4), Pseudomonas (2);
+ See foot note Table 1; ++ Total number of isolates per soil = 10.
**
See foot note Table 1;
272
P.
BHATTACHARYYA
et al.
Table 4. Amount of mineral nitrogen in the rhizosphere soils of rice, treated with different manures, and the same in the rhizosphere of wheat, due to residual manurial effect* Treatment
Amount of mineral nitrogen (mgjlOOg dry soils) (Average of duplicate sets)
Mineralization
R I **
R2
W2
WI
Mean
Incubation (Days) 7
14
14
7
Control + NH4-N NOa-N Total Min-N
15.64 6.65 22.29
7.09 2.54 9.63
2.94 1.93 2.35 12.07 5.29 14.00
FYM
NH4,N NOa-N Total Min-N
8.61 7.69 16.30
6.38 2.31 8.69
3.55 1.33 4.88
TC
NH4-N NOa-N Total Min-N
3.57 10.69 8.81 2.95 12.38 13.64
4.74 7.58 0.00 11.37 4.74 18.95
AS
NH4-N NOa-N Total Min-N
6.90 13.29 2.90 9.25 9.80 22.54
4.35 1.86 6.21
7
14
7.26 13.85 14.01 14.72 21.27 28.58
14
5.21 5.97 6.08 7.96 11.29 13.93
4.43 4.45 8.88
6.77 11.08 5.64 10.29 12.41 21.37
3.11 8.09 5.28 6.57 8.39 14.66
6.95 6.36 13.31
4.52 2.59 2.26 8.29 6.78 10.88
15.98 3.04 8.56 17.50 24.54 20.54
9.07 9.02 10.89 12.02 19.96 21.04
8.05 8.96 17.01
3.22 7.53 2.30 5.38 5.52 12.91
5.27 4.68 1.17 14.72 6.44 19.40
11.12 8.65 19.77
3.90 5.20 9.10
4.57 5.01 5.56 6.83 10.13 11.84
5.66 6.22 11.88
AS + RP NH4-N NOa-N Total Min-N
8.03 12.14 3.21 6.39 11.24 18.53
7.98 7.17 4.25 8.12 12.23 15.29
8.49 9.71 18.20
1.39 5.56 6.95
6.01 6.91 7.21 7.97 13.22 14.88
7.26 6.55 13.81
RS+AS NH4-N NOa-N Total Min-N
10.15 0.58 10.73
5.45 6.06 11.51
+ RPNH
4-N
NOa-N Total Min-N
Mean
NH4,N NOa-N Total Min-N
1.19 3.57 4.76
3.66 4.89 8.55
7.48 8.29 15.77
4.80 5.17 9.97
RS
2.92 8.77 11.69
7
5.46 1.09 6.55
3.33 6.70 1.66 4.02 4.99 10.72
5.65 4.79 12.43 15.97 18.08 20.76
3.33 4.18 6.66 6.07 9.99 10.25
7.73 8.94 4.59 4.27 12.32 13.21
4.61 5.00 1.86 8.63 6.47 13.63
8.31 5.60 9.68 10.40 17.99 16.00
4.99 6.28 6.65 7.51 11.64 13.79
17.00
12.72
Mean for rhizosphere 12.91 10.06 Mean for incubation = 7 days = 12.18; 14 days = 14.16
Mineralization of nitrogen At early tillering stage of rice the amount of nitrogen mineralized was highest in the rhizosphere soils of AS-amended series, followed by that under control (Table 4). At final harvest, it was under RS RP, followed by TC, AS RP, and control. In general, the amount of nitrogen, mineralized in the rhizosphere soils, increased with incubation time from 7 to 14 days at final harvest, while at early tillering stage, it RP, RS RP, and TC-treated series (Fig. 1). In the mineralized was true in AS, AS
+
+
+
+
Organic Manures in R ela ti on to R hizosphere Effect. I II.
273
T ab. 4 Contd. CD at 1%
CD at 5% NH 4 ·N 0. 1430 0. 1379 0.275 8 NS 2.1444 1.6211 4.2889
Rhizosph er e (R ) Treatmen t (T) Inter act ion (R X 1') Incubation (1) Interaction (1' x 1) Inter action (R X 1) Interaction (R X 1 X 1')
X0 3-N
Total }lin-N
NH, -N
N0 3-N
Total Min-N
0.2032
0.3560 0.2912 0.58 26 0.2022 0.5356 0.4048 1.071 2
0.2760 0.200 8 0,40 17 NS NS NS NS
0.3923 0.30 40 0.6085 0.1660 0.439 1 0.3320 0.8787
0.6 870 0.424 1 0.84 85 0.2923 0.7766 0.5869 1.5537
0.:W87
0.4 178 0.1145 0.3028 0.2289 0.6058
* Initial soil after 7 d ays' in cubati on NH 4N : 10.41 , N0 3,N: 1.53 ; Total : 11.94 a nd after 14 da ys' in cubati on NH4 -N : 11.32, N0 3-N: :3.04; Total: 14.36 mg per 100 g d ry soil. ** See foo t note Table 1. + See foo t note Table 1.
nitrogen, the amount of ammonia-nitrogen was more than t hat of nit rate-nitrogen, except in the series under TC at early t illering stage after 7 day's incubation, and under RP, TC, AS, AS RP, and control at fina l harvest after 14 days' incubat ion, RS where t he reverse was true. Ammonia-nitrogen, produced in the rhizos phere soils of manure-amended rice at fina l har vest , was universally more than that of control in th c order AS + RP, TC, RS + AS, RS + RP, AS, and FYM, while for nitrat e nitrogen, it was so und er AS, TC, and FYM at early tillering stage and und er RS + RP at final harvest stage.
+
+
Tab le 5. Com parison between th e response of ri ce rhizosph er e under t he direct infl uen ce and of wheat rhizosp he re under the resid ual influence of differ ent manures taken toget he r Charae te r ist ies
Ob ser ved mean differen ce
Total number of ammonifying bact er ia To tal number of oellulos e deco mposers
11.214 3 499.700
" t" value ob served
Table va lue
0.7875
3.707
1 % level 5 % level 2.44 7
Remarks on su pe rior ity of rhizosphere NS
1.7965
NS
0.4729 1.02 71
0.3473 0.8057
NS NS
4.9269 2.5472
5.79:J6* 1.4387
W heat > Ri ce NS
5.2400 4.4400
2.8200 * 0.9634
Wheat > Ri eo NS
NHrN a) 7.day -incu b.
b) 14· day -incub . N0 3·N a) 7·d ay ·incub. b) 14. day- ineub. Min eralizati on (Total) a) 7.day .in cub. b) 14.day .incu b. * Significa n t at 1 % level ; ** Significant a t 5 % level.
274
P.
BHATTACHARYYA
et a1.
With t he age of the crop, t here was a un iversal decrease in t he ammonia-nitrogen produced, while an increase was obser ved in t he nitrat e-nitrogen under RS + RP, control, TO, AS + RP, and US + AS and in the total nitrogen mine ra lized under R S + RP and TO. R esidu ally in t he wheat rhizo sphere soils, t he amount of to t al nitrogen min eralized was improved by AS alone and wit h RP and TO at fina l harvest , t hat of ammonia-nit rogen by AS at ear emergence st age an d by AS, TO, and AS + RP at fin al harvest and t hat of nit rate-nitrogen by AS at ear emergence and by AS and AS + R P at fina l harv est, as compared t o t hose in t he control. Like in t he rice rhiz osphere soils, th e amount of nitrogen min eral ized increased with incubati on ti me at final harv est and un der TO, cont rol, and RS + AS at ear emergence stage. Excep t under TO, AS, and R S + RP at ear emergence stage aft er 7 days of incubat ion an d und er TO at fin al harv est stage aft er 14 days of incubation, t he amount of nitrat enitrogen was fou nd to be higher t ha n that of ammoni a-nitrogen . With t he age of the crop t here was a uniform decrease in the production of mineral nitrogen, except under F YM, where an increase in the amo unt of ammonia-nitrogen was evident. Th e nitrogen minera lizat ion in the wheat rhizosphere soils under the residual influence of manures was higher t han that in the ri ce r hizosphere soils und er t heir direct influence (Table 5). Although t he correlation bet ween the number of ammonifyin g bacteria and amount of nitrogen minera lized was positive throughout the growth period of t he two crops, it was significant only at fin al har vest of wheat with a linear relationship y = O.12x + 3.65 (Table 6). Ta bl e 6. R elationshi p among different parameters ! R ela t ionsh ip between
N umber of a m m on ify ing ba ct eri a (x ) and to tal Min. N (y) 7-da y· incub.
Gr ow th stage of cro p
" r" va lue ob ser ved
R 1 ** R2 WI
0.66 05 0.6 933 0.6854 0.8494*
\" 2
T able valu e 5%
1%
R egr ession E qua ti on
0.754 0.874
Y
=
0. 12 X
+ 3.65
* Signifi ca nt at 5 % level. + "r" va lues o f only t he sig ni fica n t a nd nearly sig nifica nt rela t ionsh ips are given. ** See foot n ot e T a bl e 1.
Discussion Gree n ma nur ing (cont rol) itse lf had an inciting influ ence on t he proliferation of am monify ing bact eria and cellulose-decomposing microorgan isms in t he rhizosp here, sustaining earl ier reports (VO.JINOVIC and PETROVIC 1963). The manur es might have cont ained ingredients in hibit ory to t he ammonifying bac t eria , as evidenced from t heir lower number t hroughout t he gro wt h of ri ce and following wheat (Ta ble 1). Th e negati ve rh izosphere effect, du e to AS in t he rice rh izosphere, was t o some extent understandable, since the ap plication of a m mo niu m sulp hate obvio usly cutshort t he need of t hese ba cte ria t o produ ce ammoniacal n itrogen . Th e increase in t he number of t hese ba cte ria, residually in this series in t he rhizosphere of wheat, may be at tribu t ed t o t heir seconda ry prolifera t ion after t he exhaustion of ammo nium sulp hat e. Spontaneous incorpora t ion of additional organ ic matt er t hrough pl an t remain s, root st ubbles etc . of t he previous rice crop might be anot her fact or , apa r t, from bette r aeration on redu cti on of moisture level. Th e pr esence of RP to some ext ent counted t he effect of AS.
Org ani c Manures in R elat ion to Rhizospher e Effect . III.
275
St imulating influ en ce of AS and RP on cellulose decomp osers, as reported earl ier (TODOROVA 1972), at ea rly t iller ing stage of ri ce was due t o bet t er nutriti on of nitrogen and phosphorus. Their numer ical prepond erance in the RS- treated series at t his stage was presu mably du e t o addit ion of cellulosic materials, which was furt her ac centu ated with AS at final harvest , sust aining earlier views (ALEXANDER 1977). Decrease in the number of these organisms in the other amended ser ies with time, excepting wit h TC, indicat ed that the need of their ser vice was gradually lessened wit h the reduct ion of cellulosic mater ial wit h t ime. A second flu sh in number of t hese organ isms in t he rhizospher e of wheat un der t he residual influence of t he manures at ear emerge nce st age may be asc ribed t o t he fr esh pr ovision of cellulosic mat er ial s in t he for m of plant remains and root stu bbles of th e previous r ice crop. Despi t e t he genera l pr edominan ce of a m monifyi ng B acill us and t he speci fic maint enan ce of Sarcina wit h AS alone or with R S or RP, t he prolifera tion of M icrococcus, Serratia , and Proteus was t he resul t of rh izosphere effect , AS with eit her RP or RS aid ing Prot eus an d suppressing Serratia (Ta ble 3). Thi s sup port s a com mon observation of suppression of some bacteria by am monium-containing fert ilizers (ALEXANDER 1977). Cont ra d ict ing ASATSUMA et al. (1976), Pseudomonas was not st imula ted in t he r ice r hizosp here . T he factor migh t very well be exc ess of wat er , as it is stimulated un ifor mly in t he rh izosphere of t he following wheat. Increase in t he amount of nitrogen mineralized in t he rhi zosphere soils as comp ared to t hat in t he initial soil is alm ost certainly attributable to t he rhi zosphere effect on t he process, as al so suggeste d by earl ier workers (BARTHOLOMEW and CLARK 1950 ; L EGG and STANFORD 1967 ; NATH 1974). Oth er ma nures, ex cepting AS, appreciably redu ced t he rhizosphere effect of gree n manure at ea rly t illering stage of r ice. Greate r prod uction of minera l nitrogen in the series, treated wit h AS alone or wit h RP over green manure (.F ig. 1), ma y be asc ribed t o t he primary act ion of mineral nitrogen on the mineraliza tion of nitrogen in soil, as obs er ved by earlie r worker s (BROADBENT 1965; LEGG an d STANFORD 1967) with t agged mineral n itrogen. In t his regard, beneficial effect of R P at lat er stages was also ev idenced in t he RS RP series at final harvest of rice. Such effect of TC may be ascribed t o t he degrad ation of inhibit or y components with time. AS, eit her alone or with RP, also residu ally en ha nced t he minera l nitrogen produ cti on in t he rh izosphere of wheat a nd mollified to some extent t he depressing effect of RS. Th ese result s are in kee ping wit h those by ASAl\II (1971). L ike earlier rep or t s (ROVIRA 1965 ; K ATZNELSON 1965 ; J ANSSON 1971), age of plant s had no essential effect on ult imat e net t ra nsformation of nit rogen. The a mou nt of nitrogen minera lized was essentially related to the numbel' of am monifyi ng bacteria (KATZNELSON 1960). H owever, R S + RP was fou nd to produ ce better stimulation on the growt h rather t han on the efficiency of the a.mmon ifiers, Th e great er accu mulation of am monia at ea rly t illering stage of rice sup por te d the fin din gs by P ONNAMPERUMA (1964), who suggested t ha t the am monia release holds the key to produ ct ivit y of rice soils. Th e a mou nt decreased at the fin al har vest stage, indicati ng that t he condition was in fa vour of n itrifica t ion . Th e better ment of a mmonia-nitrogen content of t he wheat rhizosphere soils, resid ually , at ear eme rgence stage, indicated tha t at t his stage t here migh t have been a recovery of some nitrogen, im mobi lized at final har vest stage of r ice. Th e effect was more pronoun ced in t he ser ies under AS, which also st imulated t he a mmoni fyi ng ba ct eria at t his stage. As suggest ed by GORING and CLARK (1948), a less amount of nitrate-nitrogen, available in the rhiz osphere of R S + AS treated ser ies than that obt ained in initial soil, was not du e t o a ny in hibit ion of nitr ification, but rather to an imm obiliz at ion of nitrogen by t he enhanced microorganisms in the rh izosphere soils.
+
276
P. BHATTACHARYYA et al.
A similar explanation may be given for FYM. Universal accumulation of lesser amount of nitrate in the water-saturated rhizosphere soils of rice at early tillering stage may be explained in accordance to HULPOI et al. (1970), who emphasized that, with the high moisture content, nitrification depended on both ammonium nitrogen supply and degree of aeration and that such ammonium nitrogen, remaining after incubation under inadequate aeration, was not recoverable as nitrate. This was amply proved indirectly from the accumulation of greater amount of nitrate-nitrogen in the drier rhizosphere soils of rice at final harvest. This event of better nitrification owing to better aeration was more conspicuous in the rhizosphere soils of wheat on further reduction of moisture content, where the residual effects of the manures were studied. However, the significant difference in the nitrate-nitrogen content between the wheat rhizosphere soil and that of rice sustained the views of earlier workers (PLHAK and VICHERKOVA 1967, 1970; DEY and ROYCHOWDHURY 1976), who observed that the production of nitrate is different in soils under different crops. From the foregoing discussion, it can be concluded that the most favourable conditions for nitrogen mineralization in the rhizosphere of rice and wheat, grown in succession, could be provided by AS and RP amendments, either alone or together, in addition to green manure.
References ALLEN, O. N.: In: Experiments in Soil Bacteriology. Minneapolis, Minnesota (U.S.A.) 1957. ALEXANDER, M.: Introduction to Soil Microbiology. 2nd ed., New York 1977. ASAMI, T.: Immobilization and mineralization of nitrogen compounds in paddy soils. 4. Effect of plant residues on the immobilization and mineralization of nitrogen compounds in paddy soil, incubated under submerged or upland conditions.•1. Sci. Soil Manure 42 (1971), 97-102. [Soils Fert. 37 (1974),1700]. ASATSUMA, K., USHIGOSHI, A., and FURUSAKA, C.: A survey of the bacterial flora of paddy soils treated with different amounts of manure. Science Reports of the Research Institute Tohoku University. 27 (1976), 1-21 [Soils Fert. 40 (1973), 4891. BARTHOLOMEW, W. V., and CLARK, F. E.: Trans. 4th Intern. Congr. Soil Sci., Amsterdam 1950, 2 (1950), 112. Quoted from .JANSSON, S. L. (1971). BERGEY'S Manual of Determinative Bacteriology. 8th ed. (BUCHANAN, R. E., and GIBBONS, N. E., eds.] Baltimore 1974. BROADBENT, F. E.: Soil Sci. Soc. Amer, Proc. 29 (1965), 692. Quoted from JANSSON, S. L. (1971). DEY, B. K., BANIK, S., and N ATH, S.: Residual effect of organic manures on the microbial population and phosphate-solubilizing power of wheat (Triticum aestivum L.) rhizosphere soils. Indian Agric. 20 (1976), 245-249. - and ROYCHOWDHURY, S.: The influence of previous cultivation of crop plants on soil nitrification. Indian Agric. 20 (1976), 121-126. GORING, C. A. 1., and CLARK, F. E.: Influence of crop growth on mineralization of nitrogen in the soil. Soil Sci. Soc. Amer. Proc. 13 (1948), 261-266. HULPOI, N., DAKESIAN, S., ELIADE, G. H., and GHINEA, L.: The effect of soil-physical conditions on the nitrification of ammonium (NH4 ) . PI. Soil. 32 (1970), 468-477. JANSSON, S. L.: Use of 15N in studies of soil nitrogen. In: Soil Biochemistry, Vol. 2 (McLAREN, A. D., and SKUJINE, .1., eds.) New York 1971, 129-166. KATZNELSON, H.: Proc, 7th Intern. Con gr. Soil Sei., Wisconsin 1960, 537-544. Quoted from ROVIRA, A. D. (1965). Nature and importance of the rhizosphere. In: Ecology of soil-borne pathogens - Prelude to Biological Control (BAKER, K. F., and SNYDER, W. C., eds.) Univ. of California Press, Berkley 1965, 187-209. LOCHHEAD, A. G., and TIMONIN, M. 1.: Soil microorganisms and the rhizosphere, Bot.an, Rev. 14 (1948), 543-587.
Organic Manures in Relation to Rhizosphere Effect. III.
277
LEGG, J. 0., and STANFORD, G.: Soil Sci. Soc. Amer. Proc, 31 (1967), 215. Quoted from .JA~SSOX, S. L. (1971). NATH, S.: Effect of organic manures on mineralization of nitrogen in and nitrogen-fixing power of rice rhizosphere soils. M. Sc. (Ag.) Thesis, Calcutta University (1974). PLHAK, F., and VICHERKOVA, M.: Untersuchungen tiber Faktoren, die sich bei Folgekultivierung der Pflanzen geltend machen. BioI. Plant 9 (1967), 122-134. - - The influence of previous plant cultivation on soil nitrification. PI. Soil 32 (1970),50-60. PONNAMPERFMA, F. N.: The chemistry of submerged soils in relation to the growth and yield of rice. Unpublished Ph. D. Thesis, Cornell University, Ithaca, N.Y. (1955). Quoted from The Mineral Nutrition of the Rice Plant. Proc. Symp, Intern. Rice Research Inst., February 1964. Baltimore, Maryland, 1965. ROVIRA, A. D.: Interactions between plant roots and soil microorganisms. A. Rev. Microbiol. 19 (1965), 241-2(;(i. TODoRovA, B.: Effect of fertilization on the activity of cellulolytic bacteria and breakdown of cellulose in the soil. Proc. Syrnp. Soil Microbiology Symp. BioI. Hungarica II (1972), 139-142 [Soils Fert. 36 (1975), 4074J. VOJINOVIC, Z., and PETROVIC, V.: Effect of fertilizers on the microflora and microbiological processes in soil. Zemlj. Bilj. 12 (1963), 349-355. Authors' addresses: Dr. P. BHATTACHARYYA, Research Officer (Microbiology), Pulses and Oilseeds Research Station, Government of West Bengal, Berhampore Murshidabad, PIN 742101, West Bengal; Prof. Dr. B. K. DEY, Bidhan Chandra Krishi Viswa Vidyalaya, West Bengal; S. NATH and S. BANIK, College of Agriculture, Calcutta University, West Bengal, India.
Organic Manures in Relation to Rhizosphere Effect III. Effect of Organic Manures on Population of Ammonifying Bacteria and Mineralization of Nitrogen in Rice and Succeeding Wheat Rhizosphere Soils") P.
BHATTACHARYYA, B.
K.
DEY,
S.
NATH,
and S.
BANIK
With 1 Figure
Summary Ammonifying bacteria were stimulated in the rhizosphere of rice with green manuring (control), while depressed with town compost (TC) at early tillering stage. The growth of cellulose decomposers was greatest with ammonium sulphate (AS). Except under control and farm yard manure (FYM), the number increased with time and with the age of the crop at final harvest, the highest was with rice straw (RS) + rock phosphate (RP). At this stage, the abundance of cellulose decomposers was greatest with RS + AS. AS induced the best residual effect on the proliferation of ammonifying bacteria and RS + RP on cellulose decomposers in the rhizosphere of wheat. Green manuring (control) promoted the growth of ammonifying Bacillus, which was further intensified with TC in the rice rhizosphere. Residually Pseudomonas was stimulated by the manures in the wheat rhizosphere. At early tillering stage of rice, mineralization of nitrogen in the rhizosphere decreased with the manures, excepting AS and AS + RP. Barring under RS + RP, the transformation declined with the age of the crop. AS and RP, either alone or together, also provided a favourable condition for nitrogen mineralizat.ion, residually in the rhizosphere of wheat. In this respect, the rhizosphere effect of wheat under the residual influence was superior to that of rice under the direct influence of the manures. The process was positively correlated with the number of ammonifiers, being significantly in the wheat rhizosphere at final harvest.
Zusammenfassung Tm fruhen Entwicklungsstadium des Reises wurden in der Reis-Rhizosphare die Ammonifikanten durch alleinige Grundungung (Kontrolle) stimuliert, durch Stadtkompost (TC) jedoch herabgesetzt. Die Vermehrung der Zellulosezersetzer war am hochsten bei Verabreichung von Ammonsulfat (AS). Au/3er bei Griindiingung und Stalldung (FYM) stiegen die Keimzahlen im Laufe der Zeit und mit dem Alter der Pflanzen an, die hochsten wurden in der Variante Reisstroh (RS) Rohphosphat (RP) nachgewiesen. Zum Erntetermin war die Zahl der Zelhrlosezerset.zer in der Variante RS -I-- AS am hochsten, In der Rhizosphure des nachfolgenden Weizens stimulierte AS die Ammonifikanten und RS + RP die Zellulosezersetzcr am stiLrksten. Unter Reis wurde der Ammonifikant Bacillus durch Grundungung und TC stimuliert. Unter Weizen wurde Pseudomonas durch die Dunger stimuliert. Au/3er bei AS und AS + RP wurde irn fruhen Entwicklungsstadium des Reises die N-Mineralisation in der Rhizosphare durch die Dungungen herabgesetzt. Sie ging auch zur Ernte hin zuruek. Unt.er Wei zen sehufen AS und RP, allein o der kombiniert, gunstige Bedingungen fur die N-Mineralisation, wie uberhaupt die Nachwirkung der Dunger dem direkten Einflu/3 uberlegen war. Die N-Mineralisation korrelierte mit der AmmonifikantenKeimzahl.
+
1) Part of Ph.D. Thesis, submitted by the senior author to the University of Calcutta. 18"
268
P. BHATTACHARYYA et a!'
Mineral nitrogen is produced in soils as a by-product of microbial metabolism. A stimulation in the growth and activity of microorganisms carrying out mineralization of nitrogen and decomposition of cellulosic materials in the rhizosphere of ri ce, am end ed with different manures, followed by a subsequent increase in the proliferation of the ammonifiers, and a decline of the cellulose decomposers in the rhizosphere of the succeeding wheat, grown without further am endment have been found (N ATH 1974 ; DEY et al. 1976). The present work was undertaken to gather information regarding the dir ect effect of organic manures, applied after green manuring, on the numerical preponderan ce of ammonifying ba ct eria and cellulose-decomposing organisms, generic distribution of t he ammonifiers and th e mineralization of nitrogen in the rhizosphere soils of rice (Oryza 8ativa L. cv. IR-8) and their residual effect on the same in the rhizosphere of wheat (Tritic7lm aestioum L. cv, Sonalika). For better understanding, the data have been compared and correlated.
Materials and Methods The ex p er im en t al field conta in ed a g angetic alluvial (Fluv aquent, DOLUI 1977) silty cla y so il, h a ving pH 7.5, EC 0.2 m m hoe/em, org a n ic carbon 0.75 %' t ot al nitrogen 0.057 %' C : N r atio 13 : I, available P 0.0012 %' a v a ilab le K 0.0048 %' a n d CEC 22.0 5 m eq/l00 g soil. The la y-out w as in a randomised bl ock des ign with 5.5 X 5.3 m pl ots a fter green manuring with 6- wk old S esbania aculeata P el's. pl ant s , g rown with a seed rate of 45 kgjha. The following tre atments were d on e at t he time of puddl ing for ri ce wi th four replicati ons : (a) (b) (c) (d) (e) (f) (g)
n o m anure - con trol farm y ard manure (FY"l) with 0.93 % N - p er 40 kg N /h a ; t own co m p ost (TC) wi th 0.64 % N - p er 40 kg N /ha; ammo n ium sulp hate (A S) wi th 20 % N - p er 40 kg N /ha; ri ce str aw (R S) wi th 0.4 % N - p er 40 kg N /ha plus rock p hosph ate (R P ) wi th 25 % P 20S pe r 30 kg P 20 5/ha; AS - p er 40 kg N /ha plus RP - p er 30 kg P 205 /ha; R S - p er 20 kg N /ha plus A S - p er 20 kg N /h a.
-
\ Vith a hill to row spa cing of 15 c m b y 20 em , 30-day-old ri ce seed ling s were transplant ed, two pl an t s p et· hill. After h arvesting of rice, wheat seeds were s own by drilling with a spacing of 15 cm b etween t he rows without further a p plicat ion of m anures. Usual ag ro no m ic pra ctices were follow ed for cultiv at ion of the cr op s. Rhizosphere so ils were coll ected as p er s tandard m ethods (KATZNELSON et a l. 194 8), for rice at early t ille ring stag e, 24 days after transpl anti ng, and at final harvest, 105 d ays after transplanting, and for wheat a t ear emergence stage after 58 d ays of growth, and at f in al h arvest after 102 days of grow th and those from all the replicated plots of the same treatment wer e p ool ed together. The initial so il sam p le was a composite one an d collected from all the pl ots after green manuring b efore ap plication of manures, prior to the cu lt ivat ion of rice crop. The soils were immediately u sed wi thou t drying to attain a n effect a t field condition, keeping s id e b y si de sets for estimati on of m oisture con t en t . T ot al number of ammonify ing b a ct eri a w a s estimate d in REMY' S nutrient agar m edium, a do p t in g pl ate count m ethod foll owing seria l d ilution technique, and t hat of cell ulose-d ec o m p osi n g m icroorg anisms in D UBOS' m ediu m , a dop t ing mo st prob able nu mber t ech n iq ue (ALLEN 1957). T he isol a t ed a m m on ify ing b a ct eri a were id entified up to gen eric lev el accor d ing to the k ey pro vided in B ER G EY 'S Manu al o f D et ermin a ti v e B a cteriol og y (8th ed , 1974). The amo unt of ni trog en min er ali zed w a s estim at e d b y d et ermining t h e am mon ia ni trogen (NHa-N) a n d nitrate-nitrogen (NOa-N ) content of t he initi al an d rh izosp here so ils after in cubating 100 g lot s of soil s in 100 m l b e ak er s at 30 °C, m aintaining the mo is t u re lev el at field co ndition . After 7 an d 14 d ay s of incub a ti on, 10 g of soi l w as extracted twice in a 25 0 ml conical fl ask, each t ime wit h 15 0 ml of 10 % K Cl so lution b y shak in g in a mech anical s haker for 30 mi n . The con ten ts w ere fil t er ed t h ro ugh What .man No. 42 filt er p aper. Fro m t he fil t r ate, 100 m l of t h e K CI e xtract w as taken t o a di still ation fl a sk wi th 400 ml of distilled w at er a n d d istilled wi th 3 g of pow d er ed
269
Organic Manures in Relation to Rhizosphere Effect. III.
ignited magnesium oxide, collecting 250 ml distillate for ammonia-nitrogen. The residue, after cooling and adding 250 ml of distilled water, was distilled with 0.5 g of DEVARDA'S alloy for nitrate nitrogen.
Results Enumeration of ammonifying bacteria and cellulose-decomposing organisms As compared to initial soil, the number of the ammonifying bacteria increased in the rhizosphere soil of rice, except under TC, AS alone, or with RS or RP at early tillering stage and FYM at final harvest (Table 1). Only RS + RP could increase the Table 1. Total number of ammonifying bacteria present in the rhizosphere of rice, treated with different manures, and their number in the rhizosphere of wheat due to residual manurial effect*) Treatment
Total number of ammonifying bacteria (X 105 ) per g dry soil ++ R I **
Control> FYM TC AS RS + RP AS + RP RS + AS Mean S.Em. ±
161 62 19 35 80 54 35 63.7 17.91
R2
WI
79
112 93 51 130 94 57 86 89.0 10.60
5:~
57
n
83 62 72 69.8 3.85
W2 85 43 45 121
n
52 48 66.7 10.82
Mean 109.2 62.7 45.5 89.7 82.5 56.2 60.2 72.3 8.43
S.Em.
±
23.29 8.88 9.98 22.12 4.37 3.35 11.50 5.70
* Initial number: Ammonifying bacteria = 54 X 105 ; ** R I = Rhizosphere soils of rice, collected at early tillering stage after 24 days of transplanting; R 2 = Rhizosphere soils of rice, collected at final harvest after 105 days of transplanting; WI = Rhizosphere soils of wheat, collected at ear emergence after 58 days of growth; W 2 = Rhizosphere soils of wheat, collected at final harvest after 102 days of growth; + Control = Green manure alone; FYM = Farm yard manure; TC = Town Compost; AS Ammonium sulphate; RS = Rice straw; RP = Rock phosphate; ++ Average of duplicate plates.
number of control series in the rhizosphere of rice and that, too, at final harvest, while AS could do so residually throughout in the wheat rhizosphere. With the age of the crop the number decreased uniformly in the wheat rhizosphere, while in the rice rhizosphere an increase was observed under TC, AS alone, or with RS or RP and RS + RP (Fig. 1). The overall difference in the number in rice and wheat rhizosphere was not significant (Table 5). The number of cellulose decomposers in the rhizosphere of rice was in general more than the initial number, except under AS and FYM at final harvest (Table 2). AS, AS + RP, RS + RP, TC, FYM, and RS + AS at early tillering stage and RS + AS, TC, RS + RP, AS + RP at final harvest could increase the number of control series in the rice rhizosphere, while RS + RP and AS + RP had that effect as residue in the wheat rhizosphere at ear emergence stage and AS at final harvest. With the age of the crop the number of cellulose decomposers increased in the rhizosphere of rice, getting RS + AS and TC and in the rhizosphere of wheat getting AS. Despite a gradual variation, the overall difference in the number in rice and wheat rhizosphere was not significant (Table 5).
270
P.
BHATTACH AR Y Y A
et al,
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N u m ber of ammonifie rs a nd tot al mineralized n itrogen in ric e a nd su cceeding wheat rhi zosphere so ils .
Gene ric distribution o f ammo n if y i n g bacteri a A positive rhizosphere effec t towards Bacillus, the most pr evalent ammonifying bacteria in t he rhizosphere was observed in the series under TC and control at early t illering stage and under F YM and TC at final harvest of rice (Ta ble 3). With the age of the crop , B acill us was enha nced un der FYM, while AS with RS or RP or alone dep ressed t he nu mber at both t he stages. Sarcina was stimulated wit h RS + AS and AS + RP at early t illering stage and AS alone at final har vest. P seud omonas was elimi nated from t he rice rhizosp here, except unde r RS + RP at final har vest , while it was incited in the wheat rhizosphere u nd er TC and RS + RP at ear emergence stage and wit h FYM, TC, and control at final har vest. P roteus was stimulated wit h AS + RP, RS + AS, an d AS alone in t he rhizosph ere of both ri ce and wheat an d with RS + RP in t he lat t er. FYM at early t illeri ng stage an d TC and RS + RP at final harvest incit ed Serratia in t he rhizosphere of ri ce. Micrococcus was stimulated only in t he rhiz osphere of wheat ex clusively under t he residual influence of F YM.
271
Organic Manures in Relation to Rhizosphere Effect. III.
Table 2. Most probable number of cellulose-decomposing organisms in rice rhizosphere, as affected by different organic manures, and in wheat rhizosphere, due to their residual effect* Most probable number ( X 103) per g dry soil
Treatment
R 1 ** MPN Control + FYM TC AS RS + RP AS + RP RS + AS Mean S.Em. ±
R2 MPN
p**
116 8.98 212 0.02 270 0.11 3,460 2.25 1,090 2.73 1,500 10.83 188 0.12 976.6 460.35
155 94 792 45
esr
P 0.08 0.07 10.73 14.62 0.01 0.14 0.14
210 2,780 672.4 367.35
W2
W1
MPN P
Mean
MPN
0.11 18.14 14.99 0.14 0.01 0.11 0.01
308 38 56 237 2,023 1,453 283 628.3 295.85
S.Em.
±
P
27 0.11 21 0.14 45 0.99 278 0.14 17 0.04 23 0.01 17 0.04 61.1 :36.32
151.5 91.2 290.7 1005.0 940.2 796.5 817.0 584.6 82.38
58.64 29.53 174.92 619.80 422.57 :394.56 656.64 190.90
* Initial most probable number of cellulose-decomposing organisms = 115 X 103 pcr g dry soil, P value = 0.84; ** See foot note Table 1; + See foot note Table 1; ++ P = Percent of time identical results would be obtained with similar experiments.
Table 3. Distribution of the predominant genera of ammonifying bacteria in the rhizosphere of rice, treated with different manures, and their distribution in the rhizosphere of wheat due to residual manurial effect* Genus
Bacillus
Rhizosphere
Number of strains ++ Control r
FYM
TC
AS
RS
R 1 ** R2
5 4 4 :3
4 5 3 :3
6 5 4 3
2 2 2 2
4 3 :3 2
RI R2
2 2 1
1 1 2 1
1 2 1
WI W2
Micrococcus
WI W2 Proteus
Pseudomonas
RI R2
WI W2 RI R2
Sarcina
2
1 1 1 1
2 1 1 2 3 :3 3
2 2 2 :3
2
:3 3 3 3
2 :3
2 4
:3 4
2 1
1 3 2
2
2 2
RI R2
1 1
2 1
1
4 5 4 3
3 1 1 1
5 4 4 2
6 4 3 3
2
:3 2 3 2
2 3 2 3
1 1
2 3 3 3
RI
R2 WI
W2
*
1 2 1 1
WI W2 WI W2
Serratia
+ RP AS + RP RS + AS
2 :3 3
Initial soil contained: Bacillus (4), Sarcina (4), Pseudomonas (2);
+ See foot note Table 1; ++ Total number of isolates per soil = 10.
**
See foot note Table 1;
272
P.
BHATTACHARYYA
et al.
Table 4. Amount of mineral nitrogen in the rhizosphere soils of rice, treated with different manures, and the same in the rhizosphere of wheat, due to residual manurial effect* Treatment
Amount of mineral nitrogen (mgjlOOg dry soils) (Average of duplicate sets)
Mineralization
R I **
R2
W2
WI
Mean
Incubation (Days) 7
14
14
7
Control + NH4-N NOa-N Total Min-N
15.64 6.65 22.29
7.09 2.54 9.63
2.94 1.93 2.35 12.07 5.29 14.00
FYM
NH4,N NOa-N Total Min-N
8.61 7.69 16.30
6.38 2.31 8.69
3.55 1.33 4.88
TC
NH4-N NOa-N Total Min-N
3.57 10.69 8.81 2.95 12.38 13.64
4.74 7.58 0.00 11.37 4.74 18.95
AS
NH4-N NOa-N Total Min-N
6.90 13.29 2.90 9.25 9.80 22.54
4.35 1.86 6.21
7
14
7.26 13.85 14.01 14.72 21.27 28.58
14
5.21 5.97 6.08 7.96 11.29 13.93
4.43 4.45 8.88
6.77 11.08 5.64 10.29 12.41 21.37
3.11 8.09 5.28 6.57 8.39 14.66
6.95 6.36 13.31
4.52 2.59 2.26 8.29 6.78 10.88
15.98 3.04 8.56 17.50 24.54 20.54
9.07 9.02 10.89 12.02 19.96 21.04
8.05 8.96 17.01
3.22 7.53 2.30 5.38 5.52 12.91
5.27 4.68 1.17 14.72 6.44 19.40
11.12 8.65 19.77
3.90 5.20 9.10
4.57 5.01 5.56 6.83 10.13 11.84
5.66 6.22 11.88
AS + RP NH4-N NOa-N Total Min-N
8.03 12.14 3.21 6.39 11.24 18.53
7.98 7.17 4.25 8.12 12.23 15.29
8.49 9.71 18.20
1.39 5.56 6.95
6.01 6.91 7.21 7.97 13.22 14.88
7.26 6.55 13.81
RS+AS NH4-N NOa-N Total Min-N
10.15 0.58 10.73
5.45 6.06 11.51
+ RPNH
4-N
NOa-N Total Min-N
Mean
NH4,N NOa-N Total Min-N
1.19 3.57 4.76
3.66 4.89 8.55
7.48 8.29 15.77
4.80 5.17 9.97
RS
2.92 8.77 11.69
7
5.46 1.09 6.55
3.33 6.70 1.66 4.02 4.99 10.72
5.65 4.79 12.43 15.97 18.08 20.76
3.33 4.18 6.66 6.07 9.99 10.25
7.73 8.94 4.59 4.27 12.32 13.21
4.61 5.00 1.86 8.63 6.47 13.63
8.31 5.60 9.68 10.40 17.99 16.00
4.99 6.28 6.65 7.51 11.64 13.79
17.00
12.72
Mean for rhizosphere 12.91 10.06 Mean for incubation = 7 days = 12.18; 14 days = 14.16
Mineralization of nitrogen At early tillering stage of rice the amount of nitrogen mineralized was highest in the rhizosphere soils of AS-amended series, followed by that under control (Table 4). At final harvest, it was under RS RP, followed by TC, AS RP, and control. In general, the amount of nitrogen, mineralized in the rhizosphere soils, increased with incubation time from 7 to 14 days at final harvest, while at early tillering stage, it RP, RS RP, and TC-treated series (Fig. 1). In the mineralized was true in AS, AS
+
+
+
+
Organic Manures in R ela ti on to R hizosphere Effect. I II.
273
T ab. 4 Contd. CD at 1%
CD at 5% NH 4 ·N 0. 1430 0. 1379 0.275 8 NS 2.1444 1.6211 4.2889
Rhizosph er e (R ) Treatmen t (T) Inter act ion (R X 1') Incubation (1) Interaction (1' x 1) Inter action (R X 1) Interaction (R X 1 X 1')
X0 3-N
Total }lin-N
NH, -N
N0 3-N
Total Min-N
0.2032
0.3560 0.2912 0.58 26 0.2022 0.5356 0.4048 1.071 2
0.2760 0.200 8 0,40 17 NS NS NS NS
0.3923 0.30 40 0.6085 0.1660 0.439 1 0.3320 0.8787
0.6 870 0.424 1 0.84 85 0.2923 0.7766 0.5869 1.5537
0.:W87
0.4 178 0.1145 0.3028 0.2289 0.6058
* Initial soil after 7 d ays' in cubati on NH 4N : 10.41 , N0 3,N: 1.53 ; Total : 11.94 a nd after 14 da ys' in cubati on NH4 -N : 11.32, N0 3-N: :3.04; Total: 14.36 mg per 100 g d ry soil. ** See foo t note Table 1. + See foo t note Table 1.
nitrogen, the amount of ammonia-nitrogen was more than t hat of nit rate-nitrogen, except in the series under TC at early t illering stage after 7 day's incubation, and under RP, TC, AS, AS RP, and control at fina l harvest after 14 days' incubat ion, RS where t he reverse was true. Ammonia-nitrogen, produced in the rhizos phere soils of manure-amended rice at fina l har vest , was universally more than that of control in th c order AS + RP, TC, RS + AS, RS + RP, AS, and FYM, while for nitrat e nitrogen, it was so und er AS, TC, and FYM at early tillering stage and und er RS + RP at final harvest stage.
+
+
Tab le 5. Com parison between th e response of ri ce rhizosph er e under t he direct infl uen ce and of wheat rhizosp he re under the resid ual influence of differ ent manures taken toget he r Charae te r ist ies
Ob ser ved mean differen ce
Total number of ammonifying bact er ia To tal number of oellulos e deco mposers
11.214 3 499.700
" t" value ob served
Table va lue
0.7875
3.707
1 % level 5 % level 2.44 7
Remarks on su pe rior ity of rhizosphere NS
1.7965
NS
0.4729 1.02 71
0.3473 0.8057
NS NS
4.9269 2.5472
5.79:J6* 1.4387
W heat > Ri ce NS
5.2400 4.4400
2.8200 * 0.9634
Wheat > Ri eo NS
NHrN a) 7.day -incu b.
b) 14· day -incub . N0 3·N a) 7·d ay ·incub. b) 14. day- ineub. Min eralizati on (Total) a) 7.day .in cub. b) 14.day .incu b. * Significa n t at 1 % level ; ** Significant a t 5 % level.
274
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BHATTACHARYYA
et a1.
With t he age of the crop, t here was a un iversal decrease in t he ammonia-nitrogen produced, while an increase was obser ved in t he nitrat e-nitrogen under RS + RP, control, TO, AS + RP, and US + AS and in the total nitrogen mine ra lized under R S + RP and TO. R esidu ally in t he wheat rhizo sphere soils, t he amount of to t al nitrogen min eralized was improved by AS alone and wit h RP and TO at fina l harvest , t hat of ammonia-nit rogen by AS at ear emergence st age an d by AS, TO, and AS + RP at fin al harvest and t hat of nit rate-nitrogen by AS at ear emergence and by AS and AS + R P at fina l harv est, as compared t o t hose in t he control. Like in t he rice rhiz osphere soils, th e amount of nitrogen min eral ized increased with incubati on ti me at final harv est and un der TO, cont rol, and RS + AS at ear emergence stage. Excep t under TO, AS, and R S + RP at ear emergence stage aft er 7 days of incubat ion an d und er TO at fin al harv est stage aft er 14 days of incubation, t he amount of nitrat enitrogen was fou nd to be higher t ha n that of ammoni a-nitrogen . With t he age of the crop t here was a uniform decrease in the production of mineral nitrogen, except under F YM, where an increase in the amo unt of ammonia-nitrogen was evident. Th e nitrogen minera lizat ion in the wheat rhizosphere soils under the residual influence of manures was higher t han that in the ri ce r hizosphere soils und er t heir direct influence (Table 5). Although t he correlation bet ween the number of ammonifyin g bacteria and amount of nitrogen minera lized was positive throughout the growth period of t he two crops, it was significant only at fin al har vest of wheat with a linear relationship y = O.12x + 3.65 (Table 6). Ta bl e 6. R elationshi p among different parameters ! R ela t ionsh ip between
N umber of a m m on ify ing ba ct eri a (x ) and to tal Min. N (y) 7-da y· incub.
Gr ow th stage of cro p
" r" va lue ob ser ved
R 1 ** R2 WI
0.66 05 0.6 933 0.6854 0.8494*
\" 2
T able valu e 5%
1%
R egr ession E qua ti on
0.754 0.874
Y
=
0. 12 X
+ 3.65
* Signifi ca nt at 5 % level. + "r" va lues o f only t he sig ni fica n t a nd nearly sig nifica nt rela t ionsh ips are given. ** See foot n ot e T a bl e 1.
Discussion Gree n ma nur ing (cont rol) itse lf had an inciting influ ence on t he proliferation of am monify ing bact eria and cellulose-decomposing microorgan isms in t he rhizosp here, sustaining earl ier reports (VO.JINOVIC and PETROVIC 1963). The manur es might have cont ained ingredients in hibit ory to t he ammonifying bac t eria , as evidenced from t heir lower number t hroughout t he gro wt h of ri ce and following wheat (Ta ble 1). Th e negati ve rh izosphere effect, du e to AS in t he rice rh izosphere, was t o some extent understandable, since the ap plication of a m mo niu m sulp hate obvio usly cutshort t he need of t hese ba cte ria t o produ ce ammoniacal n itrogen . Th e increase in t he number of t hese ba cte ria, residually in this series in t he rhizosphere of wheat, may be at tribu t ed t o t heir seconda ry prolifera t ion after t he exhaustion of ammo nium sulp hat e. Spontaneous incorpora t ion of additional organ ic matt er t hrough pl an t remain s, root st ubbles etc . of t he previous rice crop might be anot her fact or , apa r t, from bette r aeration on redu cti on of moisture level. Th e pr esence of RP to some ext ent counted t he effect of AS.
Org ani c Manures in R elat ion to Rhizospher e Effect . III.
275
St imulating influ en ce of AS and RP on cellulose decomp osers, as reported earl ier (TODOROVA 1972), at ea rly t iller ing stage of ri ce was due t o bet t er nutriti on of nitrogen and phosphorus. Their numer ical prepond erance in the RS- treated series at t his stage was presu mably du e t o addit ion of cellulosic materials, which was furt her ac centu ated with AS at final harvest , sust aining earlier views (ALEXANDER 1977). Decrease in the number of these organisms in the other amended ser ies with time, excepting wit h TC, indicat ed that the need of their ser vice was gradually lessened wit h the reduct ion of cellulosic mater ial wit h t ime. A second flu sh in number of t hese organ isms in t he rhizospher e of wheat un der t he residual influence of t he manures at ear emerge nce st age may be asc ribed t o t he fr esh pr ovision of cellulosic mat er ial s in t he for m of plant remains and root stu bbles of th e previous r ice crop. Despi t e t he genera l pr edominan ce of a m monifyi ng B acill us and t he speci fic maint enan ce of Sarcina wit h AS alone or with R S or RP, t he prolifera tion of M icrococcus, Serratia , and Proteus was t he resul t of rh izosphere effect , AS with eit her RP or RS aid ing Prot eus an d suppressing Serratia (Ta ble 3). Thi s sup port s a com mon observation of suppression of some bacteria by am monium-containing fert ilizers (ALEXANDER 1977). Cont ra d ict ing ASATSUMA et al. (1976), Pseudomonas was not st imula ted in t he r ice r hizosp here . T he factor migh t very well be exc ess of wat er , as it is stimulated un ifor mly in t he rh izosphere of t he following wheat. Increase in t he amount of nitrogen mineralized in t he rhi zosphere soils as comp ared to t hat in t he initial soil is alm ost certainly attributable to t he rhi zosphere effect on t he process, as al so suggeste d by earl ier workers (BARTHOLOMEW and CLARK 1950 ; L EGG and STANFORD 1967 ; NATH 1974). Oth er ma nures, ex cepting AS, appreciably redu ced t he rhizosphere effect of gree n manure at ea rly t illering stage of r ice. Greate r prod uction of minera l nitrogen in the series, treated wit h AS alone or wit h RP over green manure (.F ig. 1), ma y be asc ribed t o t he primary act ion of mineral nitrogen on the mineraliza tion of nitrogen in soil, as obs er ved by earlie r worker s (BROADBENT 1965; LEGG an d STANFORD 1967) with t agged mineral n itrogen. In t his regard, beneficial effect of R P at lat er stages was also ev idenced in t he RS RP series at final harvest of rice. Such effect of TC may be ascribed t o t he degrad ation of inhibit or y components with time. AS, eit her alone or with RP, also residu ally en ha nced t he minera l nitrogen produ cti on in t he rh izosphere of wheat a nd mollified to some extent t he depressing effect of RS. Th ese result s are in kee ping wit h those by ASAl\II (1971). L ike earlier rep or t s (ROVIRA 1965 ; K ATZNELSON 1965 ; J ANSSON 1971), age of plant s had no essential effect on ult imat e net t ra nsformation of nit rogen. The a mou nt of nitrogen minera lized was essentially related to the numbel' of am monifyi ng bacteria (KATZNELSON 1960). H owever, R S + RP was fou nd to produ ce better stimulation on the growt h rather t han on the efficiency of the a.mmon ifiers, Th e great er accu mulation of am monia at ea rly t illering stage of rice sup por te d the fin din gs by P ONNAMPERUMA (1964), who suggested t ha t the am monia release holds the key to produ ct ivit y of rice soils. Th e a mou nt decreased at the fin al har vest stage, indicati ng that t he condition was in fa vour of n itrifica t ion . Th e better ment of a mmonia-nitrogen content of t he wheat rhizosphere soils, resid ually , at ear eme rgence stage, indicated tha t at t his stage t here migh t have been a recovery of some nitrogen, im mobi lized at final har vest stage of r ice. Th e effect was more pronoun ced in t he ser ies under AS, which also st imulated t he a mmoni fyi ng ba ct eria at t his stage. As suggest ed by GORING and CLARK (1948), a less amount of nitrate-nitrogen, available in the rhiz osphere of R S + AS treated ser ies than that obt ained in initial soil, was not du e t o a ny in hibit ion of nitr ification, but rather to an imm obiliz at ion of nitrogen by t he enhanced microorganisms in the rh izosphere soils.
+
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P. BHATTACHARYYA et al.
A similar explanation may be given for FYM. Universal accumulation of lesser amount of nitrate in the water-saturated rhizosphere soils of rice at early tillering stage may be explained in accordance to HULPOI et al. (1970), who emphasized that, with the high moisture content, nitrification depended on both ammonium nitrogen supply and degree of aeration and that such ammonium nitrogen, remaining after incubation under inadequate aeration, was not recoverable as nitrate. This was amply proved indirectly from the accumulation of greater amount of nitrate-nitrogen in the drier rhizosphere soils of rice at final harvest. This event of better nitrification owing to better aeration was more conspicuous in the rhizosphere soils of wheat on further reduction of moisture content, where the residual effects of the manures were studied. However, the significant difference in the nitrate-nitrogen content between the wheat rhizosphere soil and that of rice sustained the views of earlier workers (PLHAK and VICHERKOVA 1967, 1970; DEY and ROYCHOWDHURY 1976), who observed that the production of nitrate is different in soils under different crops. From the foregoing discussion, it can be concluded that the most favourable conditions for nitrogen mineralization in the rhizosphere of rice and wheat, grown in succession, could be provided by AS and RP amendments, either alone or together, in addition to green manure.
References ALLEN, O. N.: In: Experiments in Soil Bacteriology. Minneapolis, Minnesota (U.S.A.) 1957. ALEXANDER, M.: Introduction to Soil Microbiology. 2nd ed., New York 1977. ASAMI, T.: Immobilization and mineralization of nitrogen compounds in paddy soils. 4. Effect of plant residues on the immobilization and mineralization of nitrogen compounds in paddy soil, incubated under submerged or upland conditions.•1. Sci. Soil Manure 42 (1971), 97-102. [Soils Fert. 37 (1974),1700]. ASATSUMA, K., USHIGOSHI, A., and FURUSAKA, C.: A survey of the bacterial flora of paddy soils treated with different amounts of manure. Science Reports of the Research Institute Tohoku University. 27 (1976), 1-21 [Soils Fert. 40 (1973), 4891. BARTHOLOMEW, W. V., and CLARK, F. E.: Trans. 4th Intern. Congr. Soil Sci., Amsterdam 1950, 2 (1950), 112. Quoted from .JANSSON, S. L. (1971). BERGEY'S Manual of Determinative Bacteriology. 8th ed. (BUCHANAN, R. E., and GIBBONS, N. E., eds.] Baltimore 1974. BROADBENT, F. E.: Soil Sci. Soc. Amer, Proc. 29 (1965), 692. Quoted from JANSSON, S. L. (1971). DEY, B. K., BANIK, S., and N ATH, S.: Residual effect of organic manures on the microbial population and phosphate-solubilizing power of wheat (Triticum aestivum L.) rhizosphere soils. Indian Agric. 20 (1976), 245-249. - and ROYCHOWDHURY, S.: The influence of previous cultivation of crop plants on soil nitrification. Indian Agric. 20 (1976), 121-126. GORING, C. A. 1., and CLARK, F. E.: Influence of crop growth on mineralization of nitrogen in the soil. Soil Sci. Soc. Amer. Proc. 13 (1948), 261-266. HULPOI, N., DAKESIAN, S., ELIADE, G. H., and GHINEA, L.: The effect of soil-physical conditions on the nitrification of ammonium (NH4 ) . PI. Soil. 32 (1970), 468-477. JANSSON, S. L.: Use of 15N in studies of soil nitrogen. In: Soil Biochemistry, Vol. 2 (McLAREN, A. D., and SKUJINE, .1., eds.) New York 1971, 129-166. KATZNELSON, H.: Proc, 7th Intern. Con gr. Soil Sei., Wisconsin 1960, 537-544. Quoted from ROVIRA, A. D. (1965). Nature and importance of the rhizosphere. In: Ecology of soil-borne pathogens - Prelude to Biological Control (BAKER, K. F., and SNYDER, W. C., eds.) Univ. of California Press, Berkley 1965, 187-209. LOCHHEAD, A. G., and TIMONIN, M. 1.: Soil microorganisms and the rhizosphere, Bot.an, Rev. 14 (1948), 543-587.
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LEGG, J. 0., and STANFORD, G.: Soil Sci. Soc. Amer. Proc, 31 (1967), 215. Quoted from .JA~SSOX, S. L. (1971). NATH, S.: Effect of organic manures on mineralization of nitrogen in and nitrogen-fixing power of rice rhizosphere soils. M. Sc. (Ag.) Thesis, Calcutta University (1974). PLHAK, F., and VICHERKOVA, M.: Untersuchungen tiber Faktoren, die sich bei Folgekultivierung der Pflanzen geltend machen. BioI. Plant 9 (1967), 122-134. - - The influence of previous plant cultivation on soil nitrification. PI. Soil 32 (1970),50-60. PONNAMPERFMA, F. N.: The chemistry of submerged soils in relation to the growth and yield of rice. Unpublished Ph. D. Thesis, Cornell University, Ithaca, N.Y. (1955). Quoted from The Mineral Nutrition of the Rice Plant. Proc. Symp, Intern. Rice Research Inst., February 1964. Baltimore, Maryland, 1965. ROVIRA, A. D.: Interactions between plant roots and soil microorganisms. A. Rev. Microbiol. 19 (1965), 241-2(;(i. TODoRovA, B.: Effect of fertilization on the activity of cellulolytic bacteria and breakdown of cellulose in the soil. Proc. Syrnp. Soil Microbiology Symp. BioI. Hungarica II (1972), 139-142 [Soils Fert. 36 (1975), 4074J. VOJINOVIC, Z., and PETROVIC, V.: Effect of fertilizers on the microflora and microbiological processes in soil. Zemlj. Bilj. 12 (1963), 349-355. Authors' addresses: Dr. P. BHATTACHARYYA, Research Officer (Microbiology), Pulses and Oilseeds Research Station, Government of West Bengal, Berhampore Murshidabad, PIN 742101, West Bengal; Prof. Dr. B. K. DEY, Bidhan Chandra Krishi Viswa Vidyalaya, West Bengal; S. NATH and S. BANIK, College of Agriculture, Calcutta University, West Bengal, India.