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Phenols in rice soils
PHENOLS
IN RICE SOILS
A.V. RAO,K. P. RAJARAM,R.SIDDARAMAPPA N. SETHUNATHAN* Central
Rice Research (Acqtrd
Institute,
Cuttack-6,
Orissa.
and India
24 October 1974)
Summary-In a laboratory study on the distribution of phenols in rice soils, a humus-rich acid sulphate soil liberated more alkali-extractable phenols than an alluvial soil under both flooded and nonflooded conditions. Nonflooded conditions appeared to favour the release of phenols from the acid sulphate soil whereas in the alluvial soil no difference was apparent under the two water regimes. The addition of calcium carbonate to the acid sulphate soil further increased the production of phenols. Analysis of the solvent extract of the soils by thin-layer chromatography showed that both the soils contained vanillic acid and two unidentified phenols.
INTRODUCTION Decomposition of organic matter in predominantly anaerobic flooded soil liberates organic acids. These organic acids appear to persist in flooded soil (Steven-
son, 1967). Organic acids play an important role in the solubilization, mobilization and transport of mineral matter in the soil. Organic acids retard rice growth particularly at pH levels below 6.0. It has been demonstrated that even in neutral soils, organic acids can inhibit rice growth (Chandrasekaran and Yoshida, 1973). Until recently, in studies on organic acid accumulation in rice soils, great emphasis was given to the formation of aliphatic acids. The aliphatic acids reported to occur in flooded rice soils are formic, acetic. propionic, butyric, iso-butyric, valeric, lactic and succinic acids (Takai, 1958; Takijima, 1964 a and b; Wang rt al., 1967 a and b; Chandrasekaran and Yoshida, 1973). Wang et al. (1967~) reported that many agricultural soils of Taiwan under different crops contained phenolit acids at concentrations that can inhibit the growth of plants such as corn, rice, soybeans, sugarcane and wheat in culture solution. However, little is known on the distribution ofaromatic acids in rice soils although recently two phenolic acids, ferulic and sinnapic, have been detected in flooded soil solutions (Anon., 1970). This paper reports the phenolic acid contents of two acid rice soils of India under both flooded and nonflooded conditions. One of the soils used is a unique peaty soil from Kerala, India, and is known as Kari soil. This acid sulphate soil is characterized by high organic matter, extremely low pH, and high electrical conductivity. Despite these characteristics, rice is cultivated in this problem soil after suitable amendments such as flooding and subsequent draining and liming. The effect of liming on phenolic acid distribution in the acid sulphate soil was also investigated. MATERIALS AND METHODS
Procedure The air-dried soils (an alluvial soil from the Institute farm, pH 6.0, organic matter 1.35 per cent and the acid sulphate Kari soil from Kerala, pH 3.1, organic matter * Dr. N. Sethunathan
is corresponding
author. 227
26.15 per cent) were passed through a 2 mm sieve after grinding and placed in large test tubes (200 x 25 mm) in 20 g amounts. In one treatment, the soils were maintained at 75 per cent of water holding capacity and in the other, the soils were flooded with 25 ml of water to provide about 5cm of standing water. To determine the effect of liming on phenolic acids in the acid sulphate soil, 600 mg CaC03 were added to 20 g soil and the soils were flooded. The pH of the soil increased from 3.1 to 6.5 on addition of CaCO,. During incubation, the pH of the acid sulphate soil increased from 3.1 to 4.2 in 45 days after flooding whereas in samples amended with CaCO,, the pH rose to 6.8 during the same period. From 45 to 75 days, no further variations in pH were noticed. The pH of alluvial soil also increased by one unit in 45 days. Two replicate tubes were removed periodically for the extraction of phenolit acids. E.utraction Phenolic acids were extracted from the soils following the procedure of Henderson (1959) with certain modifications. For flooded soils, the contents of each tube were transferred to a 250 ml Erlenmeyer flask and were shaken for 30 min in a wrist action shaker with 30 ml 4% (w/v) NaOH + 5 ml H20 to provide a final 27: alkali concentration. The nonflooded soil samples were treated with 60 ml 2:/O (w/v) NdOH. In the case of the alluvial soil, the alkali-treated soil samples which were less colloidal in nature than the acid sulphate soil after alkali treatment were filtered. The alkali treatment of the soil samples was repeated again using 40 ml 2% NaOH. The alkali extracts were pooled and acidified to about pH 2.0 with concentrated HCl. Following acidification, a thick gelatinous precipitate was formed, in the peaty acid sulphate soil evidently due to the precipitation of humic substances; the precipitation was negligible in the alluvial soil. The extracts were filtered and the filtrate was made up to lOOm1 before extraction of phenols with ether. The precipitate formed following acidification of alkali extract of acid sulphate soil was treated with 2% NaOH, acidified with HCl and filtered. This was repeated 4-5 times. The filtrate of each treatment was made up to 100 ml and extracted with ether separately.
A. V. RAO rt d.
228
Twenty-five millilitre aliquots of the filtrate were extracted with ether twice with a I:2 filtrate-ether ratio followed by three extractions with a 1 :I ratio. The ether extracts were evaporated to dryness at room temperature and then the residues were dissolved in 3 ml methanol for determination of total phenols and individual phenols after separation by chromatography. Chromatography
RESULTS
of phenols)om
acid sulphate
1. Total phenols recovered from an acid sulphate soil during
Filtrate* First Second Third Fourth Fifth
-:
repeated
extractions
with alkali
/tg Phenol/20 g soil (catechol equivalent) Flooded Flooded + CaCO, 600 258 184 142 143
6
E 2
Alluvial
0
soil
I IO
I 20
Incubation
I 30
I 40
I 50
days
Fig. 1. Total phenols (ng/2Og soil in catechol equivalent) recovered from acid sulphate and alluvial soils. (0 -0) Nonflooded soil; (*------0) Flooded soil; (+. 0) Flooded soil + CaCO,.
soil
In the extraction method employed, the alkali extract of the soil was acidified to about pH 2.0. In order to determine whether the precipitate formed in the acid sulphate soil during acidification still contained some more phenols, the precipitate was treated with alkali, acidified and filtered. This was repeated 4 times. The phenols in the acidified filtrate of each extraction were extracted with ether. The results are presented in Table 1. The extraction of phenols from the acid sulphate soil was not evidently improved in the presence of CaCO,. The amount of phenol recovered by extraction immediately after CaCO, incorporation was comparable to that obtained without CaCO, (Table 1). Studies clearly indicated that repeated extractions from the precipitate appeared to be necessary for the recovery of the soil phenols from organic soils rich in humic materials such as the acid Table
‘\ e-
qf’phenols
The phenolic acids in the soil extracts were separated by thin-layer chromatography (tic). For separation, IO&150 ~1 of the solvent extract were spotted on 300,~m thick silica gel-G plates alongside authentic standards of phenols. The solvent extract of the soils was separated by tic in developing solvents such as benzeneemethanol-acetic acid (45:8:4), butanollpyridine-saturated aqueous NaCl (1: 1: 2tupper organic layer, and ethanollNHjPH,O (125: 20: 10). The plates were developed for a distance of IS cm in various solvents. After separation, the phenolic acids in the samples were compared with authentic vanillic, ferulic, hydroxybenzoic acids and catechol for their reaction to chromogenic agents such as 1% FeCl, solution, a solution of 0.5% diazotized sulfanilic acid (DSA) in 10% Na,C03 solution or Folin-Ciocalteau reagent (Bray and Thorpe, 1954; Randerath. 1966) or examined for U.V. fluorescence at 254 nm.
Extraction
‘\
I200
648 292 155 129 141
* Alkali-treated soil extract was acidified and filtered to provide the first filtrate. The precipitate formed during acidification was treated with alkali, acidified and filtered. This was repeated four times to provide the second, third, fourth and fifth filtrates.
sulphate soil used in this study. Cumulative values of total phenols only for 3 extractions are taken into consideration. Phenols
under,flooded
md rzor$looded
conditions
The total phenols in the acid sulphate and alluvial soils under hooded and nonllooded conditions are prcsented in Fig. 1. Generally, the phenolic acids in both soils increased until 18 days after incubation and then declined. Acid sulphate soil liberated more phenols than the alluvial soil under both water regimes at all incubation periods. In the acid sulphate soil, nonflooded soil yielded more phenols than the flooded soil, particularly at 30 and 50 days. A reverse trend was noticed with the alluvial soil except at 18 days. although the difference in the phenolic levels between flooded and nonflooded conditions was not very striking.
.Ejjkt qf’liming Addition of CaCO, to the acid sulphate soil increased the yield of total phenols at all incubation periods. The amount of phenols recovered from 20 g soil on the 30th day under flooded condition was I534 pg for soils amended with C&O, and 831 pg for unamended soils. Despite a decline in the phenolic levels in the amended soils between 30 and 50 days, the amended soil still yielded more total phenols than the unamended soil on the 50th day. Nature
of phenols
Chromatograms of solvent extracts of all soils revealed 3 spots (r.f. 0,00,0.32.0-66) under U.V. of which 2 spots (0.00,0.66) also gave positive reaction to Folin Ciocalteau reagent. Chromatography with authentic
Phenols
phenols showed that the r.f. value for vanillic acid was 0.73 when spotted singly, but its r.f. was lowered to 0.66 when spotted in combination with the solvent extract of the soil. Evidently the compound (r.f. 0.66) present in the soil extract was identical to vanillic acid. In addition, the compound (r.f. 0.66) in the sample and authentic vanillic acid gave a yellow reaction to DSA. Other compounds (0.00, 0.32) detected in the chromatograms of soil were not identified. Ferulic acid with r.f. close to vanillic acid turned pink to DSA sprays. No pink spot was visible in the chromdtograms sprayed with DSA indicating the absence of ferulic acid. p-Hydroxybenzoic acid and p-coumaric acid, which were known to occur in the soils, were also not detected in the chromatograms. The analysis showed little qualitative differences in the phenols of acid sulphate and alluvial soils under both flooded and nonflooded conditions. However, the acid sulphate soil contained higher concentrations of vanillic acid than the alluvial soil. Moreover, the acid sulphate soil amended with CaCO, released more vanillic acid than the unamended soil. DISCUSSION
The anaerobic conditions of flooded soils are known to favour the formation and accumulation of organic acids during the decomposition of organic matter (Stevenson, 1967). Leachates of peaty paddy soils of Japan have been shown to contain aliphatic acids and other substances that inhibited root development of rice (Takijima, 1962). According to our data (Fig. 1) soil extracts of the humus rich acid sulphate peaty soil contained more quantities of phenols than the alluvial soil with low organic matter content. Moreover, higher quantities of phenols were found in the acid sulphate soil under nonflooded conditions than under flooded conditions. This is in contrast to the accumulation of organic acids in flooded soil environment by several workers (Stevenson, 1967). As in the case of other biochemical substances. a rapid turnover of phenol can occur when the microbial activity is intense (Stevenson, 1967). The fact that the application of CaCO, to the flooded acid sulphate soil increased the phenol accumulation apparently due to optimal pH conditions favouring microbial activity is additional evidence for the probable role of soil microorganisms in phenol formation in peaty soil. We have evidence that application of CaCO, to the acid sulphate soil boosted up
229
in rice soils
the bacterial numbers in the soil leachates as well as in the rhizosphere of rice (Kaur and Kuruvila, unpublished data). The bacterial numbers in the leachates from unamended and amended soils were 300 and 27.300 g- ’ soil respectively.
Ack,lo~~/rciyrnlr,ItsThe authors thank Dr. S. Y. Padmanabhdn. Director. for critically going through the mdnuscript and Dr. R. Sridhar, Pool Officer. Plant Pathology Division. for helpful discussion. The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi, for financial assistance. K. P. Rajaram and R. Siddaramappa were supported by a grant from the International Atomic Energy Agency. Vienna, Austria.
REFEREKCES
ANONYMOUS (1970) Annual Report, International Rice Research Institute, Los Banos. Laguna, Philippines. BRAY H. G. and THOKP~ W. V. (1954) Analysis of phenolic compounds of interest in metabolism. Meth. Biochem Aimlysi.5 1, 27752. CHAU~RASLXARAN S. and Y~SHIDA T. (1973) Effect of organic acid transformations in submerged soils on growth of the rice plant, Soil Sci. Plunt Nutr. 19, 39-45. HENDERSON M. E. K. (1959) Release of aromatic compound from birch and spruce sawdust during decomposition by white rot fungi. Nururr. Land. 175, 634635. RANDEKA.~H K. (1966) Thin-ltrtvr Chro/nafogruphy. Academic Press. New York. STEVENSON.F. _I.(1967) Organic acids in soil. In Soil Biochrmist~y (A. D. McLaren and G. H. Peterson, Eds.) 1, pp. 119- 146, Marcel Dekker, New York. TAKAI Y. (1968) On quantitative analysis or organic acids in paddy soil. J. Sci. Soil MUIIUI.B. Tokyo 28, 7-10. TAKIJIM Y. (1962) Nippon Dojo-Hiryogaku Zuzzhi. 33, 415, cited by Stevenson F. J. ( 1967). TAKUIMA Y. (1964a) Studies on organic acids in paddy held soils with reference to their inhibitoryetfectson the growth of rice plants-l. Soil Sci. Pht Nfm. IO, 1421. TAKIJMA Y. (1964b) Studies on organic acids in paddy field soils with reference to their inhibitory effects on the growth of rice plants-~- 2. Soil SC;. Phr. Nutr. IO, 22-29. WANG T. S. C.. CH~NG S. Y. and T~JZIG H. (1967a) Extraction and analysis of soil orgamc acids. Soil Sci. 103, 36& 366. WAXG T. S. C.. CHLNC; S. Y. and TUNC; H. (1967b) Dynamics of soil organic acids. Soil Sci. 104, I38 144. WANG T. S. C.. YANG T. K. and Crrr ANC;T. T. (1967~) Soil phenolic acids as plant growth inhibitors. Soil Sci. 103, 239-246.
IN RICE SOILS
A.V. RAO,K. P. RAJARAM,R.SIDDARAMAPPA N. SETHUNATHAN* Central
Rice Research (Acqtrd
Institute,
Cuttack-6,
Orissa.
and India
24 October 1974)
Summary-In a laboratory study on the distribution of phenols in rice soils, a humus-rich acid sulphate soil liberated more alkali-extractable phenols than an alluvial soil under both flooded and nonflooded conditions. Nonflooded conditions appeared to favour the release of phenols from the acid sulphate soil whereas in the alluvial soil no difference was apparent under the two water regimes. The addition of calcium carbonate to the acid sulphate soil further increased the production of phenols. Analysis of the solvent extract of the soils by thin-layer chromatography showed that both the soils contained vanillic acid and two unidentified phenols.
INTRODUCTION Decomposition of organic matter in predominantly anaerobic flooded soil liberates organic acids. These organic acids appear to persist in flooded soil (Steven-
son, 1967). Organic acids play an important role in the solubilization, mobilization and transport of mineral matter in the soil. Organic acids retard rice growth particularly at pH levels below 6.0. It has been demonstrated that even in neutral soils, organic acids can inhibit rice growth (Chandrasekaran and Yoshida, 1973). Until recently, in studies on organic acid accumulation in rice soils, great emphasis was given to the formation of aliphatic acids. The aliphatic acids reported to occur in flooded rice soils are formic, acetic. propionic, butyric, iso-butyric, valeric, lactic and succinic acids (Takai, 1958; Takijima, 1964 a and b; Wang rt al., 1967 a and b; Chandrasekaran and Yoshida, 1973). Wang et al. (1967~) reported that many agricultural soils of Taiwan under different crops contained phenolit acids at concentrations that can inhibit the growth of plants such as corn, rice, soybeans, sugarcane and wheat in culture solution. However, little is known on the distribution ofaromatic acids in rice soils although recently two phenolic acids, ferulic and sinnapic, have been detected in flooded soil solutions (Anon., 1970). This paper reports the phenolic acid contents of two acid rice soils of India under both flooded and nonflooded conditions. One of the soils used is a unique peaty soil from Kerala, India, and is known as Kari soil. This acid sulphate soil is characterized by high organic matter, extremely low pH, and high electrical conductivity. Despite these characteristics, rice is cultivated in this problem soil after suitable amendments such as flooding and subsequent draining and liming. The effect of liming on phenolic acid distribution in the acid sulphate soil was also investigated. MATERIALS AND METHODS
Procedure The air-dried soils (an alluvial soil from the Institute farm, pH 6.0, organic matter 1.35 per cent and the acid sulphate Kari soil from Kerala, pH 3.1, organic matter * Dr. N. Sethunathan
is corresponding
author. 227
26.15 per cent) were passed through a 2 mm sieve after grinding and placed in large test tubes (200 x 25 mm) in 20 g amounts. In one treatment, the soils were maintained at 75 per cent of water holding capacity and in the other, the soils were flooded with 25 ml of water to provide about 5cm of standing water. To determine the effect of liming on phenolic acids in the acid sulphate soil, 600 mg CaC03 were added to 20 g soil and the soils were flooded. The pH of the soil increased from 3.1 to 6.5 on addition of CaCO,. During incubation, the pH of the acid sulphate soil increased from 3.1 to 4.2 in 45 days after flooding whereas in samples amended with CaCO,, the pH rose to 6.8 during the same period. From 45 to 75 days, no further variations in pH were noticed. The pH of alluvial soil also increased by one unit in 45 days. Two replicate tubes were removed periodically for the extraction of phenolit acids. E.utraction Phenolic acids were extracted from the soils following the procedure of Henderson (1959) with certain modifications. For flooded soils, the contents of each tube were transferred to a 250 ml Erlenmeyer flask and were shaken for 30 min in a wrist action shaker with 30 ml 4% (w/v) NaOH + 5 ml H20 to provide a final 27: alkali concentration. The nonflooded soil samples were treated with 60 ml 2:/O (w/v) NdOH. In the case of the alluvial soil, the alkali-treated soil samples which were less colloidal in nature than the acid sulphate soil after alkali treatment were filtered. The alkali treatment of the soil samples was repeated again using 40 ml 2% NaOH. The alkali extracts were pooled and acidified to about pH 2.0 with concentrated HCl. Following acidification, a thick gelatinous precipitate was formed, in the peaty acid sulphate soil evidently due to the precipitation of humic substances; the precipitation was negligible in the alluvial soil. The extracts were filtered and the filtrate was made up to lOOm1 before extraction of phenols with ether. The precipitate formed following acidification of alkali extract of acid sulphate soil was treated with 2% NaOH, acidified with HCl and filtered. This was repeated 4-5 times. The filtrate of each treatment was made up to 100 ml and extracted with ether separately.
A. V. RAO rt d.
228
Twenty-five millilitre aliquots of the filtrate were extracted with ether twice with a I:2 filtrate-ether ratio followed by three extractions with a 1 :I ratio. The ether extracts were evaporated to dryness at room temperature and then the residues were dissolved in 3 ml methanol for determination of total phenols and individual phenols after separation by chromatography. Chromatography
RESULTS
of phenols)om
acid sulphate
1. Total phenols recovered from an acid sulphate soil during
Filtrate* First Second Third Fourth Fifth
-:
repeated
extractions
with alkali
/tg Phenol/20 g soil (catechol equivalent) Flooded Flooded + CaCO, 600 258 184 142 143
6
E 2
Alluvial
0
soil
I IO
I 20
Incubation
I 30
I 40
I 50
days
Fig. 1. Total phenols (ng/2Og soil in catechol equivalent) recovered from acid sulphate and alluvial soils. (0 -0) Nonflooded soil; (*------0) Flooded soil; (+. 0) Flooded soil + CaCO,.
soil
In the extraction method employed, the alkali extract of the soil was acidified to about pH 2.0. In order to determine whether the precipitate formed in the acid sulphate soil during acidification still contained some more phenols, the precipitate was treated with alkali, acidified and filtered. This was repeated 4 times. The phenols in the acidified filtrate of each extraction were extracted with ether. The results are presented in Table 1. The extraction of phenols from the acid sulphate soil was not evidently improved in the presence of CaCO,. The amount of phenol recovered by extraction immediately after CaCO, incorporation was comparable to that obtained without CaCO, (Table 1). Studies clearly indicated that repeated extractions from the precipitate appeared to be necessary for the recovery of the soil phenols from organic soils rich in humic materials such as the acid Table
‘\ e-
qf’phenols
The phenolic acids in the soil extracts were separated by thin-layer chromatography (tic). For separation, IO&150 ~1 of the solvent extract were spotted on 300,~m thick silica gel-G plates alongside authentic standards of phenols. The solvent extract of the soils was separated by tic in developing solvents such as benzeneemethanol-acetic acid (45:8:4), butanollpyridine-saturated aqueous NaCl (1: 1: 2tupper organic layer, and ethanollNHjPH,O (125: 20: 10). The plates were developed for a distance of IS cm in various solvents. After separation, the phenolic acids in the samples were compared with authentic vanillic, ferulic, hydroxybenzoic acids and catechol for their reaction to chromogenic agents such as 1% FeCl, solution, a solution of 0.5% diazotized sulfanilic acid (DSA) in 10% Na,C03 solution or Folin-Ciocalteau reagent (Bray and Thorpe, 1954; Randerath. 1966) or examined for U.V. fluorescence at 254 nm.
Extraction
‘\
I200
648 292 155 129 141
* Alkali-treated soil extract was acidified and filtered to provide the first filtrate. The precipitate formed during acidification was treated with alkali, acidified and filtered. This was repeated four times to provide the second, third, fourth and fifth filtrates.
sulphate soil used in this study. Cumulative values of total phenols only for 3 extractions are taken into consideration. Phenols
under,flooded
md rzor$looded
conditions
The total phenols in the acid sulphate and alluvial soils under hooded and nonllooded conditions are prcsented in Fig. 1. Generally, the phenolic acids in both soils increased until 18 days after incubation and then declined. Acid sulphate soil liberated more phenols than the alluvial soil under both water regimes at all incubation periods. In the acid sulphate soil, nonflooded soil yielded more phenols than the flooded soil, particularly at 30 and 50 days. A reverse trend was noticed with the alluvial soil except at 18 days. although the difference in the phenolic levels between flooded and nonflooded conditions was not very striking.
.Ejjkt qf’liming Addition of CaCO, to the acid sulphate soil increased the yield of total phenols at all incubation periods. The amount of phenols recovered from 20 g soil on the 30th day under flooded condition was I534 pg for soils amended with C&O, and 831 pg for unamended soils. Despite a decline in the phenolic levels in the amended soils between 30 and 50 days, the amended soil still yielded more total phenols than the unamended soil on the 50th day. Nature
of phenols
Chromatograms of solvent extracts of all soils revealed 3 spots (r.f. 0,00,0.32.0-66) under U.V. of which 2 spots (0.00,0.66) also gave positive reaction to Folin Ciocalteau reagent. Chromatography with authentic
Phenols
phenols showed that the r.f. value for vanillic acid was 0.73 when spotted singly, but its r.f. was lowered to 0.66 when spotted in combination with the solvent extract of the soil. Evidently the compound (r.f. 0.66) present in the soil extract was identical to vanillic acid. In addition, the compound (r.f. 0.66) in the sample and authentic vanillic acid gave a yellow reaction to DSA. Other compounds (0.00, 0.32) detected in the chromatograms of soil were not identified. Ferulic acid with r.f. close to vanillic acid turned pink to DSA sprays. No pink spot was visible in the chromdtograms sprayed with DSA indicating the absence of ferulic acid. p-Hydroxybenzoic acid and p-coumaric acid, which were known to occur in the soils, were also not detected in the chromatograms. The analysis showed little qualitative differences in the phenols of acid sulphate and alluvial soils under both flooded and nonflooded conditions. However, the acid sulphate soil contained higher concentrations of vanillic acid than the alluvial soil. Moreover, the acid sulphate soil amended with CaCO, released more vanillic acid than the unamended soil. DISCUSSION
The anaerobic conditions of flooded soils are known to favour the formation and accumulation of organic acids during the decomposition of organic matter (Stevenson, 1967). Leachates of peaty paddy soils of Japan have been shown to contain aliphatic acids and other substances that inhibited root development of rice (Takijima, 1962). According to our data (Fig. 1) soil extracts of the humus rich acid sulphate peaty soil contained more quantities of phenols than the alluvial soil with low organic matter content. Moreover, higher quantities of phenols were found in the acid sulphate soil under nonflooded conditions than under flooded conditions. This is in contrast to the accumulation of organic acids in flooded soil environment by several workers (Stevenson, 1967). As in the case of other biochemical substances. a rapid turnover of phenol can occur when the microbial activity is intense (Stevenson, 1967). The fact that the application of CaCO, to the flooded acid sulphate soil increased the phenol accumulation apparently due to optimal pH conditions favouring microbial activity is additional evidence for the probable role of soil microorganisms in phenol formation in peaty soil. We have evidence that application of CaCO, to the acid sulphate soil boosted up
229
in rice soils
the bacterial numbers in the soil leachates as well as in the rhizosphere of rice (Kaur and Kuruvila, unpublished data). The bacterial numbers in the leachates from unamended and amended soils were 300 and 27.300 g- ’ soil respectively.
Ack,lo~~/rciyrnlr,ItsThe authors thank Dr. S. Y. Padmanabhdn. Director. for critically going through the mdnuscript and Dr. R. Sridhar, Pool Officer. Plant Pathology Division. for helpful discussion. The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi, for financial assistance. K. P. Rajaram and R. Siddaramappa were supported by a grant from the International Atomic Energy Agency. Vienna, Austria.
REFEREKCES
ANONYMOUS (1970) Annual Report, International Rice Research Institute, Los Banos. Laguna, Philippines. BRAY H. G. and THOKP~ W. V. (1954) Analysis of phenolic compounds of interest in metabolism. Meth. Biochem Aimlysi.5 1, 27752. CHAU~RASLXARAN S. and Y~SHIDA T. (1973) Effect of organic acid transformations in submerged soils on growth of the rice plant, Soil Sci. Plunt Nutr. 19, 39-45. HENDERSON M. E. K. (1959) Release of aromatic compound from birch and spruce sawdust during decomposition by white rot fungi. Nururr. Land. 175, 634635. RANDEKA.~H K. (1966) Thin-ltrtvr Chro/nafogruphy. Academic Press. New York. STEVENSON.F. _I.(1967) Organic acids in soil. In Soil Biochrmist~y (A. D. McLaren and G. H. Peterson, Eds.) 1, pp. 119- 146, Marcel Dekker, New York. TAKAI Y. (1968) On quantitative analysis or organic acids in paddy soil. J. Sci. Soil MUIIUI.B. Tokyo 28, 7-10. TAKIJIM Y. (1962) Nippon Dojo-Hiryogaku Zuzzhi. 33, 415, cited by Stevenson F. J. ( 1967). TAKUIMA Y. (1964a) Studies on organic acids in paddy held soils with reference to their inhibitoryetfectson the growth of rice plants-l. Soil Sci. Pht Nfm. IO, 1421. TAKIJMA Y. (1964b) Studies on organic acids in paddy field soils with reference to their inhibitory effects on the growth of rice plants-~- 2. Soil SC;. Phr. Nutr. IO, 22-29. WANG T. S. C.. CH~NG S. Y. and T~JZIG H. (1967a) Extraction and analysis of soil orgamc acids. Soil Sci. 103, 36& 366. WAXG T. S. C.. CHLNC; S. Y. and TUNC; H. (1967b) Dynamics of soil organic acids. Soil Sci. 104, I38 144. WANG T. S. C.. YANG T. K. and Crrr ANC;T. T. (1967~) Soil phenolic acids as plant growth inhibitors. Soil Sci. 103, 239-246.