SOIL SCIENCE AND MICROBIOLOGY

SOIL SCIENCE

PRE-SEMI-DWARF ERA

Prior to the introduction of semi-dwarf high-yielding varieties, the main emphasis was to study the behaviour of submerged (waterlogged) rice soils in relation to crop production and to carry out investigations on the chemistry of rice crop nutrition. Systematic studies on chemical and physical properties of submerged rice soils were undertaken to trace changes in nutrient availability and to ameliorate conditions for successful crop production.

Changes in nitrogen and phosphate and their availability in submerged soil to crop during its life cycle, and uptake of these nutrients by varieties of different durations at various growth stages were investigated. These included water and pot culture studies to determine the form and time of application of nitrogen, phosphate and potash to rice crop, the diagnostic symptoms of nitrogen, phosphate, potash, calcium and magnesium deficiencies in rice and developing rapid methods of testing submerged soils for available nutrients and of plant tissues of nutrient reserve.

Studies on changes in nitrogen revealed that the losses of nitrogen from submerged soil occur due to oxidation of ammonium nitrogen from the surface-applied ammonium sulphate in the surface soils and its subsequent de-nitrification in the reduced sub-surface layer together amount to 30-40%. In addition, 10-12% losses of nitrogen was shown to occur in drainage waters, particularly, when there was a heavy downpour of rain within 24-48 hours after fertilizer application. It was demonstrated that these nitrogen losses can be considerably reduced by deep placement or sub-surface application of the fertilizer in the reduced layer of the soil which enhances the fertilizer efficiency, and, thereby augments rice yield. Based on this, different methods of sub-surface application under diverse soil-water conditions were recommended.

The importance of soil drying before cultivation to increase the ammonifying capacity of soil was emphasized particularly for intensive cultivation areas where two or three successive rice crops are to be grown on the same land.

Application of lime at 1500-2000 kg/ha on the surface soil, in puddle, just before transplanting was found to have mineralizing effect on soil nitrogen in lowland rice soils as it releases about 30-40 kg of easily available ammoniacal nitrogen per hectare thus augmenting rice yield by 350-400 kg/ha.

Soaking of seeds in 10-20% solution of dibasic potassium phosphate for 24 hours and drying it before sowing was found to give an yield increase of about 8-10% in direct-seeded rice.

Studies on fertility value of urea showed that its efficiency for increasing rice yields can be enhanced, if the fertilizer is thoroughly mixed up with soil 24-48 hours before flooding and puddling. Urea applied after flooding the soil did not give good response.

Fundamental studies on the chemistry of crop nutrition have provided valuable information on the appropriate time for fertilizer application for harvesting maximum rice. Varietal differences in the process of nitrogen uptake which is related to different growth phases of the plant were noticed.

Studies on the influence of nitrogen application on various plant characters revealed that the basal application improves the setting of grains and increased the grain weight.

With the launching of indica-japonica hybridization program at this institute in early fifties, comparative studies with the indica and japonica varieties were undertaken. These investigations showed that indica varieties were not able to utilize nitrogen as efficiently as the japonica varieties for grain production. The indicas gave maximum grain yield at about 20-40 ppm nitrogen over which the yield was considerably reduced, while the japonicas continued to yield better even above 60 ppm nitrogen.

SEMI-DWARF ERA

With the introduction of semi-dwarf high-yielding varieties during mid-sixties, information of basic nature on fertilizer and soil management were obtained. These have helped in working out production technology for the high-yielding varieties. Some of these practices continue to be adopted in highly productive regions in north-western and southern parts of India, where farmers have been getting high yields.

Nitrogen management

Since urea fertilizer was found to be inferior to ammonium sulphate, a new method of urea application was developed to increase its efficiency by incubating it with 5-6 times of its weight of soil for 48-72 hours. During this period, urea gets hydrolyzed to ammonium carbonate, and, thus chances of loss of nitrogen during this period are eliminated.

During eighties, more attention was paid for studies on nitrogen management in rainfed lowlands, where top dressing of nitrogen was not possible due to adverse soil-water conditions. Early application of the entire amount of nitrogen either at seeding or at 3 weeks after germination, when water begins to accumulate in the field was tested. Application of sulphur-coated urea, neem oil or neem cake-coated urea, urea super-granules or soil pre-treated urea in single dose of 40 kg N/ha at either of the two earlier stages gave grain yields (3.5 to 4.0 t/ha) which was comparable to split application of urea.

Varietal response to applied nitrogen with elite breeding lines gave production function equations of the form : y- a+bx+cx2, where 'y' is the grain yield (t/ha), 'x' is the nitrogen applied (kg/ha) and 'a', 'b', 'c' are constants. This study has shown the possibility of obtaining grain yield in the range of 3.9-5.0 t/ha with a single dose application of 40 kg N/ha, three weeks after germination.

The weekly photo-period sensitive medium and late duration varieties had a vegetative lag period from the maximum tiller number stage to panicle initiation when there was little or no increase in dry matter and nitrogen uptake. This resulted in the accumulation of mineral nitrogen in the soil during this period, which was used by the crop after panicle initiation to meet its high N requirement.

The need to build inherent fertility of the soil which could favour increased release of soil nitrogen has been pointed out. Application of compost (5 t/ha) or rice straw (10 t/ha) resulted in a build up of fertility.

Basally applied 15N was utilized for vegetative growth of the crop whereas top dressed 15N at panicle initiation stage was utilized for grain formation. For increasing the efficiency of nitrogen in rainfed lowland, several nitrogen management practices were undertaken. Use of urea super-granule (placement in the reduced zone), integrated nitrogen management using green manure (Sesbania aculeata) and urea in 1:1 proportion on nitrogen basis were found to be better.

For direct seeded rainfed lowland rice suitable nitrogen management practices involving band placement of urea in dry soil at seeding, integrated nitrogen management and USG applications were developed. Details on the transformation of fertilizer nitrogen in flooded soil-rice ecosystem were studied using 15N tagged fertilizers. Fixation of atmospheric nitrogen by green manuring species like S. aculeata and S. rostrata and suitability of these species for rice were studied in relation to nitrogen accumulation, decomposition and mineralisation in the soil and growth and dry matter production of rice. It was observed that S. aculeata was better up to 45 days of its growth, beyond this S. rostrata took over. Integrated use of bio, organic and chemical fertilizers was better than the use of chemical fertilizer alone.

Treatment

Plant recovery

Retained in soil

Unaccounted

   

Dry season

 
Prilled urea

33.9

20.3

45.8

Urea super-granules

50.4

25.3

24.2

S.E.

2.2

1.4

_

   

Wet season

 
Prilled urea

33.6

23.0

43.4

Urea super-granules

43.8

22.5

33.7

S.E.

1.8

2.2

_

Soil management

Liming acid soils for waterlogged rice was not required for their reclamation. Compacting the light textured soils to achieve bulk density of 1.58 to 1.59 gm/cc resulted in higher grain yield and better utilization of applied nutrients. In some soils, P and K were more limiting for rice production than nitrogen emphasizing the importance of soil test prior to rice cultivation for fixing the fertilizer schedule.

Phosphate management

Measures for increasing the efficiency of citrate soluble-P and insoluble-P for growing rice in different soil types were undertaken.

Phosphate rocks from middle-east and US, which had low P and high CaCO3 applied two weeks before flooding to acid soils gave comparable result to water soluble P, but these were not effective in neutral and alkaline soil. On the other hand, indigenous phosphate rocks collected from different P mines all over the country, were found unsuitable for direct use even in acid soils. Measures for increasing the efficiency of some of these phosphate rocks indicated that their thermal products with Na2CO3 were better. Application of these phosphate rocks to green manure crop and its subsequent incorporation to soil for rice cultivation with no further P application was found to increase their efficiency. However, this needs continuous application of phosphate rocks at least for four years. Acidulation of phosphate rocks to an extent of 25% for acid soils and 50% for neutral and alkaline soils were sufficient for giving comparable result with water soluble P sources. Acidulation with HCl was as good as that of H2SO4 or NHO3.

A chemical-statistical model was developed for characterizing phosphate rocks in the absence of X-ray diffraction. Based on these information, possible methods for economic utilization of these natural resources have been suggested.

Studies on potassium

Basic studies on the transformation and availability of potassium in flooded soils of different types of intensive cropping of rice show that the potassium supplying power of soil depended on the ability of the soil potassium reserve to get converted into non-exchangeable form which in turn met the potassium requirement of the crop. For the first time, the exhaustion potential of potassium for rice crop was worked out.

Secondary nutrients in rice and rice soils

Suitable methods for extraction and estimation of Si and S in rice soils were standardized. Si extracted with 0.025 M citric acid in dry soils as well as soils flooded for 20 days gave indications of Si supplying power of these soils in relation to its uptake by rice. Uptake of Si in relation to rice growth was worked out in rice varieties of different durations. This showed that with increase in duration, Si content in straw was increased but grain Si remained unaffected.

Bray's I extractant was found to be the best for extraction of available S in flooded soil. Flooding increased availability of S up to 21 days in soils. S in solution from 0-5 ppm was deficient, 5-40 ppm was sufficient and above 40 ppm was in excess to the plants.

Micro-nutrients in rice and rice soils

With the introduction of high yielding rice varieties and intensive rice cultivation, problems due to micro-nutrient deficiency or toxicity have been increasingly noticed. Suitable methods of extraction and estimation of Fe, Mn, Zn, Cu, B and Mo were developed. Micro-nutrient nutrition of high-yielding rice varieties in relation to growth and duration of the crop was undertaken.

Heavy metal pollution in rice and rice soils

Studies on this aspect intensified during mid-eighties showed that flooding decreased extractable Cd, Ni, Pb and Cr (VI), irrespective of soil types. The toxic form, Cr (VI) was totally convertible to Cr (III), a non-toxic form, within 10 days of flooding in all types of soils except in laterite soil, indicating its differential behaviour in different soils. Leachability of Cd was very low in alluvial sandy loam soil suggesting a remote possibility of ground water pollution.

Chemistry of submerged soils

Detailed and systematic studies on chemistry of submerged soils and transformation of nutrients were carried out during late sixties in twenty different types of soils, which indicated that pH of the acid soils increased and that of alkali soils decreased due to flooding. Eh of the soil decreased which was followed by a slight increase. EC of the soils increased because of flooding due to increased availability of NH4-N, K, Ca and Mg. Different prediction equations were worked out for nutrient transformation under submergence in different soil types.

Long-term fertilizer experiment

A long-term fertilizer experiment started in 1969 to study the effect of intensive cropping with the use of NPK with and without compost application on the changes in soil properties and productivity. There was response to application of N from beginning and response to compost was obtained from the fourth year of cropping. The grain yield was steady during the course of experiments. No response to P and K was obtained till date. The pH of the soil has decreased slightly. Compost application increased organic carbon of soil whereas with no compost its level decreased. Available P and K in soil in control plots and where they are not applied have decreased slightly but this was not reflected in grain yield. Nitrogen balance was positive in treatments receiving nitrogenous fertilizer.

Studies on problem soils

Intensive laboratory and pot experiments on low productive problem soils like acid sulphate soils of Kerala and acid, red and laterite soils were carried out for their reclamation for increasing rice production. Low pH, high salt content, high Fe and Mn and low P and high soil reduction were found to be the causes of low production in acid sulphate soils. The ameliorating effect of lime, MnO2 and NO3 and Fe(OH)3 on acid sulphate soils lasted for short period of only one cropping season. On the other hand, MgCO3 and Mg silicate had somewhat longer lasting ameliorating effect. Problems with acid, red and laterite soils were due to high Fe and Al in soil with flooding and also nutrient stress like P and Zn deficiency and low organic matter. The soils were reclaimed by liming and suitable nutrient management practices. Studies on Fe toxicity indicated that this problem could be overcome by balanced fertilizer application, liming, potash application and growing suitable rice varieties.

Studies on physical properties of rice soils

Results obtained from soil moisture studies indicated that moisture at 70-80% of water holding capacity after flowering was adequate for yield and N uptake of plant. Flooding of soil up to 45-60 days of planting was essential for increasing grain yield. During winter, ground water table fluctuated from 64 to 75 cm soil depth and contributed as much as 50 cm water to the 10-40 cm depth zone over a period of 52 days in loamy sandy soil. In summer when ground water table fluctuated between 80-90 cm, as much as 26 cm of water was supplied over a period of 31 days.

Experiments carried out on tillage for six years showed that after three years of continuous no tillage, rice yield declined. Under high water table condition, tillage in the wet season could be avoided. Dry season tillage increased the yield of blackgram and fodder cowpea irrespective of wet season ploughing. It is possible to have a groundnut crop after lowland rice on residual soil moisture by restricting the tillage to seed zone depth or by no tillage sowing and a subsequent tillage between the rows after seedling emergence. Puddling accelerated mineralisation of soil N and increased the concentration of K and Ca in soil solution.

Diffusibility of water to germinating seeds of cowpea and soybean decreased at zero water potential but increased at -2 and -10 bar water potential.

SOIL SCIENCE AND MICROBIOLOGY

SOIL SCIENCE

PRE-SEMI-DWARF ERA

Prior to the introduction of semi-dwarf high-yielding varieties, the main emphasis was to study the behaviour of submerged (waterlogged) rice soils in relation to crop production and to carry out investigations on the chemistry of rice crop nutrition. Systematic studies on chemical and physical properties of submerged rice soils were undertaken to trace changes in nutrient availability and to ameliorate conditions for successful crop production.

Changes in nitrogen and phosphate and their availability in submerged soil to crop during its life cycle, and uptake of these nutrients by varieties of different durations at various growth stages were investigated. These included water and pot culture studies to determine the form and time of application of nitrogen, phosphate and potash to rice crop, the diagnostic symptoms of nitrogen, phosphate, potash, calcium and magnesium deficiencies in rice and developing rapid methods of testing submerged soils for available nutrients and of plant tissues of nutrient reserve.

Studies on changes in nitrogen revealed that the losses of nitrogen from submerged soil occur due to oxidation of ammonium nitrogen from the surface-applied ammonium sulphate in the surface soils and its subsequent de-nitrification in the reduced sub-surface layer together amount to 30-40%. In addition, 10-12% losses of nitrogen was shown to occur in drainage waters, particularly, when there was a heavy downpour of rain within 24-48 hours after fertilizer application. It was demonstrated that these nitrogen losses can be considerably reduced by deep placement or sub-surface application of the fertilizer in the reduced layer of the soil which enhances the fertilizer efficiency, and, thereby augments rice yield. Based on this, different methods of sub-surface application under diverse soil-water conditions were recommended.

The importance of soil drying before cultivation to increase the ammonifying capacity of soil was emphasized particularly for intensive cultivation areas where two or three successive rice crops are to be grown on the same land.

Application of lime at 1500-2000 kg/ha on the surface soil, in puddle, just before transplanting was found to have mineralizing effect on soil nitrogen in lowland rice soils as it releases about 30-40 kg of easily available ammoniacal nitrogen per hectare thus augmenting rice yield by 350-400 kg/ha.

Soaking of seeds in 10-20% solution of dibasic potassium phosphate for 24 hours and drying it before sowing was found to give an yield increase of about 8-10% in direct-seeded rice.

Studies on fertility value of urea showed that its efficiency for increasing rice yields can be enhanced, if the fertilizer is thoroughly mixed up with soil 24-48 hours before flooding and puddling. Urea applied after flooding the soil did not give good response.

Fundamental studies on the chemistry of crop nutrition have provided valuable information on the appropriate time for fertilizer application for harvesting maximum rice. Varietal differences in the process of nitrogen uptake which is related to different growth phases of the plant were noticed.

Studies on the influence of nitrogen application on various plant characters revealed that the basal application improves the setting of grains and increased the grain weight.

With the launching of indica-japonica hybridization program at this institute in early fifties, comparative studies with the indica and japonica varieties were undertaken. These investigations showed that indica varieties were not able to utilize nitrogen as efficiently as the japonica varieties for grain production. The indicas gave maximum grain yield at about 20-40 ppm nitrogen over which the yield was considerably reduced, while the japonicas continued to yield better even above 60 ppm nitrogen.

SEMI-DWARF ERA

With the introduction of semi-dwarf high-yielding varieties during mid-sixties, information of basic nature on fertilizer and soil management were obtained. These have helped in working out production technology for the high-yielding varieties. Some of these practices continue to be adopted in highly productive regions in north-western and southern parts of India, where farmers have been getting high yields.

Nitrogen management

Since urea fertilizer was found to be inferior to ammonium sulphate, a new method of urea application was developed to increase its efficiency by incubating it with 5-6 times of its weight of soil for 48-72 hours. During this period, urea gets hydrolyzed to ammonium carbonate, and, thus chances of loss of nitrogen during this period are eliminated.

During eighties, more attention was paid for studies on nitrogen management in rainfed lowlands, where top dressing of nitrogen was not possible due to adverse soil-water conditions. Early application of the entire amount of nitrogen either at seeding or at 3 weeks after germination, when water begins to accumulate in the field was tested. Application of sulphur-coated urea, neem oil or neem cake-coated urea, urea super-granules or soil pre-treated urea in single dose of 40 kg N/ha at either of the two earlier stages gave grain yields (3.5 to 4.0 t/ha) which was comparable to split application of urea.

Varietal response to applied nitrogen with elite breeding lines gave production function equations of the form : y- a+bx+cx2, where 'y' is the grain yield (t/ha), 'x' is the nitrogen applied (kg/ha) and 'a', 'b', 'c' are constants. This study has shown the possibility of obtaining grain yield in the range of 3.9-5.0 t/ha with a single dose application of 40 kg N/ha, three weeks after germination.

The weekly photo-period sensitive medium and late duration varieties had a vegetative lag period from the maximum tiller number stage to panicle initiation when there was little or no increase in dry matter and nitrogen uptake. This resulted in the accumulation of mineral nitrogen in the soil during this period, which was used by the crop after panicle initiation to meet its high N requirement.

The need to build inherent fertility of the soil which could favour increased release of soil nitrogen has been pointed out. Application of compost (5 t/ha) or rice straw (10 t/ha) resulted in a build up of fertility.

Basally applied 15N was utilized for vegetative growth of the crop whereas top dressed 15N at panicle initiation stage was utilized for grain formation. For increasing the efficiency of nitrogen in rainfed lowland, several nitrogen management practices were undertaken. Use of urea super-granule (placement in the reduced zone), integrated nitrogen management using green manure (Sesbania aculeata) and urea in 1:1 proportion on nitrogen basis were found to be better.

For direct seeded rainfed lowland rice suitable nitrogen management practices involving band placement of urea in dry soil at seeding, integrated nitrogen management and USG applications were developed. Details on the transformation of fertilizer nitrogen in flooded soil-rice ecosystem were studied using 15N tagged fertilizers. Fixation of atmospheric nitrogen by green manuring species like S. aculeata and S. rostrata and suitability of these species for rice were studied in relation to nitrogen accumulation, decomposition and mineralisation in the soil and growth and dry matter production of rice. It was observed that S. aculeata was better up to 45 days of its growth, beyond this S. rostrata took over. Integrated use of bio, organic and chemical fertilizers was better than the use of chemical fertilizer alone.

Treatment

Plant recovery

Retained in soil

Unaccounted

   

Dry season

 
Prilled urea

33.9

20.3

45.8

Urea super-granules

50.4

25.3

24.2

S.E.

2.2

1.4

_

   

Wet season

 
Prilled urea

33.6

23.0

43.4

Urea super-granules

43.8

22.5

33.7

S.E.

1.8

2.2

_

Soil management

Liming acid soils for waterlogged rice was not required for their reclamation. Compacting the light textured soils to achieve bulk density of 1.58 to 1.59 gm/cc resulted in higher grain yield and better utilization of applied nutrients. In some soils, P and K were more limiting for rice production than nitrogen emphasizing the importance of soil test prior to rice cultivation for fixing the fertilizer schedule.

Phosphate management

Measures for increasing the efficiency of citrate soluble-P and insoluble-P for growing rice in different soil types were undertaken.

Phosphate rocks from middle-east and US, which had low P and high CaCO3 applied two weeks before flooding to acid soils gave comparable result to water soluble P, but these were not effective in neutral and alkaline soil. On the other hand, indigenous phosphate rocks collected from different P mines all over the country, were found unsuitable for direct use even in acid soils. Measures for increasing the efficiency of some of these phosphate rocks indicated that their thermal products with Na2CO3 were better. Application of these phosphate rocks to green manure crop and its subsequent incorporation to soil for rice cultivation with no further P application was found to increase their efficiency. However, this needs continuous application of phosphate rocks at least for four years. Acidulation of phosphate rocks to an extent of 25% for acid soils and 50% for neutral and alkaline soils were sufficient for giving comparable result with water soluble P sources. Acidulation with HCl was as good as that of H2SO4 or NHO3.

A chemical-statistical model was developed for characterizing phosphate rocks in the absence of X-ray diffraction. Based on these information, possible methods for economic utilization of these natural resources have been suggested.

Studies on potassium

Basic studies on the transformation and availability of potassium in flooded soils of different types of intensive cropping of rice show that the potassium supplying power of soil depended on the ability of the soil potassium reserve to get converted into non-exchangeable form which in turn met the potassium requirement of the crop. For the first time, the exhaustion potential of potassium for rice crop was worked out.

Secondary nutrients in rice and rice soils

 

Suitable methods for extraction and estimation of Si and S in rice soils were standardized. Si extracted with 0.025 M citric acid in dry soils as well as soils flooded for 20 days gave indications of Si supplying power of these soils in relation to its uptake by rice. Uptake of Si in relation to rice growth was worked out in rice varieties of different durations. This showed that with increase in duration, Si content in straw was increased but grain Si remained unaffected.

Bray's I extractant was found to be the best for extraction of available S in flooded soil. Flooding increased availability of S up to 21 days in soils. S in solution from 0-5 ppm was deficient, 5-40 ppm was sufficient and above 40 ppm was in excess to the plants.

Micro-nutrients in rice and rice soils

With the introduction of high yielding rice varieties and intensive rice cultivation, problems due to micro-nutrient deficiency or toxicity have been increasingly noticed. Suitable methods of extraction and estimation of Fe, Mn, Zn, Cu, B and Mo were developed. Micro-nutrient nutrition of high-yielding rice varieties in relation to growth and duration of the crop was undertaken.

Heavy metal pollution in rice and rice soils

Studies on this aspect intensified during mid-eighties showed that flooding decreased extractable Cd, Ni, Pb and Cr (VI), irrespective of soil types. The toxic form, Cr (VI) was totally convertible to Cr (III), a non-toxic form, within 10 days of flooding in all types of soils except in laterite soil, indicating its differential behaviour in different soils. Leachability of Cd was very low in alluvial sandy loam soil suggesting a remote possibility of ground water pollution.

Chemistry of submerged soils

Detailed and systematic studies on chemistry of submerged soils and transformation of nutrients were carried out during late sixties in twenty different types of soils, which indicated that pH of the acid soils increased and that of alkali soils decreased due to flooding. Eh of the soil decreased which was followed by a slight increase. EC of the soils increased because of flooding due to increased availability of NH4-N, K, Ca and Mg. Different prediction equations were worked out for nutrient transformation under submergence in different soil types.

 

Long-term fertilizer experiment

A long-term fertilizer experiment started in 1969 to study the effect of intensive cropping with the use of NPK with and without compost application on the changes in soil properties and productivity. There was response to application of N from beginning and response to compost was obtained from the fourth year of cropping. The grain yield was steady during the course of experiments. No response to P and K was obtained till date. The pH of the soil has decreased slightly. Compost application increased organic carbon of soil whereas with no compost its level decreased. Available P and K in soil in control plots and where they are not applied have decreased slightly but this was not reflected in grain yield. Nitrogen balance was positive in treatments receiving nitrogenous fertilizer.

Studies on problem soils

Intensive laboratory and pot experiments on low productive problem soils like acid sulphate soils of Kerala and acid, red and laterite soils were carried out for their reclamation for increasing rice production. Low pH, high salt content, high Fe and Mn and low P and high soil reduction were found to be the causes of low production in acid sulphate soils. The ameliorating effect of lime, MnO2 and NO3 and Fe(OH)3 on acid sulphate soils lasted for short period of only one cropping season. On the other hand, MgCO3 and Mg silicate had somewhat longer lasting ameliorating effect. Problems with acid, red and laterite soils were due to high Fe and Al in soil with flooding and also nutrient stress like P and Zn deficiency and low organic matter. The soils were reclaimed by liming and suitable nutrient management practices. Studies on Fe toxicity indicated that this problem could be overcome by balanced fertilizer application, liming, potash application and growing suitable rice varieties.

Studies on physical properties of rice soils

Results obtained from soil moisture studies indicated that moisture at 70-80% of water holding capacity after flowering was adequate for yield and N uptake of plant. Flooding of soil up to 45-60 days of planting was essential for increasing grain yield. During winter, ground water table fluctuated from 64 to 75 cm soil depth and contributed as much as 50 cm water to the 10-40 cm depth zone over a period of 52 days in loamy sandy soil. In summer when ground water table fluctuated between 80-90 cm, as much as 26 cm of water was supplied over a period of 31 days.

Experiments carried out on tillage for six years showed that after three years of continuous no tillage, rice yield declined. Under high water table condition, tillage in the wet season could be avoided. Dry season tillage increased the yield of blackgram and fodder cowpea irrespective of wet season ploughing. It is possible to have a groundnut crop after lowland rice on residual soil moisture by restricting the tillage to seed zone depth or by no tillage sowing and a subsequent tillage between the rows after seedling emergence. Puddling accelerated mineralisation of soil N and increased the concentration of K and Ca in soil solution.

Diffusibility of water to germinating seeds of cowpea and soybean decreased at zero water potential but increased at -2 and -10 bar water potential.

MICROBIOLOGY

BLUE-GREEN ALGAE AND AZOLLA

Nitrogen fixation by blue-green algae (BGA) and Azolla plays an important role in increasing rice production and soil fertility. Work on BGA at this Institute was started in early 1960's. In addition, investigations on Azolla work were also taken up in mid 1970's. Extensive studies covering basic aspects of these organisms and application of the findings for enhancing crop production were carried out.

Blue-green algae (Cyanobacteria)

Occurrence and distribution

A large number of N2-fixing BGA was isolated from rice fields of the Institute farm and soil samples collected from different parts of the country. Predominant among them were species of Aulosira, Anabaena, Nostoc, Cylindrospermum, Gloeotrichia and Aphanothece. Most BGA were dominant in the moist soil and Aphanothece was abundant in all water regimes whereas Aulosira preferred clean standing water. Algal biomass was highest at panicle initiation followed by late tillering, maturity and early tillering stages of rice crop.

Algal virus

Occurrence of a long-tailed LPP type virus infecting, alga, Plectonema boryanum was reported in paddy fields for the first time. N-1 virus that infected Nostoc muscorum was inactivated in distilled water, saline solution and magnesium chloride solution and stabilized by monovalent and divalent cations. Virus particles were most stable at 45 oC and at pH of 6.7-10.5. Its adsorption rate was highest at exponential growth stage and decreased with ageing of algal culture. Virus resistant mutants of N. muscorum were isolated with frequency of 3.5x10-5. Gene transfer studies showed that nif ability was transferred from parent to mutant along with resistance markers.

Cellular differentiation

Cell differentiation studies in Gloeotrichia sp. showed that calcium nitrate at high concentrations induced filament elongation, suppressed heterocyst differentiation and increased cell granulation. High concentrations of ammonium chloride also suppressed heterocyst differentiation and increased cell granulation. High concentrations of ammonium chloride suppressed heterocyst differentiation completely, but the suppression was partial at low concentrations. Amino acids like tryptophan, glutamic acid, histidine and proline at 10-20 ppm encouraged algal growth and N2-fixation, but at 80-100 ppm suppressed heterocyst differentiation and N2-fixation. Of the antibiotics tested, rifampicin produced chain of heterocysts in N. muscorm, while puromycin suppressed heterocyst differentiation. However, actinomycin-D had no effect on heterocysts. Blue and green light slightly suppressed heterocyst differentiation in Wollea bharadwajee but white, red or yellow light did not.

Sporulation and germination of spores

Laboratory studies with Fischerella muscicola showed that sporulation frequency was higher in acidic than in alkaline pH. Phosphorus deficiency or application of nitrogenous fertilizers stimulated sporulation, urea-N being more effective than ammonium and nitrate-N. Dilution of the growth medium also enhanced sporulation. Antibiotics, streptomycin and chloramphenicol, stimulated sporulation initially, but suppressed it upon prolonged incubation. Increasing concentrations of glucose and fructose up to 1000 ppm, potassium permanganate and hydrogen peroxide up to 1.0 ppm, 2,4-D up to 60 ppm and sodium chloride up to 1000 ppm enhanced sporulation and their action was concentration-dependent. Certain heavy metals (Cr, Cd, Pb and Zn) and growth hormones (IAA and GA) also increased sporulation. Application of pesticides, benthiocarb, metacid and bavistin at the recommended doses favoured BGA sporulation under field conditions. Germination of F. muscicola spores was favoured by alkaline pH, red light and application of sugars, N fertilizers, IAA and GA.

Mutational studies

Various types of mutants were isolated spontaneously and after chemical mutagenesis in several BGA. Mutants with loss of heterocysts (het-) always lacked N2-fixation (nif -). MNNG-treated, chlorate resistant (ClrR) mutants of N. muscorum and Aulosira sp., which were nitrate reductase defective (nardef) or blocked (nar-), showed heterocyst differentiation and N2-fixation in presence of nitrate. MSX resistant (MSXR) mutants of nar- N. muscorum were partially blocked in glutamine synthetase, as evident from their low GS transferase activity in comparison to nar- mutant and parent strain. MSXR mutants of Aulosira sp. also showed low GS transferase activity and fixed N in presence of ammonium. ClrRMSXR mutants of this alga were nardef GSdef and fixed N in presence of both nitrate and ammonium. Spontaneously isolated mutants of N. muscorum were characterized as het- nif- having short trichomes and abnormal cell morphology. They reverted into long forms having heterocysts but normal cell morphology was not restored. Amino acids, casein hydrolysate and vitamin B6 failed to restore cell normalcy. Some other mutants showed loss of gas vacuoles, filament formation, sporulation and variations in colony morphology.

Nitrogen fixation by unicellular BGA

Aphanothece pallida and A. castangei were reported as N2-fixers for the first time. The ARA of Gloeocapsa decarticans was higher in light, whereas A. pallida showed higher ARA in dark but required prior exposure to light. Addition of DCMU increased ARA of A. pallida under both short-term and long-term photo- incubation but decreased ARA of G. decarticans under long-term photo-incubation. Different N sources at low concentrations enhanced growth of unicellular BGA but inhibited their growth at high concentrations. Inoculation of Aphanothece sp. significantly increased rice yield and maximum algal growth and ARA were obtained at 30 kg P2O5/ha.

Growth and N2-fixing potentiality

Growth and N2-fixing potential of BGA varies with species and growth conditions. Estimation of BGA biomass in planted paddy fields showed a variation of 4-28 t fresh wt/ha and 99-822 kg dry wt/ha, containing 4-32 kg N. The ARA estimates varied from 189-259 n moles for Aphanothece sp. and 249-357 n moles ethylene /mg chl/hr for Aulosira sp.

Inoculum production

Bulk production of BGA is necessary for its large-scale use. Production techniques using GI trays, cemented tanks and flooded fields were standardized. A water depth of 5-10 cm and application of superphosphate at 20-40 kg P2O5/ha and insecticide, carbofuran at 75-90 g/ha encouraged growth of BGA. Starter culture was inoculated in clean water at the rate of 50-100 kg fresh wt or 6-10 kg dry wt/ha. Use of higher inocula was always better for quick multiplication. The BGA could be grown year round, with average monthly production of 3.3-366.5 kg dry wt/ha, but summer and early part of rainy season was most suited due to prevailing high temperature.

Release of fixed N

BGA-N becomes available to rice after decomposition of algal biomass. Its release was slow as compared to Azolla and chemical fertilizers. However, N release from fresh BGA was quicker than dry BGA and from incorporated BGA than unincorporated BGA.

Rice yield and soil fertility

Extent of grain yield increase due to algalization depends on BGA growth and availability of fixed N to rice. Field studies revealed that BGA was on par with application of 20-25 kg N/ha as chemical fertilizer. Yield increases of 15-60% over control were observed. Of different BGA tested, the highest yield was obtained with Aulosira sp. Increased rice yield was also obtained by growing it before planting. Use of BGA mixture produced higher yield as compared to single strain. Response to algalization was better in the dry season and for long duration rice varieties. Combined use of BGA and chemical fertilizer up to 30-40 kg N/ha is recommended because algal growth decreased at high doses of these fertilizers. Algalization produced a cumulative residual effect on yield of succeeding rice crop. It also increased organic C, total N and available P contents of soil.

Interaction with phosphorus

Phosphorus was often a limiting factor and its application encouraged BGA growth. The highest biomass and N yield of native and inoculated BGA were obtained at 40 kg P2O5 /ha. Split application was better than single basal application.

Interaction with nitrogen

Nitrogen fertilizers at high concentrations generally inhibited growth and N2-fixation by BGA but extent of inhibition differed among species. Growth of filamentous forms was less affected as compared to unicellular forms. Ammonium was more inhibitory than urea and nitrate. Ammonium sulphate and urea at 100 ppm or more were inhibitory for most algal species. Ammonium chloride at 10-20 ppm enhanced N2-fixation of Cylindrospermum licheniforme but inhibited its growth at 60 ppm, whereas calcium nitrate had no effect even at 300 ppm. Field experiments showed that ammonium sulphate and urea above 30 kg N/ha inhibited algal growth and N2-fixation in both planted and unplanted fields.

Interaction with other nutrients

Requirement of cobalt, molybdenum and vanadium for growth and N2-fixation of BGA was established in the laboratory studies. Application of potassium, lime and molybdenum did not prove beneficial in field experiments.

Interaction with insecticides

Gamaxene (BHC), lindane, diazinon and endrin reduced survival, growth and N2-fixation of Cylindrospermum sp., Aulosira fertilissima and Plectonema boryanum. BHC was the most toxic and at 10 ppm it was algicidal to many bloom forming BGA also. Carbofuran at 10 ppm enhanced growth and N2-fixation of N. muscorum but at higher concentrations it was inhibitory. Toxicity of carbofuran was less if the alga was grown at alkaline pH, high light intensity and high population density. Increased nutrient supply also reduced insecticidal toxicity. Insecticides were detoxified by repeated BGA culture or by prolonged incubation with BGA. Field studies demonstrated that insecticides commonly used for rice at their recommended doses were not inhibitory, although algal growth decreased when the insecticides were used at higher doses.

Interactions with herbicides

Growth of Anabaenopsis raciborskii and Mycrocystis flasaque was not inhibited by 2,4-D even at 150 ppm,. Butachlor and benethiocrb at relatively low concentrations (10-15 ppm) reduced growth, N2-fixation, uptake of phosphate and ammonium by N. muscorum. A mutant of this alga was isolated which could survive at 100 ppm of butachlor while 15 ppm was lethal for the parent alga. Effect of recommended doses of commonly used herbicides on BGA was also studied in the field. Toxicity was observed in the order of butachlor > benthiocarb > 2,4-DEE > 2,4-Dna. Inhibitory effects of the herbicides and N fertilizers could be minimized by increased application of P fertilizer. Their deleterious effects were also lowered by applying them together with insecticides.

Azolla

Azolla fixes atmospheric N in symbiotic association with the blue-green alga, Anabaena azollae. Of seven species of Azolla, A. pinnata is commonly found in India. It usually floats on the surface of idle water bodies such as ponds, canals, ditches and channels. Its occurrence was observed mostly during winter to early part of summer.

Germplasm maintenance

Ninety strains belonging to seven species of Azolla from all over the world are currently being maintained at CRRI. Wide variability was observed among these strains as regards growth and N2-fixation. Among A. pinnata strains, growth of Vietnam green and Thailand strains was better. A strain of A. caroliniana grew better and fixed more N as compared to strains of other species. It was also tolerant to several pests and diseases.

Morphology and cytology

Morphology and cytology of some Azolla species were studied in detail. Plants consist of a branched floating stem, alternately arranged bilobed leaves and adventitious roots. A. nilotica and A. filiculoides showed vertical morphology and the other species showed horizontal morphology. Rhizome surface was pubescent in A. pinnata and glabrous in A. mexicana and A. filiculoides. Nipples on the surface of dorsal lobe were rounded in species of section Euazolla and prolate in species of section Rhizosperma. Besides reproductive features, use of vegetative characters like rhizome surface, structure and distribution of trichomes and total chromatin length for species identification was suggested. Chromosome number varied among species and strains. A. mexicana, A. filiculoides, India and Vietnam green strains of A. pinnata had a chromosome number (2n) of 48, 40, 44 and 66, respectively. Meiotic behaviour of microspore mother cell was also studied in these species.

Mineral nutrition

Azolla requires all macro and micro-nutrients for its growth. Optimum levels of these nutrients in the medium for growth of different species were determined. Deficiency symptoms as well as morphological, physiological and biochemical changes caused by deficiency of various elements were studied. Phosphorus was found most important growth-limiting element. Effects of P and Ca deficiency on growth and N2-fixation were more intense than deficiency in K and Mg. Growth was better in N-free medium, with a generation time of 3-5 days. Addition of combined N decreased growth, heterocyst frequency, N2-fixation, soluble sugar and chlorophyll contents but increased amino nitrogen content.

Sporocarp formation and germination

Almost all strains of A. pinnata and some strains of A. caroliniana formed sporocarps during November to March, when day length was short and temperature was relatively low at night. A strain of A. mexicana sporulated year-round. Vietnam green strain of A. pinnata formed only male sporocarps. Phosphorus deficiency and overcrowding enhanced sporulation. Growth hormones IAA, GA and kinetin also stimulated sporulation.

A more efficient method of sporocarp collection from Azolla was developed where sporocarp balls were collected by repeated shaking and sieving of dried plant powder. Sporocarps germinated within 10-15 days after incubation. Light was required for germination. Nitrogen, phosphorus, amino acids and growth hormones like GA and kinetin increased germination. Young seedlings derived from sporocarps of different Azolla species showed interesting morphological differences.

Alga-free Azolla

Alga-free Azolla was produced by sequential treatment of antibiotics and characterized. Isolation of Anabaena was attempted. A new association between alga-free Azolla and Aphanothece sp. was developed.

Growth and N2 -fixing potentiality

Growth rate, maximum biomass and nitrogen content of Azolla provide estimate of its potential for agricultural use. Fresh weight of Azolla increased 2 to 6-fold every week. It fixed about 75 mg N/g dry wt/day. Total nitrogen content was 3-5% on dry weight basis and 0.2-0.3% on fresh weight basis. Year-round multiplication yielded a maximum biomass of 347 t fresh wt/ha which contained 868 kg N. The ARA studies have also confirmed its large N2-fixing potentiality.

Method of utilization

It could be used as both green manure and dual crop with rice, but dual cropping (inter-cropping) was more practicable and economical. Inoculation of fresh Azolla at 0.5-1.0 t biomass/ha before 15-20 days of planting for green manuring and after 7 days of planting for dual cropping is recommended. Dual cropping up to 3-4 weeks was beneficial for rice. Split application of superphosphate at 8-10 kg P2O5/ha and carbofuran at 75-90 g/ha ensured rapid fern growth. Phosphorus need of Azolla could be met from recommended dose of P for rice by applying half the dose of P during Azolla cultivation. Green manuring supplied 20-40 kg N/ha and dual cropping 20-30 kg N/ha. Using Azolla as green manuring or dual cropping was on par with application of 30 kg N/ha through chemical fertilizer and increased grain yield of rice by 0.5-1.5 t/ha over control.

Decomposition and N release

Azolla supplies nitrogen to rice after its decomposition. Investigations revealed that incorporated Azolla decomposed in a period of 8-10 days and released about 70% of nitrogen in 35 days. Decomposition of un-incorporated Azolla was relatively slow and it decomposed in about 30 days. Several fungi and bacteria helped in decomposition. High temperature also favoured decomposition. Azolla-N was released slowly as compared to N from chemical fertilizer and its availability to first rice crop was 70% of ammonium sulphate-N.

Rice yield and soil fertility

Studies carried out for over 15 years have established that Azolla green manuring or dual cropping increased rice yield to the tune of 30 kg N/ha as chemical fertilizer. Yield was generally higher with green manuring than dual cropping. Green manuring was more suited for short duration rice and dual cropping for medium and long duration rice. Experiments with different combinations of Azolla and N fertilizer revealed that green manuring plus dual cropping and green manuring/dual cropping with 30 kg N/ha were on par with 60 kg N/ha. Green manuring followed by two dual cropping was superior to 60 kg N/ha. Response of rice to Azolla was higher in the dry season. Use of Azolla also improved rice growth and crop N uptake; organic C, total N and P contents of soil; checked growth of weeds and increased yield of succeeding rice crop. Studies on economics of using Azolla indicated a much higher net return as compared to chemical N fertilizer.

Water management

About 5-10 cm water depth was most suited for Azolla growth but increase in water depth up to 30 cm had no adverse effects. Growth at increasing water depths depended on availability of essential elements.

Interaction with fertilizers

Azolla also responded maximum to application of phosphate fertilizer and its optimum level was 10-20 kg P2O5/ha. Water soluble P fertilizer was more suited and its split application was superior to single basal application. Requirement of Azolla inoculum could be substantially reduced by increasing level of P application, which also reduced inhibition of Azolla growth by N fertilizer. Fresh animal dung at 1.0-1.5 t/ha gave the effect of 10 kg P2O5/ha as superphosphate. Phosphorus enrichment of inoculum could eliminate requirement of P application during Azolla cultivation. P-enriched Azolla performed better than non-enriched Azolla when grown with application of herbicides and N fertilizers. Application of iron and molybdenum also enhanced Azolla growth.

Studies with N fertilizers revealed that fern growth in planted rice field was not much inhibited by split application of ammonium sulphate and urea at 90 kg N/ha, whereas growth in fallow field decreased with increasing N levels. Increase in rice yield due to Azolla was evident with chemical fertilizer even at 130 kg N/ha. Among different N sources, urea super-granule was most suited for Azolla followed by farmyard manure, ammonium sulphate and urea. Their sub-surface placement was superior to broadcasting. Study with different splits of N fertilizer application indicated that 25:50:25 was good for both Azolla and rice. Azolla growth was better if basal nitrogen dose of rice was met through green manure rather than chemical fertilizer. When grown with nitrate-N, A. caroliniana grew better and fixed more N than A. pinnata, due to lower nitrate uptake resulting from lower nitrate reductase activity.

Method and time of rice planting

Density of rice crop affected Azolla growth adversely and a delay in inoculation decreased growth due to shading by rice canopy. Higher Azolla biomass and rice yield were obtained by using double narrow-row planting as compared to conventional line planting. This method was more suited for late-inoculated Azolla. The fern could also be grown with randomly-planted or direct-seeded rice, although its growth was poor as compared to line planting. Rectangular planting was better than square planting. Fern growth was better if rice rows were planted in east-west direction. Growth and N2 fixation of Azolla were reduced considerably when planting was delayed.

Amount of inoculum

Inoculum of 100-300 g/m2 for Azolla multiplication and 0.5-1.0 t/ha for green manuring or dual cropping has been recommended. Higher levels of inoculum helped in rapid Azolla growth and inoculum up to 3.0 t/ha could be used without any adverse effects on rice yield, provided water level is not high enough to allow rice leaves to be covered by Azolla.

Comparison with other organic manures

Azolla was superior to Sesbania, farmyard manure and BGA as regards to its N-releasing ability. Grain yield data from field trials revealed that Azolla has a greater potential than BGA and it is on par with Sesbania.

Comparative performance of rice grown with BGA, Azolla, green manuring and organic manuring in transplanted and direct-seeded rice.

Treatment

N added

(kg/ha)

Grain yield

(t/ha)
 

Transplanted

Direct-seeded

Transplanted

Direct-seeded

Control

0

0

2.8

2.0

Urea

30

30

3.7

2.7

Urea

60

60

4.9

3.2

BGA inoculation

22

16

3.3

2.5

Azolla dual cropping twice

56

48

4.1

3.1

Azolla green manuring + dual cropping twice

79

74

4.9

3.6

Organic manuring (Eichhornia)

60

60

3.7

2.9

Green manuring (Sesbania)

60

60

4.3

3.1

Insects and diseases

Major insect pests commonly affecting Azolla are species of Chironomus, Pyralis and Nymphula. They could be effectively controlled by application of carbofuran at 75-90 g/ha. Other insecticides such as BHC, phorate and thimet were also effective. Fungal and bacterial diseases were observed occasionally. Strains having tolerance to major insects have also been identified.

Interaction with pesticides

Insecticides commonly used for rice at their recommended doses generally increased fern growth and among them carbofuran was most effective. Its efficacy could be improved by applying it along with phosphorus. Organic formulations which possess pesticidal activity such as neem oil and karanj oil also improved Azolla growth. Herbicides generally inhibited growth of Azolla. Azolla should be applied after 2-3 weeks of butachlor and benthiocarb application whereas Azolla and 2,4-D could be applied together. Inhibitory effects of butachlor and benthiocarb could be reduced by increasing level of P application and also by applying them together with insecticides.

Azolla as animal feed

Chemical analysis showed that Azolla was rich in protein and minerals. Experiments on feeding of Azolla in different proportions with commercial feed to poultry, birds revealed a saving of about 20% of commercial feed.

BACTERIAL NITROGEN FIXATION AND PESTICIDE-BACTERIA INTERACTION

Research on pesticide-soil microflora interactions was initiated in 1971 while that on nitrogen fixation by free-living bacteria was initiated in 1975.

Nitrogen fixation by free-living bacteria

Employing sensitive 15N tracers, it was demonstrated that flooded soils always exhibited higher nitrogenase activity than non-flooded soils. Soil submergence increased the population of nitrogen fixing bacteria and their nitrogenase activity. N2-fixing activity was more pronounced when associated with rhizosphere. Carbon substrates like cellulose, rice straw, glucose, succinate, acetate, butyrate and pyruvate which are commonly occurring in rice soils stimulated N2-ase, and populations of different groups of N2-fixing bacteria. High levels of combined nitrogen inhibited and low levels stimulated the nitrogen fixation in paddy soils. The inhibition due to high levels of combined nitrogen was alleviated by the addition of high level of organic matter.

Azospirillum-root association was favoured by low levels of combined nitrogen and in presence of additional organic matter. Plant genotype exhibited significant influence on microbial populations and, particularly, Azospirillum association. These studies were confirmed by 15N incorporation. Root inoculation with Azospirillum led to a significant mean yield response. Biologically fixed nitrogen was transferred from the soil and distributed in different parts of the rice plant. About 40% of the fixed nitrogen was available to the rice plant and major portion remained in the soil after harvest.

Alterations in water regimes influenced the soil nitrogenase activity. Nitrogenase activity increased following a shift from non-flooded to flooded condition. Rice plant exhibited a growth phase-dependent nitrogenase activity and the rhizosphere nitrogenase is affected by water regimes and fertilizer applications. The stimulation of nitrogenase by phosphorus varied with the source of P. Superphosphate exerted highest stimulation when compared with dicalcium and rock phosphates. Alterations in N2-fixing microbial population were observed in response to P application. Form and methods of urea application and nitrogen management practices in intermediate deep water rice significantly affected nitrogenase associated with rice rhizosphere. An increase in the plant density per unit area under intermediate deep-water situations decreased the associated nitrogenase activity. Organic amendments with green manure like Sesbania significantly enhanced the bacterial N2 fixation, the nitrogenase activity and their populations. Application of dry leaves of S. aculeata and S. rostrata effected more significant stimulation of the population of N2-fixing Azospirillum and anaerobic bacteria. The results indicate that application of green manure could beneficially affect N2 fixation by bacteria in rice soils besides contributing already fixed nitrogen in the green manure to the current rice crop.

An increase in soil salinity decreased nitrogen fixation and the population of N2-fixing bacteria in flooded and non-flooded soils. Leaching the saline soils improved the nitrogen fixation and N2-fixing bacterial populations. Results indicate that soil amelioration for salinity with leaching and organic matter addition would improve the implicated microbial populations and nitrogen fixation in salt-affected rice soils. A certain degree of salt tolerance was observed in Azospirillum isolated from salt-affected paddy soils. Cultures from highly saline soils were more resistant to salt stress than cultures from medium saline and non-saline soils with regard to the nitrogenase activity.

Commonly applied herbicides thiobencarb and butachlor at 2 and 4 ug/g soil inhibited the population of anaerobic N2 fixers and Azospirillum under non-flooded conditions. Differential behaviour of the herbicides on the select group of N2-fixing bacteria was noticed depending upon the soil type and water regime. Application of butachlor a herbicide, reduced the population of Azospirillum and anaerobic N2 fixers in a non-flooded alluvial soil. However, a combination of butachlor and carbofuran, an insecticide, greatly stimulated the populations and nitrogenase activity in the soil. A uniform stimulation of N2 fixation occurred in soils when carbofuran was applied either singly or in combination with other herbicides tested. Further investigations on the pesticide-microflora, interactions revealed that select pesticides at recommended levels enhanced rhizosphere nitrogenase activity. The stimulation/inhibition depended upon the nature, level and mode of pesticide application to the field soil.

Repeat application of carbofuran and HCH effected a significant stimulation of rhizosphere associated nitrogenase activity as a consequence of increased populations of Azospirillum sp., Azotobacter and anaerobic nitrogen-fixing bacteria. Application of herbicides (Cinmethyline, butachlor and benthiocarb) at field recommended doses under field situations caused significant increase in the rhizosphere associated nitrogenase. Anilofos when applied singly had no substantial effect on nitrogenase activity but in combination with 2,4-D, the activity was considerably enhanced with a resultant increase in the population of Azotobacter and Azospirillum.

The efficiency of nitrogen fixation of Azospirillum-asscociated with rice roots is controlled by alterations in pH, redox status and organic substrate availability in soils. Root inoculation with Azospirillum led to a significant mean yield response. Biologically fixed nitrogen was transferred from the soil and gets distributed to different parts of the rice plant with grain deriving maximum amount.

Field plots with high percolation rate and high sand contents supported higher rhizosphere nitrogenase activity under continuous flooding. In plots with low percolation rate and lower sand content, intermittent flooding favoured nitrogenase activity.

A definite partitioning of the nitrogenase activity within the root and a plant growth dependent shift in the activity associated with basal and terminal portions of the root were observed in several rice cultivars grown under field conditions. Higher population density of Azospirillum and Azotobacter occurred at the basal portion of the root while root tip had higher populations of facultative anaerobic nitrogen-fixing bacteria. Certain wild rices and trisomic lines of cultivated rice exhibited higher nitrogenase activity in the root region, and as well harboured Azospirillum sp. with greater nitrogenase activity.

Effect of pesticides on bacteria

Studies with flooded soils at this institute showed that certain pesticides, even at close to field application rates, may effect distinct changes in the microbial functions of a flooded soil. Thus, application of carbofuran to rice rhizosphere soil suspension at 10 and 100 ppm led to a distinct stimulation of Nitrosomonas-mediated ammonium oxidation. Similarly, benomyl, a fungicide, inhibited autotrophic nitrification in soil and pure cultures of Nitrosomonas sp. and Nitrobacter sp., but stimulated heterotrophic nitrification mediated by Pseudomonas sp. Benomyl also retarded the drop in redox potential of a flooded soil and prevented iron and manganese reduction. Application of commercial formulation of HCH inhibited the oxidation of sulphur by 50% at 5 ppm level. Further, two pesticides individually at sub-toxic rates, when applied in combination, can adversely affect soil microbial processes. Thus, carbofuran and HCH when applied in combination effected a drastic inhibition of nitrification in a soil, although both pesticides were individually not inhibitory at the concentrations used.

In an isotopic study, employing 15N, application of carbofuran, benomyl and gamma-HCH to submerged soils at 5 ppm level led to a significant increase in nitrogen fixation in a majority of five soils due to a stimulation of Azospirillum and anaerobic nitrogen fixers. While carbofuran consistently stimulated heterotrophic nitrogen fixation in all the soils tested, an organo-phosphorus insecticide, diazinon, inhibited the process.

Bacterial degradation of pesticides

The studies hitherto demonstrated that the dynamic aerobic-anaerobic interface of a flooded soil can serve as an effective medium for decontamination, both aerobically and anaerobically mediated, of many commonly used pesticides in rice culture, and flooded rice soils harbour a myriad of microorganisms (aerobic and anaerobic) capable of mineralizing many of these pesticides and their degradation products to harmless end products in pure cultures and soils. Thus, hexachlorocyclohexane (HCH), which was known to be recalcitrant to biodegradation under aerobic conditions, can undergo rapid aerobically mediated degradation even under flooded conditions especially after two or three applications of HCH. Thus, repeated additions of HCH to a rice field under non-flooded or flooded conditions led to a distinct development of acclimatized aerobic, and not anaerobic microorganisms capable of degrading gamma-HCH.

Carbofuran and carbaryl, the widely used carbamate insecticides also underwent rapid degradation upon their repeated additions to a flooded soil. Common cultural practices like flooding, rice straw amendment etc. enhanced degradation of these carbamate insecticides concomitant with the accumulation of their hydrolysis products. It was also demonstrated that repeated additions of carbofuran to a flooded soil held at 35oC, conditioned the soil for accelerated degradation of carbofuran by acclimatized microorganisms, an Arthrobacter sp. in particular.

Organo-phosphorus insecticides such as parathion, methyl parathion and fenitrothion can undergo both hydrolysis and nitro group reduction. A shift in the pathway of degradation of parathion from nitro group reduction after first application to essentially hydrolysis after three additions was noticed. Accelerated hydrolysis of parathion occurred also after repeated applications of its hydrolysis product, p-nitrophenol to the flooded soil. A Pseudomonas sp. isolated from parathion-treated flooded soil hydrolyzed parathion to p-nitrophenol which was further metabolized to nitrite and CO2. Related methyl analogues like methyl parathion and fenitrothion also underwent faster degradation essentially by nitro group reduction after fist application to flooded soils. However, the rate and pathway of degradation of methyl parathion in a flooded soil was temperature-dependent and an increase in the temperature not only increased the rate of degradation of methyl parathion in a flooded soil, but also altered its pathway from nitro group reduction at 28oC to hydrolysis at 35oC. Methyl parathion and to a less extent, fenitrothion, are affected by accelerated biodegradation after repeated applications in flooded soils.

In studies on soil-pesticide interactions, the organic matter content of the soil appeared to be the most important single factor responsible for the sorption of parathion, methyl parathion, three isomers (alpha, beta and gamma) of HCH, carbofuran and carbaryl. A modified Freundlich equation was developed from the relationship between soil organic matter and Freundlich K values, which would assist in predicting pesticide sorption in soils with known organic matter content. De-sorption of pesticides was, however, inversely related to organic matter content. Interestingly, soil-sorbed carbofuran was readily degraded by carbofuran-degrading enrichment culture in alluvial soil with low organic matter content. In contrast, soil sorbed HCH-isomers more readily degraded by a Pseudomonas sp. in both alluvial and Pokkali soils.

Methane emission from rice fields

Studies were initiated on measurements of methane emission from Indian rice fields under different agro-climatic conditions, moisture regimes in particular, using manual static box system. Based on the estimates at 18 locations under National Methane Campaign and at four centers under ICAR Coordinated Project, the mean emission from irrigated and rainfed lowland systems in India has been estimated to be around 4.0 Tg/year (million tons/year).

Methane flux reached the maximum at and around the reproductive stage of rice plant. Methane emission was highest in the afternoon (12 noon- 3 p.m.) and declined to minimum at around midnight. Peak emission in the afternoon and at reproductive stage was correlated with low root oxidase activity (based on alpha-napthylamine oxidation). Addition of green manure increased methane emission from flooded rice fields, even after 115 days of its application. Among the fertilizer sources, the order of methane emission was: green manure > prilled urea > farmyard manure > un-amended. There is evidence, but not conclusive, to suggest that methane emission is positively correlated with biomass produced.

Addition of single superphosphate, a widely used source of phosphorus, significantly inhibited the production of methane in laboratory incubation studies and also emission of methane from field plots. This inhibition was attributed to the presence of sulphate in the superphosphate formulation used. On the other hand, addition of K2HPO4 stimulated methane production in a P-deficient soil, but was innocuous in a P-normal soil. Cumulative methane emission from a direct-seeded rice, one of the increasingly used cultural practice which is less labour intensive, was less pronounced than in transplanted rice. Puddling of flooded soil for transplanted rice causes compaction of the soil, retards the downward percolation of water with dissolved oxygen, and hastens the reduction of the soil favouring methane emission.

Methane production was considerable in an alluvial soil under flooded conditions. But, high sulphate content almost totally inhibited the production of methane in an acid sulphate soil (Pokkali) under flooded conditions. No relationship existed between the methanogenic populations and methane production in soils. Methane production was inhibited in a flooded acid sulphate soil despite very favourable conditions of high organic matter content, low redox potential and near neutral pH, due to high population of sulfate-reducing bacteria and sulfide formed.

Methane oxidation was high in the surface and rhizosphere soil samples, concomitant with relatively high populations of methane oxidisers in these layers. Application of carbofuran, a widely used carbamate insecticide, at 5 and 10 ppm stimulated methane oxidation thereby resulting in a significant reduction in methane emitted.