CHAPTER -1

INTRODUCTION

STATEMENT OF THE PROBLEM

Hunger and disease have affected humankind since the dawn of history. An expanding world

population and urgency of eradicating hunger and malnutrition call for determined policies and

effective actions to ensure sustainable growth in agricultural productivity. According to FAO,

(1989) the goal of sustainable agriculture should be to maintain production at levels necessary

to meet the increasing aspirations of an expanding world population without degrading

environment. India is a largest country in South Asia and contains 70 percent of the total

regional population. In 1960s, after Green Revolution enormous progress with modern HYVs,

irrigation and fertilizer application has been made. But Green Revolution enhanced the chemical

fertilizer consumption from almost zero in 1950 to 17 million tones in the year 2000 (Tandon,

2004).

The rising cost of petroleum and other fossil fuel reserves indicates that the energy used

in the production of nitrogen fertilizer might have some substantial on the total fossils fuel

energy requirement of the nation. The cost of nitrogen fertilizers is going up day by day due to

sky rocketing prices of petroleum products (Singh, 1961). As Blue Green Algae is an important

factor used in Rice crop, its application reduces the dependency upon chemical fertilizers,

improve the Rice yield and also showing positive effects on other crops. But notably, there is a

general lack of awareness regarding the presence of algae in the soil, while they play an

important role in soil microbiology. However, algae are sensitive indicators in the soil and their

practical uses at field level can provide us information about possible beneficial/detrimental

effects on crop plants, which can improve our Agricultural practices and productivity in near

future.

1. ALLELOPATHY:

The concept of Allelopathy is ancient in the life science especially in Agricultural Science.

Allelopathy is derived from two Greek words “Allelon” meaning mutual and “Pathos” meaning

harmful i.e. the injurious effect of one plant upon another. Allelopathy is defined as the direct or

indirect, harmful or beneficial effects of one plant on another through the production of chemical

compounds that escape in to the environment (Rice, 1984). Organic compounds that are

inhibitory at some concentrations may be stimulatory to the same processes in different

concentrations. The compounds involved in this phenomenon are known as plant

allelochemicals (Mizutani, 1999).

Various water soluble substances are released into the environment through leaching,

root exudation, volatilization and decomposition of plant residues and are affected by several

environmental factors. The allelochemicals, which are secondary metabolites, produced by

plants and are by-products of primary metabolic processes, includes plant biochemicals that

exert their physiological/ toxicological action on plants and microbes (Rice, 1984).

These allelochemicals are present in different parts of the plant i.e. roots, rhizomes,

stems, leaves, flowers, inflorescence, pollens, fruits and seeds but leaves are the major source.

The allelochemicals cause both inter-specific and intra-specific interaction between organisms,

which are mediated through chemical process (Reigosa et al., 1999).

Although the impact of allelopathy in agriculture and forestry was recognized since 500 BC,

most of the progress occurred during 20th century. Molish (1937) coined the term Allelopathy

first time and since then, few investigations have been made on allelopathic interaction of blue

green algae (Patterson et al, 1979).

All allelochemicals added to aquatic or agro-ecosystem is either of blue green algae or

plant origin or various residues left in the fields which on decomposition get added to the soil

system. Decomposing Paddy root residual and algal material present in the field, can contribute

towards the pool of allelochemical compounds.

These can influence the seed germination and growth of next crop in those fields (Rice,

1984). Huang, (1978) studied the effect of Paddy straw and fertilizers on the growth and yield of

Paddy and concluded that the applications of nitrogen fertilizers during the two crops season

are significantly different.

1.1 RICE PLANT (ORYZA SATIVA L.)

1.1.1 Morphology: The Rice plant is probably native

to India. There are some 10,000 varieties, differing

in flavor, shape, size, and color of the grains. Under

cultivation the plant looks much like barley or oats.

Rice is an erect annual, about four feet (120 cm) tall,

with smooth tapering leaves that are enclosed in

sheaths. The leaves are one and half inch (1.3 cm)

wide and up to two feet (60 cm) long. The plant has

several stalks at the end of which are smaller stalks

called panicles on which grow spikelets. Several grains grow within each spikelet.

1.1.2 Classification:

Taxon: Oryza sativa L.

Kingdom Plantae

Subkingdom Tracheobionta

Superdivision Spermatophyta

Division Magnoliophyta

Class Liliopsida- Monocotyledon

Subclass Commelinidae

Order Cyperales

Family Poaceae

Genus Oryza

Species Oryza sativa L.

1.1.3 Production: Rice is a major food staple and a

mainstay for security. It is mainly cultivated by small

farmers in holdings of less than 1 hectare. Rice is also

a wage commodity for workers in the cash crop or non-

agricultural sectors. Rice is vital for the nutrition of

much of the population in Asia, as well as in Latin

America and the Caribbean and in Africa; it is central to

the food security of over half of the world population.

Developing countries account for 95% of the total

production, with China and India alone responsible for

nearly half of the world output.

1.1.4 Description of Rice crop: Rice is one of the

widely cultivated major food crops of the world

belonging to family Poaceae. It grows under a variety

of agro-climatic conditions much wider than for other major crops. Rice, the one among cereals

takes pride of place in that production and area of cultivation in Indian agriculture and cultivated

since ancient era (10,000 BC in Ganga valley). It accounts for 29% of the total calories intake by

population of developing countries (Shunmugavalli et al., 1999).

Among annual crops the Rice and Wheat are widely practiced food crops providing

meal, income and employment to millions of rural and urban growers and consumers (Paroda et

al., 1994; Dwivedi et al., 2003). Rice is an important food crop globally, as more than 40% of the

world‟s population depends on Rice as a major source of calories. The Green Revolution

Production of Rice by country — 2010

(million metric ton) People's Republic of China 197.2

India 120.6

Indonesia 66.4

Bangladesh 49.3

Vietnam 39.9

Myanmar 33.2

Thailand 31.5

Philippines 15.7

Brazil 11.3

United States 11.0

Japan 10.6

Cambodia 8.2

Source: Food and Agriculture

Organization, 2002

technology including high yielding varieties, massive use of chemical fertilizer and irrigated area

expansion, increased the yield of both Wheat and Rice crops leading the extent of 23% in total

food grain production of India (Dwivedi et al., 2003).

Rice covers 41.6 million hectares of land area which is about 37% of the total area

utilized for the production of cereals. The nitrogen fertilizer required for Rice alone is 60 lakh

tones of the total available, 140 lakh tones fertilizer nitrogen with presence level of consumption

(150 kg N ha-1). The best nitrogen use efficiency of the applied nitrogen fertilizer is 40% and

therefore about 60% of applied nitrogen is lost either through leaching or volatilization. To meet

the nitrogen deficiency and to manage its efficient use for sustainable agriculture, biological

renewable resources could be a better way in future (Venkataraman, 1980).

Since both Rice and Wheat crop utilize the plant nutrients at very high rates, on the other

hand removal of nutrients in substantial amount taken place due to high annual productivity that

often replenish through manures and fertilizers ultimately results in to deterioration in soil fertility

which ultimately leading to decline in productivity (Hegde and Dwivedi, 1992). In this regard,

farmers have started to use greater than required rates of chemical fertilizers, especially N2

fertilizers to maintain the crop yield (Yadav, 1998).

Nitrogen is the key nutrient in Rice production. Most actual and potential microbiological

soil management of wetland Rice refers to the N2 cycle. The oldest technology of employing N2

fixing microorganisms in Rice fields is utilization of Azolla. The use of Azolla as a green manure

dates back to the 11th century in Vietnam and at least to the 14th century in China (Lumpkin and

Plucknett, 1982). The causative agent of the beneficial effect of Azolla inoculation was identified

by Strasburger, (1873) as a N2 fixing blue green alga (Anabaena azollae) but progress in Azolla-

A. azollae biotechnology, in particular recombination and sexual hybridization, is very recent

(Lin et al., 1988; Lin and Watanabe, 1988; Wei et al., 1986).

1.1.5 Rice field eco-system: Flooding and crop growth lead to the differentiation of macro-

environments differing in physicochemical and trophic properties like: floodwater, surface-

oxidized soil, reduced soil, Rice plants (submerged parts and rhizosphere), plough layer and

subsoil as denoted in figure given bellow-

Macro-environments of the wetlands Rice field eco-system

From a microbiological point of view, the Rice plant provides two environments for the

microflora- submerged plant parts and the rhizosphere. Submerged portions of Rice shoots (and

aquatic plants) are colonized by epiphytic bacteria and algae. Epiphytes are ecologically

important in deep-water Rice for which the submerged plant biomass, including nodal roots, is

very large. Epiphytic biological N2 fixation can be agronomically significant in deep-water Rice

(Kulasooriya et al., 1980).

The rhizosphere is a nonphotic environment in which redox conditions are determined by the

balance of the oxidizing and reducing capacities of Rice roots, and production of chemical

compounds by roots provides energy sources for microbial growth. Major activities in the

rhizosphere include: (1) associative heterotrophic biological N2 fixation, (2) nitrification-

denitrification, and (3) sulfate reduction (Watanabe and Furasaka, 1980).

The agronomic potential of N2 fixing Blue Green Algae was recognized by De (1939),

who attributed the natural fertility of wetland Rice fields to biological N2 fixation by blue-green

algae. Biological nitrogen fixation, an alternative source of nitrogen, assumes greater

significance to bridge the gap between demand and supply of nitrogen fertilizer. Annual turnover

of nitrogen in „Biosphere‟ vary from 10 to 200 million tons (Burns and Hardy 1975; Burris, 1977;

Subbarao, 1977). Implication of biological nitrogen fixation is reviewed from time to time by

many workers (Hardy, 1976; Kaushik, 1985; Vaishampayan et al., 2001).

1.1.6 Tested variety of Rice (Oryza sativa L.): In the present study PR 114 (1999) Rice variety

was selected. It is a successful cultivated variety of Rice in the agricultural fields of nearby area

of Dayalbagh, Agra District.

It is semi-dwarf variety, having dark green, erect leaves. Its average plant height is

about 107 cm. It matures in about 132 days after seedlings. It possesses long slender clear

translucent grains with good cooking quality. It is resistant to most of pathotypes of bacterial

blight pathogen prevalent in Panjab. Its average Paddy yield is about 28.5 quintal per acre (Gill

and Mahindra, 2011).

1.2 BLUE GREEN ALGAE (CYANOBACTERIA)

Blue Green Algae also known as Cyanobacteria; belong to division Cyanophyta and class

Cyanophyceae or Myxophyceae. This division includes a large number of algae, which are

characterized by a low state of cell organization. The cell lacks the well-defined nucleus that is

met within the other division of algae. These organisms are characterized by a blue green

coloration of the cell being chief pigments chlorophyll „a‟, carotenes, xanthophylls, c-

phycocyanin and c-phycoerythrine. These organisms lack flagellated reproductive bodies and

there is a total lack of sexual reproduction (Manickavelu et al., 2006).

1.2.1 Role and distribution in Paddy fields: The Blue Green Algae are of ecological

importance in Paddy fields for maintaining soil fertility and reclaiming of alkaline soil. They are

ubiquitous in nature and could contribute 25-30 kg N2 per season. Cyanobacteria or BGA are

prokaryotic photosynthetic microorganism that produces a wide array of substances, including

plant growth regulators. In the case of growth regulators- gibberellin, auxin, cyatokinin, ethylene

and jasmonic acids have been detected in Cyanobacteria. (Stirk et al., 1996; Ordog and Pulz,

1996).

BGA were among the first N2 fixing agents recognized to be active in flooded Rice soils.

Many trials have been conducted to increase Rice yield by inoculating the soil with BGA. This

practice, also called algalization, a terminology introduced by Venkataraman (1972), has been

reported to have a beneficial effect on grain yield in different agro-climatic conditions.

Cyanobacteria which occupy unique position in continuum of life are common of the algal flora

of Indian Rice fields and of water rich in organic matter. They constitute one of the largest sub

groups of gram negative prokaryotes. The flooded Rice soil ecosystem is characterized by

aerobic and anaerobic zones in three major ecological layers, the flood water and the surface of

the soil, the anaerobic flow layer and the rhizosphere. The flood water and the surface of the

soil provide the sites for aerobic phototrophic nitrogen fixation by free living Cyanobacteria

(Roger and Kulasooriya, 1980) and the Azolla-Anabaena symbiotic N2 fixing complexes (Singh,

1978; Singh and Bisoyi, 1993).

The concept of utilizing Cyanobacteria as fertilizer for fixing nitrogen in Rice field has

been developed by De, (1939) and this was later confirmed by Singh (1961). In India, IARI, New

Delhi has developed means and methods for culturing inoculum of nitrogen fixing Cyanobacteria

for using them as bio-fertilizer (Watanabe et al., 1951, Roger and Kulasooriya, 1980; Kaushik,

1996).

De, (1939) showed nitrogen fixing Blue Green Algae (Cyanobacteria) as abundant and

widely distributed in Indian Paddy fields. Singh, (1961) experimentally demonstrated the

possible role of BGA in the productivity of Rice. The transfer of the fixed nitrogen to the plants

was demonstrated by Stewart, (1967). Such demonstration gave support to the earlier reports of

Singh, (1961); and Allen, (1956) for the role of Cyanobacteria in soil fertility. Apart from this,

much works have also been done on Indian soil (Subrahmanyan and Sahay, 1964;

Subrahmanyan et al., 1965; Venkataraman et al., 1961; Kaushik, 1996; Mishra and Pabbi,

2004) and there is a similar information from Japan (Watanabe and Yamamoto, 1971); Australia

(Bunt, 1961); America (Schields and Durrel, 1964); Philippines (McRoc and Castro, 1967;

Yoshida and Ancajas, 1973).

The nitrogen fixing Cyanobacteria are often referring to as Paddy organism, because of their

abundance in Paddy fields. Moreover 125 strains of Cyanobacteria are known to possess N2

fixing capacity and such N2 fixing strains occur in all typological groups (Rippka et al., 1971).

These cyanobacteria are found all around the year and prefer slightly alkaline pH, water

logged/moist reducing condition for their luxuriant growth and multiplication (Fogg et al., 1973).

1.2.2 Effect of BGA on soil property: Effect of Cyanobacteria on the physico- chemical

properties of soils has been reported by a number of workers (Singh, 1961; Thomas, 1977). In

certain tropical soils nitrogen fixation by Cyanobacteria can undoubtedly make a major

contribution to soil fertility and improving the soil quality. Various kinds of evidence indicated

that there are the principal agents responsible for maintaining the nitrogen status of the soil.

Peterson, (1935) concluded that they are useful; (i) in addition of organic matter, (ii) they may

play direct or indirect role in nitrogen fixation.

Soil nitrogen pool in Paddy fields is principally maintained by N2 fertilizer and biological

nitrogen fixation (Kundu and Ladha, 1995). Among nitrogen fixers in Paddy fields,

Cyanobacteria are important contributors to N2 fixation (Roger and Ladha, 1992).

Cyanobacterial trophic independence makes them suitable for being used as bio-fertilizers

(Irisarri, 2006). Paddy is mainly grown under irrigated conditions where nitrogen fertilizer

efficiency is low due to large N2 losses from flooded soils (De Datta and Buresh, 1989), in such

conditions the crop utilizes only about one third amount of nitrogenous fertilizers have been

applied, so it is essential to develop such a system, which not only supplements the supply of

nitrogen but also enables the crops to utilize most of the applied nitrogen fixing Blue Green

Algae.

1.2.3 Other effects on Rice: Besides increasing N2 fertility, BGA have been assumed to benefit

higher plants by producing growth-promoting substances (Roger and Kulasooriya, 1980). This

hypothesis is based on the additive effects of BGA inoculation in the presence of N2 fertilizers.

More direct evidence of hormonal effects has come primarily from treatments of Rice seedlings

with algal cultures or their extracts. It has also been established that algal growth-promoting

substances are beneficial to other crops besides Rice and that the production of such

substances is not confined to BGA. Whether, these substances are hormones, vitamins, amino-

acids or any other components are still unknown. Possible effects as Phosphate solubilizers or

as antagonists of Rice pathogens have not yet been demonstrated.

1.2.4 Favourable conditions for BGA growth: Under natural conditions, BGA grow

preferentially in environments that are neutral to alkaline; which explains that in Paddy field

direct correlations occur between:

1. Water pH and BGA number (Okuda and Yamaguchi, 1956);

2. Soil pH and number of spores of N2 fixing BGA in the soil during the dry season (Watanabe,

1959);

3. Soil pH and BGA growth (Okuda and Yamaguchi, 1952);

4. Soil pH and N2-fixing algal biomass (Roger and Reynaud, 1977).

In this regard pH is one of most important factor which affects the presence of BGA in a

habitat. Saline and Sodic (alkali) nature of soils significantly reduce the value and productivity of

affected lands. Salt affected soil are divided into three groups depending on the total soluble

salts (measured in the terms of Electrical Conductivity E. C.), soil pH and Exchangeable Sodium

percentage. Usar soils are grouped into two divisions Saline (Solanchak) and Alkalines

(Solonetz). These soils are characterized by impermeability, extreme hardness and occasional

presence of undesirable salt on the surface, all of which affect adversely the plant growth and

locally known as, „Usar‟ or „Reh‟ in U.P. These soils are so far regarded as unsuitable for

perennial irrigation and cropping. They occur generally in patches from a few acres to a few

square Km. in extent amidst well derived fertile soil. These soils have been studied by various

workers (Dhar and Mukherjee, 1939; Desai and Sen, 1953; Singh, 1950; Lamond and Whitney,

1992; Pandey et al., 2005).

The dominant algal species in acidic and alkaline soils often differ, i.e. the growth of

Chlorophyceae is favored by low pH values and that of BGA by higher values (Pandey, 1965).

In acidic soils of Kerala (pH 3.6-6.3), application of lime increased available N2 and promoted

the growth of N2 fixing BGA; in the untreated plots, predominant algae were Chlorophyceae

(Amma et al., 1966). Low pH and phosphorus deficiency most commonly limit the nitrogen fixing

Cyanobacteria in Rice field soil (Chen, 1974; Tilman and Kilham, 1976).

In most soils phosphorus availability is maximum in the pH range of 5.5 to 7.0 and

decrease at the pH drops below 5.5 or rises above 7.0 (Tisdel and Nelson, 1956; Moore, 1980).

Nitrogen remains in different states, either elemental or inorganic, but none is more important

than the nitrogen present in ionic form (NO2-, NO3

-, NO4+ etc), which constitutes the bulk of soil

nitrogen and is nutritionally more important for plants (Black, 1968).

High organic matter of soil favors cyanobacterial distribution (McRoc and Castro, 1967).

Alimagnob and Yoshida, (1978) have found that high fertility level reduces the population of

nitrogen fixing Cyanobacteria.

Mitra, (1951) indicated that manuring with cow dung increased the proportion of

cyanophyceae to that of chlorophyceae. Over 125 strains or types of such algae are known to

possess nitrogen fixing capacity and such nitrogen strains occur in all major typological groups

(Rippka et al., 1979). An extensive knowledge of the indigenous population is necessary. The

knowledge of the native cyanobacterial population in Rice field has so for exclusively been

achieved by means of cultivation based analysis followed by morphological identification of

individual isolates (Tiwari et al., 1991).

The number of Cyanobacteria phytotypes showed a seasonal variation and reached a peak

in September, some cyanobacterial sequences were only present during the Rice growth

season, while others were only found after harvest (Song et al., 2005). Over 90% of the

heterocytous filamentous Cyanobacteria isolates from the Rice Paddy fields belonged to two

genera, Anabaena and Nostoc (Kim and Lee, 2006).

It has been reported in earlier studies that Blue Green Algae changes the nitrogen fixing

potential in the soil (Chou and Lin, 1976; Rice et al., 1981; Ghawana, 1993; Gopalasowamy et

al., 2002; Satsangi et al., 2002). Nitrogen is the most important major nutrient required by Paddy

crop as compared to phosphorus, potash and other essential nutrients contributing towards

significant yield improvement. But nitrogen efficiency is only 30% in the case of low land Paddy;

it can be improved by blue green algae, which are the dominating forms that increase the yield

during the life period of Paddy crops. Therefore, it is worthwhile to study the effect of blue green

algae/ algal leachates as whole and individually on the growth parameters of Paddy.

The negative results of algal inoculation on Rice yield have also been reported in pot

experiments in the presence of ammonium sulfate. It was concluded that this depressive effect

may be due to a competition for nitrogen between the inoculated alga (Tolypothrix tenius) and

the crop (EL-Fadal, 1964). Watanabe, (1973) reported that Aulosira fertilissima widely used as

an inoculum in India, failed to achieve the desired effects in Japanese soils. Yamaguchi, (1980)

conducted a field experiment to investigate the effect of algalization for 5 years.

A statistical analysis of the results revealed that inoculation had no significant effect

except for a slight increase of N2 uptake by the Rice plant. Algalization was reported to be

ineffective under widely different agro-climatic conditions (Ahmad and Venkataraman, (1973);

Cole, (1977); Huang, (1978); Okuda and Yamaguchi, (1952); Mori, (1937), Subrahmanyan et

al., (1965) and Watanabe, (1961) respectively.

1.3 NEED OF THE STUDY

Presently, only little information is available on algal allelopathy, regarding the ecological

aspects in relation to soil, where Cyanobacteria are extensively used as bio-fertilizer. An urgent

need, therefore, exists to relate in vitro experimental studies with field level (In-vivo)

experimental studies to promote suitable recommendations to agriculturists on the use of

specific algal strain as bio-fertilizers.

Thus, in present investigation entitled “Allelopathic Responses of Some Selected

Strains of Cyanobacteria on the Growth and Yield of Paddy (Oryza sativa L.) in

Dayalbagh.” main emphasis was given on to study the allelopathic effect of isolated

Cyanobacteria (from Paddy fields) on the growth parameters (In- vitro) and yield attributes

(under field conditions) of Paddy and their effect on Rice grain quality (protein, amino acids and

carbohydrate). Extraction and identification of allelochemicals explored from leachates of

selected Cyanobacteria was also done at the end of study.

1.4 OBJECTIVES

1. Isolation, identification and culturing of Cyanobacteria from different Paddy fields of

nearby areas.

2. To analyze the physical and chemical parameters of soil from Paddy fields viz., water

holding capacity, texture, electrical conductivity, pH and C/N ratio.

3. To find out the effect of algal leachates on seed germination and seedling growth of

Paddy (In vitro).

4. To study the effect of BGA on growth and yield attributes of Paddy crops (Under field

conditions).

5. Estimation of protein, carbohydrate, amino acids in the grains of Paddy grown with or

without cyanobacterial application.

6. Extraction and characterization of allelochemicals explored from algal extract.

1.5 LIMITATIONS

1. During present study only three strains (i.e., Nostoc sp., Anabaena variabilis, Aulosira

fertilissima) were selected to check their allelopathic effect under lab and field condition.

2. Only methanolic extracts of tested BGA strains were analyzed for Identification of

allelochemicals.

3. Only identification and quantification of allelochemicals with the GC-MS instrument was

done. More time efforts and experimental strategies are required to fulfill the complete

characterization of BGA allelochemicals.

**********