Essay: Effect of biofilm biofertilizer on growth and yield of rice

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EFFECT OF BIOFILM BIOFERTILIZER ON GROWTH AND YIELD OF RICE (Oriza sativa L.)

CHAPTER 1
INTRODUCTION
1.1. Background
Rice (Oryza sativa L.) is the staple food of Sri Lankans. In Sri Lanka, around 34% of the total cultivated land area (0.88 million ha) is under paddy and around 1.8 million farm families are engaged in paddy cultivation producing nearly 5,000 MT annually. About 45% of total calories and 40% of total protein requirement of Sri Lankans are provided by the rice (Rice Research and Development Institute (RRDI), 2017). Therefore it is very important to enhance its productivity with sustainable and eco-friendly agricultural practices.
However, in Sri Lanka around 520,000 MT of chemical fertilizers (CF) used in 2016/2017 for rice, at a cost of over Rs. 3.0 billion (Ministry of Agriculture, 2017). Continuous use of such excessive amount of CF has caused serious environmental issues such as climatic change and ground water pollution etc. Besides the import of fertilizers is a drain on limited foreign exchange and adds to the cost of production. Due to the contamination of food chain and water bodies from this excessive CF application, human health is also affected. Therefore, remedial measures should be taken to avoid further serious environmental and socio economic problems. Hence there is a great need of having alternative eco-friendly bio fertilizers which can enhance crop productivity while maintaining environmental sustainability. Therefore agriculture today should shift from fossil-based inputs to bio-based inputs.
Biofertilizer is defined as a substance which contains living micro-organisms as mono culture or mixed culture and is known to help with expansion of the root system, better seed germination and plant growth (Chen, 2006). As a novel advancement in research field, Fungal-Bacterial Biofilms (FBBs) have been developed in-vitro and tested as biofertilizers. Application of such developed microbial communities in the mode of biofilm is called Biofilm biofertilizers (BFBFs).
Biofilm is assembling of microorganisms adherent to each other and/or biotic or abiotic surfaces and embedded in a matrix of polymers (Morris and Monier 2003; Seneviratne et al., 2007). Such biofilm consists of microbial cells (fungal, algal, bacterial and/or other microbial) and extracellular polymeric substances (EPS), which are secreted by themselves, to have biochemical and structural protection from adverse environmental stresses such as extreme pH, UV radiation, desiccation and osmotic shock etc. (Seneviratne et al., 2009).
Studies carried out have shown that BFBF application has restored the soil beneficial microbial community and enhanced crop growth and development while improving soil fertility and soil liability. BFBF facilitates to biological N2 fixation in non-legumes (rice, tea, wheat and vegetables) acting as ‘Pseudo nodules’ on the plant root while increasing solubility and availability of P and other macro, micro nutrients which required for crop growth (Seneviratne et al., 2007; Seneviratne and Indrasena, 2006).
Past studies have shown that application of BFBF can cut down the use of CF as per the 2001 recommendation by Department of Agriculture for rice by 50% with better crop growth, yield and soil condition than when 100% CF application was made (Weeraratne et al., 2012). But there is no critical evaluation for BFBF application with new CF recommendation (2013) for rice. And 50% of new recommendation (2013) provides only 36% of N received under the 2001 recommendation, which is inadequate for optimal plant growth and activation of BFBF.
Therefore studies were carried out to find out the optimum level of CF to be applied along with BFBF under the new fertilizer recommendation to obtain optimum yield of rice.

1.2. Problem Identification
Past studies showed that BFBF + 50% of the recommended CF level produced yields higher than that obtained with 100% of the recommended level of CF as per 2001 DOA recommendation for rice (Weeraratne, et al., 2012). But 50% of the new chemical fertilizer recommendation (2013) provides only 36% of N received under the 2001 recommendation up to 7 weeks after sowing, which is inadequate for optimal growth of rice and also not sufficient for better activation of BFBF. Therefore, it is necessary to find out the optimum level of CF to be applied along with BFBF under the new fertilizer recommendation to obtain optimum yield of rice.
1.3. Objectives
1.3.1. General objective
To determine the fertilizer replacement value of BFBF under the new fertilizer recommendation for paddy.
1.3.2. Specific objectives
To determine the effect of BFBF on soil nutrient improvement
To determine the effect of BFBF on plant growth
To examine the effect of BFBF on grain filling
To determine the effect of BFBF on yield

CHAPTER 2
LITERATURE REVEIW
2.1 Importance of rice in Sri Lanka
Rice (Oryza sativa) can be known as central to the lives of billions of people all around the world. Possibly it should be the oldest domesticated grain (nearly 10,000 years), and the staple food for 2.5 billion people. Growing rice is the largest single use of land for producing food, covering 9% of the earth’s arable land (Archive.gramene, 2013). As an agricultural country, rice has become one of the most important and leading crop in both food and socio economic sectors in Sri Lanka.
Social importance
In Sri Lanka, 34% of total cultivated land area (0.88 million ha) is devoted for rice production. Approximately 560,000 ha are cultivated during Maha and 310,000 ha are cultivated during Yala seasons, as a wetland crop in all the districts, making the average annual extent sown with rice to about 870,000 ha. Around 1.8 million farm families are engaging in paddy cultivation island-wide in Sri Lanka (RRDI, 2017). About 30% from total labor force and employment is also represent by the agricultural sector (Central Bank, 2016a).
Nutritional importance
Rice provides 21% per capita energy and 15% of per capita protein in global human population. Calories from rice are important especially in Asia, providing two third from total Requirement (Archive.gramene, 2013). Rice provides 45% of total calories and 40% of total protein requirement as an average in Sri Lanka (RRDI, 2017).

Economic importance
The contribution of the total agricultural sector on Gross domestic production (GDP) in Sri Lanka is 7.1% and from that, rice represents 0.6% (Central Bank, 2016b). Current annual rough rice production in Sri Lanka is 2.7 million tons and it is enough to satisfy around 95% of the domestic requirement, with Rs. 8.57 per kg cost of production (RRDI, 2017).
Within next three decades, there is a need to increase the rice production by 2.9% per annum to meet increasing demand for rice (by 1.1% per annum), as the result of global population increment. The per capita consumption of rice fluctuates around 100 kg per year depending on the price of rice, wheat flour and bread. Increment of cropping intensity and national average yield are the effective alternations that available to achieve this production target (RRDI, 2017).
Nutrient requirement of rice
As all other crops, rice also requires sufficient and balanced supply of plant nutrients throughout the growing season to achieve optimum growth and yield. The Response for fertilizer of rice depend on variety, environment condition and farmer practices (Hach and Nam, 2006).
Old varieties verse newly improved varieties
With the green revolution, high yielding rice varieties were developed to meet the increasing food requirement, as increasing global population. So farmers adopted to cultivate newly improved rice varieties which provide higher yield than traditional or old improved varieties. Many of old improved or traditional rice varieties showed low response for the CF to increase their yield or other performance (Jayawardena, n.d.). Although newly improved rice varieties supply more yield than traditional varieties, they are directly sensitive to the inorganic petrochemical fertilizers, especially for the N fertilizers. Therefore these high yielding rice varieties require intensive application of inorganic fertilizers to have better yield while traditional varieties possess minimum sensitivity for fertilizers (Taylaran et al., 2009)

N requirement

Nitrogen (N) is the single most important soil nutrient that necessary for the plant growth and yield especially in intensive agricultural systems. It is known as motor of plant growth. N makes 1 – 4% of total dry mater of a plant, because it is important to increase the plant height, leaf area, number of panicles, number of spikelets and number of filled grains, which largely determine the yield capacity of a rice plant (Harrell and Saichuk, n.d.). Being essential component in protein, N involves to all most all the major plant development and yield formation processes. Satisfactory soil N availability is important to uptake the other nutrients and N is most limiting element in soil at many times (Food and Agriculture Organization (FAO), 2000).
N in the soil can be available in both ammonium (NH4+) and nitrate (NO3′) forms. Although rice plants capable to use both forms of soil N, ammonium consider as the main source of N rather than nitrate, with the flooded nature of rice cultivation. Nitrate is unstable and can be lost simply by de-nitrification and leaching under anaerobic, flooded conditions, when ammonia is more stable and available for plants (Agricultural National Service and Statistics, 2014).
P requirement

Soil phosphorous (P) is another macro nutrient which is important to have better growth of rice plants,especially in early stages. It promotes early plant growth and development, facilitating to have a strong root system. P is important because it increases the tillering, root development, early flowering, and ripening of rice plants. P contributes 0.1 – 0.4% of the dry matter of plant. It plays a key role in plant’s energy transformation, while being an essential element for photosynthesis and many of other chemical and physiological processes (FAO, 2000)
Soil P available in both organic and inorganic forms, and organic P is slowly available to the plants. Flooding and draining rice cultivation system is greatly impact on P availability or lost from the soil. However flooding increases the availability of soil P, and when soil drain and aerated, it decreases the P available level (Harrell and Saichuk, n.d.). Soil P mostly available for plants as phosphate (PO43-) ions but it is greatly loss due to leaching under flooded conditions.
K requirement
Another important soil nutrient for rice is Potassium (K) responding 1- 4% of dry mater of plant as N. Some of modern high yielding rice varieties response for K than other nutrients. It is essential for building the leaves biomass at first, then for the culms and later for the grains. K is responsible to active more than 60 enzymes governing metabolism of life. K is also a vital part in synthesis of protein and carbohydrates. K is closely associated with the ability of disease resistance. It improve water regime and increase drought, frost and salinity tolerance ability of plants. K is also known as the quality nutrient because it greatly effects on quality factors such as size, shape, colour, taste, shelf life, fiber quality and other quality measurements. K is less affected by flooding and draining than N and P (FAO, 2000).
In addition to these major nutrients, there are so many other macro and micro nutrients which are critical for rice growth and yield. Deficiency or toxicity of any single nutrient, limits the rice plant growth and development in different ways and it finally reduces the rice yield also. Therefore, better management of soil nutrient level is one of main factor to have optimum plant growth and yield. Famers have to use external sources of fertilizers, to manage soil fertility level with the removal of these nutrients from soil by plant consumption. It is not enough to have natural soil nutrient only for plant growth, especially in agricultural lands. But the most considerable condition should be the management of nutrient level of the soil, than application of chemical fertilizers without any consideration.
Fertilizer application in rice
Fertilizer recommendation
Table 2.2: Department of Agriculture Sri Lanka, fertilizer recommendation (2013) for rice in kg/ac.
Source Basal Top 1 Top 2 Top 3 Top 4 Total Notes
2001 DOA recommendation:
120 Bushels/acre
Dry Zone
3 1/2M paddy Urea 5 + TSP 35
+ MOP 15 Urea 30 Urea 50 Urea 20 + MOP 15 – Urea 105 + TSP 35 + MOP 30 Add ZnSO4 2kg/acre in Maha season
2013 DOA recommendation:
Irrigated
Dry & Intermediate Zone
3 1/2M paddy TSP 22 Urea 20 Urea 30 + MOP 10 Urea 26 + MOP 14 Urea 14 Urea 90 + TSP 22 + MOP 24 Add ZnSO4 2 kg/ac in Maha season

Amount of fertilizer applied in rice cultivation in Sri Lanka

Sri Lankan government has been subsidizing on fertilizer since more than four decades with the objectives of increasing production of agriculture sector and giving support to livelihood of majority of people (Ekanayake, n.d.). But there are many argue on effectiveness and sustainability of that effort, when concerns about yield increments and excess use of fertilizers, the highlights of different literature sources have shown that providing fertilizer subsidy makes less efficient in increasing production of agriculture sector (Rodrigo, 2015). However it badly influenced on indiscriminate use of CF which caused for many negative consequences faced by today agricultural sector.
Sri Lanka becomes highest agro chemical user and 8th highest chemical fertilizer user in the world (World Health Organization, 2013). The government has to spend over Rs.50 billion in importing fertilizer and Rs.40 billion as fertilizer subsidy (Central Bank, 2014). This is consider as one of especial burden of the government budget.
Out of other agricultural practices, government gives special attention on paddy sector,as it is our staple food and engaging majority of people with paddy cultivation. Quantity of total CF required in the paddy sector is around 500,000 MT in 2016/2017 at a cost of over 3 billion SLRs (Ministry of Agriculture, 2017; Chatura and Katsuhiro, 2017). Avarage use of urea, Murate of pottash and triple supper phosphate is 434, 151 and 253 (kg/ha) respectively in Sri Lankan paddy sector (Ministry of Agriculture, 2017).
Continuous use of such excess amount of CF made agrarian lands unsuitable for natural vegetation and, at present farmers cannot continue their farms without fertilization. So they require more and more CF and other agro chemicals without caring of its serious environmental issues and heavy expenditure by way of foreign exchange. In many farm lands around 70% of chemical fertilizer does not get used and leaches to underground or surface water bodies. Therefore, Sri Lanka has become trapped in a destructive chemical loop.
Environmental consequences
Although it is necessary, the use of fertilizers to have high agricultural production, due to its misuse by amounts, types and times of application, today it is negatively affect on environment, human and animal health. As a result of this improvident use of CF, today global agricultural sector is standing on alarming condition.
Continuous application of CF depletes the natural fertility of soil. So more and more application of fertilizers do not help to replenish soil nutrients. And over use of specific type of nutrient element makes imbalances in soil nutrient conditions and it is negatively affect to the equilibrium of a stable soil.
Although fertilizers help to faster growth of plants, they may be not healthy and strong, as plants which are grown under natural conditions. Because plants become more susceptible to the pest and diseases due to having poor immune system and breakdown of their natural resistance capacity under high fertilizer management systems.
One of another major issue is contamination of ground water table by chemical elements, especially with the use of N fertilizers. It also leads to accumulation of nitrate in surface water bodies causing to the eutrophication which is unsuitable for all living beings (Taher et al., 2014; Sustainable baby steps, 2017)
Excess CF application is directly harmful to the microbial community of the soil and it suppresses most of the beneficial microbes such as N2 fixing bacteria and P solubilizing organisms. It reduces the natural microbial contribution on plant growth and natural soil recycling system. Many of CFs change the pH level of soil, resulting extreme acidic or basic conditions, and it is adversely impact on microbial community, because it inhibits their activities, growth and development. Therefore finally, soil may become death and infertile, due to the loss of microbes in soil who serve as heart of a fertile soil (Bagyaraj and Revanna, 2016)
Emission of greenhouse gaseous compounds to the air is another global environmental problem that we have to face due to this excess fertilizer application. Global warming is another major issue related to this situation. These are become first and most primitive threats on human lives today.
Salinization of soil, harden of soil and accumulation of heavy metals are other different consequences raised due to excess CF application. .
Health issues
Improvident application of CF is directly affects on human and animal health as environment. Because the use of contaminated air and water causes to numerous short term and long term hazardous on their health. This alters the immune, endocrine and nervous systems of living beings. Scariest effect of CF is known as methemoglobinemia or blue baby syndrome. In addition that, gastric cancers, birth malformations, hypertension, stomach cancers, kidney diseases etc. become common issues in today (Sustainable Baby Steps, 2017).
Chronic kidney disease (CKD) is one of critical health issue in Sri Lanka today. There is a big criticism in society showing that excess use of CF and use of low quality fertilizer cause to CKD. However the government reports there are 50,000 kidney patients in the country, who are mostly agricultural workers and farmers and around 20,000 people have died because of kidney failure in the country (Chatura and Katsuhiro, 2017).
Economic issues
CF application contributes largely to increase the cost of production. Although it increases some yield, final profit of paddy sector may reduce and sustainability of this agriculture sector may be broken down in all the ways.
Biofertilizer
A biofertilizer is an agricultural input, which contains living microorganisms exerting direct or indirect beneficial effects on plant growth, productivity and soil sustainability through various mechanisms (Vessey, 2003).
Biofertilizers consist selective micro-organisms like bacteria, fungi or algae as their mono culture or mixed culture forms. For the production biofertilizers, commonly use the effective strains of microbes that can fix the atmospheric N2 or/and solubilize the P (Chen, 2006).
Need for biofertilizers
Present view of society, more concerns on the detrimental side effects of the misuse of agrochemicals, and it has caused to investigate another way of gaining better crop production. As a result of that an increasing trend can be seen towards the use of biofertilizers. According to the many researchers and scientists, biofertilizer can be used as the best solution, to re-correct the many adverse impacts that occurred due to over use of CF on environment and living beings.
Biofertilizers are different from organic or CF, because it does not directly supply nutrients to the soil or plants. It re-organizes the microbial community in the soil, enhancing rhizobacterial species which colonized in the rhizosphere or plant surfaces and facilitate to have fertile soil for plant growth (Trujillo and Ramirez, 2016).
These beneficial micro-organisms promote the plant growth, yield and crop quality, by different mechanisms. Production of plant growth promoting hormones, biological fixation of atmospheric N2 and bio-solubilization of P are the major roles play by those microbes (Seneviratne et al., 2007). Other than that, they regulate the dynamics of organic matter decomposition and availability of macro, micro nutrients in the soil (Chen, 2006). These beneficial microbes secrete antibiotic compounds and organic acids. This is important to adjust soil pH and to suppress the pest and diseases (Jayasinghearachchi and Seneviratne 2006b). Due to the organic acid production and soil acidity, they can facilitate to rock weathering as well (Seneviratne and Indrasena 2006).
In addition, biofertilizers can be used as an economic input, due to increase of yield, reducing the cost of production at same time. Therefore, it is an economically viable support for many of rural, small or marginal farmers. Not like CFs, biofertilizers can be effectively used in long terms, without having any negative impact. Another most important role of biofertilizers are management of natural recycling systems and viability of soil. Biofertilizers can be consider as the most suitable supplement for the CFs to have sustainable agriculture system (apicol.co.in., n.d).

Types of biofertilizers in rice
Biofertilizers are environmentally safe, eco-friendly and sustainable agricultural input. As one of large cultivating crop in the world, any single effect on rice field, becomes a serious issue at the global level. Hence today, to have sustainable future for the agriculture through the rice cultivation, world is shifting gradually to replace the chemical fertilizers by biofertilizers. The biofertilizers used for rice production mainly consist with Azospirillum, Phosphobacteria, Blue green algae, azolla and Mycorhiza like selective microbes (Blackseagrain, 2016).
Azospirillum
Azospirillum is one of important symbiotic bacteria, used in biofertilizers for rice. Azospirillum can use as a seed, seedling or field treatments. They successfully colonize in root zone of rice, and capable of fixing more atmospheric N2. Also they can solubilize the phosphorous and other macro micro nutrients up to some extent. It facilitates, drought resistance of plants when rainfall or irrigation is delayed (Amal Raj, 2002).
Phosphobacteria
Phosphobacteria is another important group of microbe used as biofertilizer. They are central of solubilizing phosphates in soil. Bacillus megatherium var phosphaticum and Pseudomonas fluorescens are bacteria that have strong ability of dissolving phosphates. Phosphobacteria can be used in both lowland and upland rice cultivation. Use of phosphobacteria as biofertilizer, can be reduced depending upon the native phosphorus content of the soil (Amal Raj, 2002).
Blue green algae/Azolla
Cyanobacteria as a beneficial microbe plays a major role in sustainable enhancement of agriculture productivity. Cyanobacteria are photosynthetic organism group which can easily survive under minimum requirements, and naturally occur in several agro eco-systems (Woese, 1987; Castenholz, 2001). They are used successfully as biofertilizer for rice cultivation worldwide, as mono or mixed cultures. Cyanobacteria can fix the atmospheric N2 and produce some bioactive compounds which promote the plant growth and soil nutrient state. As well as they are important to degrade several toxic compounds (Cohen, 2006). Important species of Cyanobacteria are, Blue Green Algae (BGA), Anabaena, Nostoc and Tolypothrix (Mishra and Pabbi, 2004).
BGA is important species of algae which can fix the N2 over 15 Kg/ha per season. BGA facilitate vigorous plant growth by producing vitamin Biz and growth factors. The algal mat in paddy fields protects soil from moisture losses, oxygenate the water and they excretes organic acids that can solubilize the phosphorous (Amal Raj, 2002).
Azolla is a water fern which present commonly as an algal symbiosis, called Anabaena-Azolla. This symbiotic association can fix the atmospheric N2 and Azolla excretes organic nitrogen in water during its growth. It prevents weed growth in rice fields while enhancing availability of nitrogen, potassium and organic carbon (Amal Raj, 2002).
Mycorhiza
Mycorrhiza is also a symbiotic association between fungus and roots of a host plant. This occurs naturally in low and up land rice fields. Mycorrhiza can metabolize the phosphorous required by plant growth (Amal Raj, 2002).
Biofilm biofertilizers/ BFBF
Biofilm is an assemblage of microorganisms adherent to each other and/or biotic or abiotic surfaces and embedded in a matrix of polymers (Morris and Monier 2003; Seneviratne et al., 2007). Biofilm consists with microbial cells (algal, fungal, bacterial and/or other microbial) and extracellular biopolymers (extracellular polymeric substances) produced by themselves which provides structural and biochemical protection from adverse environmental conditions such as extreme pH conditions, UV radiation, dehydration, osmotic shock, antimicrobial substances, predators, etc. (Vandevivere and Kirchman 1993; Seneviratne 2003; Romanova et al., 2006).
There are three major types of biofilms in the soil. They are bacterial biofilms, fungal biofilms and fungal-bacterial biofilms (FBBs). Although FBBs are generally formed by attaching to an abiotic surface of soil, they can be formed by adhering bacteria to the biotic surface of the fungi as well. When fungi is non filamentous, both bacteria and fungi act as the biotic surface. FBBs are more active and show better results compare to their mono and mixed cultures (Seneviratne et al., 2006; Seneviratne et al., 2008).

Biofilm naturally available in soil, plants, animals and environment. These communities may be harmful or beneficial to the agro ecosystem (Morikawa 2006). Naturally occurring these beneficial microbial communities generally attach to the plant roots of some crops and create favorable surround for their growth and development in different ways (Seneviratne, 2003). And naturally occurring biofilms activate preferably in N deficient conditions incorporating N2 fixers to ecosystem. But availability of these beneficial biofilms in natural agro ecosystem is not enough to have significant effect on crop production. Therefore, the development of such biofilms in-vitro and application as biofertilizers are essential for augmenting agricultural productivity (Bandara et al. 2006).
Prospects of using BFBF as a biofertilizer in rice
As a novel advancement in research field, Fungal-Bacterial Biofilms (FBBs) have been developed in-vitro and tested as biofertilizers. Application of such developed microbial communities in the mode of biofilm is called Biofilm biofertilizers (BFBFs). It can be known as next generation bio-fertilizer. Application of this BFBF could enhance the plant-associated nitrogenase activity, rhizoremediation, plant and soil carbon sequestration, and many of other plant growth promoting activities (Seneviratne et al., 2009).
BFBF restore the soil beneficial microbial community and enhanced crop growth and development while improving soil fertility and soil liability than the application of their monoculture forms conventional biofertilizers.

Figure 2.3: Conceptual model showing the association established between the root and the biofilm when the BFBFs were applied to a root of a non-legume.
This BFBF inoculants significantly increase the biological N2 fixation in legume plants improving plant-bacterial symbiosis compare to the conventional rhizobial mono culture inoculants (Jayasinghearachchi and Seneviratne, 2004b). Not only that BFBF can act as pseudo-nodules on the roots and fix the N2 in non-legumes also (Seneviratne et al., 2009).
BFBF can be effectively used to bio-solubilize of rock phosphate (Jayasinghearachchi and Seneviratne 2006a; Seneviratne and Indrasena 2006) increasing the availability of soil P for plant growth. This is one of critical characteristic of BFBF because limitation of soil P which directly associated with plant growth and yield in many fields. BFBF able to increase the soil K by chelating action and increase uptake of other macro and micro nutrients as well. Apart from the major nutrients, plant growth promoting inoculum enhances the micro nutrients such as Zn, Cu and Fe uptake by plants (Bashan 1998).
Many studies showed that BFBF application increases the colonization of beneficial endophytic microbes in plant tissues and facilitate high dry matter accumulation of plants, due to the release of organic acid and plant growth promoting substances such as indole acidic acid over the monoculture inoculants (Seneviratne et al., 2008; Bandara et al., 2006). The higher acidity of soil because of that acid production, important to suppress the pathogens, providing natural bio control for plants and to mineralize the soil nutrients as well (Jayasinghearachchi and Seneviratne 2006b).
One of major problem that faced by conventional microbial technologies is poor survival rate of inoculated microbes in natural system, with the diverse natural environmental stresses. But there were many evidences that proved BFBF can successfully tolerate this environmental issues. BFBF can survive with high level of salinity and tannin (Seneviratne et al., 2007), low pH and chromium levels, pathogenic microbes or some predators (eathworms) (Matz et al., 2004).
Not only that BFBF responsible to reduce the cost of production by reducing agro chemical requirement during the growing season.
Because of these primitive characteristics of BFBF, we can use this successfully for rice production. Rice cultivation in Sri Lanka should be more sustainable and productive, as the largest single land use and as largest livelihood in Sri Lankans. At present, the soil fertility and livability of Sri Lankan paddy lands become very poor and unsatisfied, due to the excess application of fossil fuel derived CF since few decades. BFBF gives new hopes to correct this helpless situation by their beneficial behavior.

Studies done in Sri Lanka
There are series of studies done by scientists to examine the effect of BFBF on growth and development of different plants including both legumes and non-legumes.
Past studies showed that application of BFBF in rice can cut down the CF application by 50% successfully with the yield higher than that obtain by 100% CF application as per 2001 recommendation (Weeraratne et al., 2012). Application of BFBF with 50% of CF could increase the plant growth, dry matter accumulation without affecting to the yield and grain quality of rice. Compare to the 100% CF application, BFBF +5 0% CF application was achieved higher plant bio mass by 55% (Seneviratne et al., 2009). Therefore, BFBF application is not only a fertilization it is a soil treatment that helps to improve it decreased quality while enhancing crop productivity.
Jayasinghearachchi and Seneviratne (2006b) have shown that Pleurotus ostreatus’Pseudomonas fluorescens biofilm (FBB) application for Tomato has increased endophytic colonization (Lycopersicon lycopersicum) by P.fluorescens, a biocontroling agent and enhance the bio control of diseases. This clearly concluded application of BFBF could be able to facilitate enhancement of natural disease resistance ability of plants,
In related to the tea plant also, BFBF with 50% of CF application has provided maximum leaf yield and shoot / root ratio compare to the 100% CF due to optimum root growth (Seneviratne et al., 2009). BFBF including four microbes (three bacteria + one fungus) application for Anthurium with 50% of CF in an inert particle medium has given relatively higher plant growth rate over 100% CF application (Seneviratne et al., 2009).
As legume plant in soybean (Glycine max) Fungal-Rhyzobium biofilm inoculant significantly increases N fixation by 30%, compared to the conventional rhizobium-only inoculant (Jayasinghearachchi and Seneviratne 2004b). For maize (Zea mays L.) also application of BFBF with 50% of CF has showed better yield and soil improvement under greenhouse pot experiment (Buddhika, et al., 2012).

Studies done in other countries
Other than Sri Lankan condition, use of biofilms as biofertilizer to have sustainable crop production has been studied in other countries.
A group of Indian scientists have examined the effect of different types of in-vitro developed biofilms on wheat crop growth and soil condition. They have shown that application of such biofilms improve the soil fertility while enhancing plant growth, N fixing potential and nutrient uptake by plants ( Karivaradharajan et al., 2013).
Trichoderma viride and Anabaena torulosa based biofilm application for Macrophomina phaseolina (Tassi) Goid infected cotton crop was evaluated to determine the effect of biofilm than monoculture in terms of plant growth, soil improvement and bio-control parameters. And they concluded there are significant beneficial activity by biofilms than monoculture inoculants (Triveni et al., 2015).
Gaps in knowledge and studies needed
Here series of studies showed that BFBF application can cut down the 50% of CF (2001 recommendation) for rice without any reduction of grain yield. But there is no sufficient studies carried out on BFBF application along with the new CF recommendation (2013). Due to the altering of fertilizer levels in 2013 recommendation, now 50% of CF only provides 36% N received under 2001 recommendation up to 7 weeks after sowing, which insufficient to have better rice plant growth and optimum activation of BFBF. So it is important to have critical evaluation to find out most effective CF level of 2013 DOA recommendation to be couples with BFBF. Because application of BFBF altered the soil micro flora and increase nutrient cycling compare to CF along application. It is also important to have understood about the crop response to BFBF under famer field level with different environmental conditions.
As staple food in Sri Lanka, having increment of growing rice with environmental friendly and human healthy manner is one of main national level requirement. But today paddy cultivation lands become death condition due to the improvident agricultural practices of farmers since few decades. So immediate taken of corrective measures to have sustainable future for paddy cultivation is must. At same time productivity improvement also must be considered. Application of BFBF may provide new hope for reducing CF usage and restoring the damaged soil by conventional agricultural practices.
This study will be targeted to evaluate BFBF application with different doses of new fertilizer recommendation (2013) under farmer field level to find the CF replacement value by BFBF examining plant growth, yield and some soil parameters.

CHAPTER 3
METHODOLOGY
Experimental design and treatments
Experiment was conducted by randomized complete block design (RCBD) during Yala season with six replicates, four at Dehiaththakandiya Mahavali Thoda farm and other two at Rice Research and Development Institute, Bathalegoda research field. BG 360 and BG 300 rice varieties were cultivated in each field respectively.
Six treatments were applied with the control as followed in both fields. Chemical fertilizer rated were selected according to the new fertilizer recommendation (2013) of Department of Agriculture for rice, to determine the effective BFBF + CF combination which can provide optimum plant growth and yield.
100% CF application (T1)
80% CF application only (T2)
80% CF + BFBF application (T3)
65% CF application only (T4)
65% CF + BFBF application (T5)
Control ‘ only soil (T6)

Field layout
Slope
Replicate 1 Replicate 2
Tr 2 Tr 1 Tr 4 Tr 3 Tr 6 Tr 5
Tr 3 Tr 5 Tr 6 Tr 1 Tr 4 Tr 2

One replicate as followed

Fertilizer application to the fields
CF were applied at each and every application times, with the dose of selected ratios from department recommendation. But BFBF was applied only at first and third top dressing periods in addition to the application of CF. It was applied with 500 ml per acre dose.
Time schedule of fertilizer application as followed.
Table 3.1: Fertilizer application schedule for the experiment fields.
Fertilizer application step Time of application
Basal dressing Before sowing
First top dressing 3 weeks after sowing
Second top dressing 5 weeks after sowing
Third top dressing 7 weeks after sowing
Fourth top dressing 8 weeks after sowing

Sample collection for soil and plant analysis
Plants with soil in rhizosphere were collected from the fields at two different growth stages to analyze the plant and soil parameters.
Tillering stage
Flowering stage
Analysis of plant samples
Two rice plants were randomly uprooted from each and every plot of the fields according to zig-zag pattern, at both tillering and flowering stages. Soil was removed carefully from plants without any damage to the root system. Then shoot length, root length and root volume were recorded and number of tillers and number of panicles per hill were counted. Root and shoot dry mass per hill were measured at the each stage.
Chlorophyll content of flag leaf was recorded in the field using a chlorophyll meter (SPAD-502-Chlorophyll meter).

Analysis of soil physical and chemical properties
Initial analysis
As initial soil analysis, soil pH and moisture content were measured at each sampling stage, and fresh soil samples which collected from rhyzosphere were analyzed for available nitrate, available ammonium and available phosphate according to the Tropical Soil Biology and Fertility (TSBF) handbook.
Determination of soil pH
The soil pH was measured in distilled water at a 1:2.5 soil-to-water volume ratio with a pH meter (Orion Star A215).
Determination of soil moisture content
Soil moisture content was measured using general standard method (TSBF).
Soil Moisture Content (%)=(W2-W3)/(W3-W1)*100
Where,
W1 – empty crucible weight (g)
W2 – crucible weight with fresh soil (g)
W3 – crucible weight with dried soil (g)
Determination of soil available nutrients
As available soil nutrient parameters soil nitrate, ammonium and bicarbonate extractable phosphate were measured in fresh soil using colorimetric method by UV spectrophotometer (Shimadzu Spectrophotometer, UV-2450) under the guidelines of TSBF handbook.

Extractable Nitrate or Ammonium or Phosphate (”g /g soil)= (C*V)/W
Where,
C – Corrected concentration (”g/ml)
V – Extract Volume (ml)
W – Weight of soil sample (g)
Final analysis
Soil collected from rhizosphere was air dried, ground and sieved (0.5mm) for total soil nutrients analysis.
Determination of total nutrients
Total nutrients also determined under the guidelines of TSBF handbook. Sieved soil digested for total nitrogen and phosphorous analysis. Total nitrogen was determined using Kjeldhal method. Total phosphorous and total carbon were analyzed using colorimetric method with UV spectrophotometer (Shimadzu Spectrophotometer, UV-2450). Exchangeable soil potassium was analyzed using atomic absorption spectrophotometer with modified Morgan extraction method.
Analysis of yield parameters
Three plants were collected from field under each treatment and each replicate at one week before harvest to analyze the yield parameters.
Number of tillers, number of panicles, total grain weight, 100 grain weight and seed moisture content were analyzed from collected samples.
Seed analysis
Rice grains were dehusked, ground and sieved (0.15 mm) before start the analysis. Prepared rice grain powder samples were digested for nitrogen and phosphorous analyses. To determine the protein content of rice grains, rice grain nitrogen content was determined by Kjeldhal method (TSBF). Total phosphorous content of rice grains also was determined using UV spectrophotometer (Shimadzu Spectrophotometer, UV-2450) (TSBF).
Potein content(g)=nitrogen content(g)*6.25
Data analysis
While experiment in progress 2 replicates out of 4 at Thoda farm affected by irrigation problem. Therefore we analyzed 4 replicates (2 from Thoda farm and another 2 from Bathalegoda farm) together to determine treatment effect.
Data were analyzed statistically by ANOVA using Minitab 16 version. ANOVA was conducted separately for each sampling stage.

CHAPTER 4
RESULTS AND DISSCUSSION
Analysis of rhizosphere soil parameters
Soil pH
Soil pH at both stages showed significant difference among treatments. At tillering stage, 80% CF + BFBF treatment showed significant reduction of soil pH than 100% CF application, while both 80% CF + BFBF and 65% CF + BFBF showed significant reduction at flowering stage compared to the control (Table 4.1).
Table 4.1: Rhizosphere soil pH at tillering and flowering stages.
Treatment Soil pH
Tillering stage Flowering stage
100% CF 6.55a 6.40ab
80% CF 6.13ab 6.44ab
80% CF+BFBF 5.81b 6.17b
65% CF 6.39ab 6.83a
65% CF + BFBF 5.92ab 5.93b
Control 6.22ab 6.80a
Pooled SD 0.2885 0.2772
Mean values in the same column followed by different letters are significantly different at P = 0.05 level, (Tukey’s, family error rate grouping test).

Biofilm inoculum produces organic acids such as Indole Acetic Acids, which are important for plant growth and development. This causes to reduce soil pH of rhizosphere. Also this acidity of rhizosphere is important for pathogen suppression and mineralization of soil nutrients (Jayasinghearachchi and Seneviratne, 2006a).

Rhizosphere soil available nutrients
Soil ammonium, nitrate and phosphate were determined as available soil nutrient parameters. Nitrate availability at tillering stage was significantly lower in BFBF applied treatments compared to the control. Even though other parameters were not significantly different among treatments at 5% probability level, there was a clear declining trend with BFBF application (Table 4.2).
Table 4.2: Rhizosphere soil available ammonium, nitrate and phosphate levels at both stages.
Treatment Soil NH4+ (”g/g soil) Soil NO3- (”g/g soil) Soil PO4-3 (”g/g soil)
Tillering Flowering Tillering Flowering Tillering Flowering
100% CF 1.45a 4.42a 3.59ab 4.29a 12.92a 7.02a
80% CF 1.46a 2.92a 3.39ab 4.64a 13.51a 7.59a
80% CF+BFBF 0.38a 1.77a 1.68b 2.55a 9.65a 3.80a
65% CF 0.53a 3.55a 2.84ab 5.18a 11.62a 6.51a
65% CF+BFBF 0.26a 1.48a 1.41b 2.43a 8.52a 3.69a
Control 1.13a 2.69a 4.09a 6.24a 12.22a 7.33a
Pooled SD 0.8724 2.931 1.068 2.864 4.784 2.754
P value 0.223 0.728 0.012 0.400 0.659 0.194
Mean values in the same column followed by different letters are significantly different at P = 0.05 level, (Tukey’s, family error rate grouping test).

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