Chapter 1INTRODUCTION1.1                     BackgroundPakistan is suffering from poverty therefore it is necessaryto increase crop production for the reduction of hunger and insecurity of food.Organic fertilizers and minerals are beneficial for increasing the cropproduction. However farmers do not use mineral fertilizer for variety ofreasons. Besides this it is necessary to improve the production of crop in aneco-friendly manner. This has led to the promotion of commercial, biologicaland chemical products that can enhance the organic matter content of soils.

Nutrients such as nitrogen (N), phosphorus (P) and sulphur(S) are released asresult of decomposition of organic matter using soil microorganism. This is thereason microorganism are important component of soil biodiversity andintegrated nutrient management systems.1.1.1       LocationTaxila, Punjab Pakistan  Chapter 2Literature Review1.2                     BIOFERTILZERSIn contrast to chemical fertilizer, biological fertilizermake use of microbes for improving of crop production and this can be doneeither by applying directly to the soil or on the seeds before planting.

Microorganismthat help plants in uptake of nutrients through their interaction in the rhizosphere(Soil that surrounds and is influenced by the roots of plant). They speed upcertain microbial processes in the soil which increase the extent ofavailability of nutrients in the form easily utilized by plants for theirgrowth.Most of the times microorganism cannot be replicated in thenatural environment because of diverse nature of environment (temperature, pH,etc.) and therefore artificially multiplied culture of microorganism play animportant role to speed up their microbial activity. As use of biofertilizers iscost effective so it is one of the important component of Integrated Nutrient Management(INM).

           Microbial FertilizerIt is defined as the addition or injection of microbialpopulation into the soil to enhance soil productivity. For example fungi,nitrogen fixing bacteria, phosphorus solubilizing bacteria etc.1.

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3                     Nitrogenfixing bacteria as microbial fertilizersAlthough nitrogen is abundantly present in atmosphere yet itis the most limiting nutrient to the growth of plants. As plant cannot directlyuptake nitrogen from atmosphere therefore nitrogen fixing bacteria present inthe roots of plants, play role in uptake of nitrogen. These bacteria areassociated with plants in three different ways:·        Some nonsymbiotic (freeliving) nitrogen fixing bacteria are present in soil·        Symbiotic nitrogen fixingbacteria; others have suggested endophytic association with plant·        Others are associated withrhizosphere and don’t form close endophytic symbioses.

The large amount of nitrogen is fixed by using these threesystems in spite of change in environmental conditions or difference incombination of plant-microbe. Fixed nitrogen is beneficial for minimizingleaching and denitrification. There are different organism helpful in easy accessof plants to the nitrogen in atmosphere and use them as biofertilizers andthese are discussed below.1.3.

1       Non symbioticnitrogen fixing bacteriaNon symbiotic bacteria such as azotobacter, clostridium alsohelp in fixing nitrogen present in atmosphere. If environmental conditions arefavourble then Frankia (nodulating bacterial symbionts) can also fix N2 innon-symbiotic association 1. Frankia can be present in rhizosphereof non-host plants and help in nitrogen fixation. Record has shown Frankia ispresent in rhizosphere of Betula Pendula2 and in soil with noactinorhizal plants3 . N2 fixation cannot be estimatedby free living bacteria. There are different methods of inoculating bacteria ingreen house experiment such as leaf spray, seed soaking and side dressing.

Thegrowth of cucumber and barley plants was stimulated by inoculation ofBeijerinckia mobilis and clostridium sp.4 . However, it remainedunclear about the mechanism of growth. The growth is not only stimulated byinoculating bacteria but it can also be due to plant growth hormones.

1.3.2       Symbiotic nitrogen-fixingbacteriaRhizobiaRhizobia family (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium)5is the best known and most exploited symbiotic nitrogen-fixing bacteria. Thiskind of bacteria cause infection in legumes. We should consider rhizobia-host compatibilitywhile selecting bio fertilizer. For N2 fixing, the capability ofrhizobia varies up to 450 Kg N.ha-1 among strains of bacteria and host plantspecies.

Rhizobial strains that are considered as practical applications as biofertilizers must have some additional characteristics like they must havefixation rate of Nitrogen and can compete with naturally occurring rhizobia.Survival and easy production of inoculum is necessary in field application whentarget seeds are inoculated. Inoculation is of prime importance when rhizobialpopulations of resident soil are absent. For example, acidic and basic soilcontain low population density of alfalfa rhizobial symbiont (Sinorhizoniummeliloti) and low inoculum potential of Brady rhizobium sp.

respectively 6.It is highly advantageous to inoculate compatible rhizobia under suchconditions. Evaluating the need for inoculation and performing cost benefitanalysis is necessary before initiating massive scale inoculation program withrhizobia. Inoculation is useful for productivity of crops if rhizobialpopulation density is less than 100 rhizobia/gram of soil 7.

Inoculation would prove cost effective in presence of low population densitiesin spite of indigenous rhizobia nitrogen fixation efficiency.CyanobacteriaCyanobacteria find its importance in ecology e.g. an aquaticcyanobacterium (Trichodesmium) contributes 36% of global nitrogen fixationaccording to estimate. 8 Nitrogen fixation using cyanobacteria hasproved beneficial in rice cultivation. Another nitrogen source for ricecultivation in China was Azolla Anabaena until end of 1970s. Cyanobacteriastill have key role in rice fields in various parts of Asia.

Furthermore we canremediate arid soils with cyanobacteria and nitrogen fixation. However theproduction and utilization is yet poorly developed 9. Forsupporting sustainable agriculture activities in different environments thereis serious need to consider cyanobacteria as biofertilizer. 1.4                     Bacteriaassociated with nitrogen fixingThere are less associated nitrogen fixing bacteria arepresent that can be used for improving crop production. For example acetobacterDiazotrophicus and Herbaspirillum spp. are associated with sorghum, maize andsugarcane 10. The use of genus Azospirillum to increase the growthof crops is not bound by host specificity.

In addition to wheat and rice itincludes carrot, tomato, sumflower, peeper, eggplant and cotton 11.The use of Azospirillum cause large increase in crop yield almost in 70 percentcases according to a general consensus12. This increase in growthis not due to nitrogen fixation but due to increase growth promotingsubstance’s production 13. Sugarcane nitrogen requirement can befixed upto 70% by Acetobacter diazotrophics 14. The environmentalconditions and plant genotype specify the amount of nitrogen fixed.

1.5                     PhosphorusSolubilizing BacteriaThe second most limiting nutrient for plants is phosphorus(P) 15. Phosphorus is present in large amount in soil but plantcannot uptake it. There are some bacteria that are able to mobilize phosphorusso that plants can easily uptake it. Bacillus and Pseudomonas are most leadingphosphorus-solubilizing bacteria (PSB) in this regard 16. Now weanalyse the potential importance of PSB using field experiment. One of thefield experiment was done by Sundara et al.

(2002) and he concluded that theapplication of PSB increase 12.6% yield of sugarcane. The phosphorusrequirement is reduced to 25% by applying PSB in combination with phosphorusfertilizer. In addition, cost effective rock phosphate can replace 50% ofexpensive superphosphate. It was observed that production of sugar and juice qualitywas also improved using PSB 17.

It also allows the use of cheapsource of phosphorus.1.6                     PhosphorusMobilizing BiofertilizerEndosymbionts of the fungi of the genera Glomus, Gigasporaand Endogone are responsible for the controlled transformation of nutrients(Phosphorus, zinc & sulphur) from soil to the roots of plants. Fungimobilize the phosphorus so that it can be easily uptake by plants.

The mostcommon genus is glomus having many species present in soil. It is beneficialwhen experimental conditions are applied however it is difficult to obtainAM(Arbuscular Mychorrhiza) fungi pure culture when we need production on largescale.1.7                     Productionof Plant Hormones by BacteriaBacteria can also increase the growth of plants by producingthe plant hormones including nitrogen fixation and solubilizing phosphorus.High amounts of gibberellins are produced by Bacillus pumilus and B.licheniformis 18. Some important bacteria that increase theproduction of hormones are Paenibacillus polymyxa (nitrogen fixation, phosphorussolubilizing and production chitinase and other hydraulic enzymes, increasesoil porosity), taxa (N2 fixation and plant hormones production) 19.

1.8                     LiquidBiofertilizersThe liquid bio fertilizers provide nitrogen and phosphorusto the plants by fixing nitrogen and solubilization of phosphorus such as Rhizobium, Azospiriliumand Phosphobacteria. These fertilizers are more effective for sugarcane,cotton, vegetable, millets and other crops. RhizobiumThis bacteria forms the root nodules for reduction ofnitrogen (Molecular) to ammonia which is used to produce vitamins, proteins andother nitrogenous compounds. Properties of liquid Rhizobium·        Dull white in colour ·        No bad smell·        No foam formation, pH6.8-7.5AzospirillumThese bacteria are fix the large amount of nitrogen 20-40kgN/ha in non-leguminous plants like cotton, oil seeds, millets etc. By use of Azospirillumthe amount of nitrogen fertilizers can be saved upto 25-30%.

Azospirillum isable to perform the reduction of nitrate to nitrite and denitrification. Properties of liquid Azospirillium·        Colour of liquid (blue ordull white)·        Quality of product can beconfirmed by formation of yellow gummy colour·        Acidic pH assure theabsence of Azospirillum bacteria·        Improper liquid formulationis confirmed by bad odourAzobacterAzobacter is nitrogen fixing bacteria and work in aerobic conditions.Azobacter biofertilizer are applied to the non-leguminous plants including rice,vegetable, cotton etc. Excess amount of Azobacter cell is present inrhizospheric region. The growth of organic matter is dependent upon the amountof organic matter.                                                                                                                   Properties of liquidAzobacter                                                                                                       Azobacter produce pigmentation ofmelanin in old culture because of the oxidation process that is carried by coppercontaining enzyme tyrosinase.

AcetobacterThis bacteria is associated with sweet sorghum, sugarcaneand sweet potato plants. Its nitrogenFixation ability is 30kgs/ N/ ha year. It is used oncommercial scale for production of sugarcane crop.

The yield of crop isenhanced upto 10-20t/acre and sugar content almost 10-15%.