NL2013072B1 - Fertilizer comprising protozoa. - Google Patents

Abstract

The invention relates to the fields of agriculture and horticulture, in particular to organic fertilizers in granular, powder or pelleted form. Provided is a fertilizer composition comprising organic sources of nitrogen, phosphate and potassium; spores or cysts of Plant-Growth Promoting Rhizobacteria (PGPR); and encysted protozoa. Also provided is a method for growing a plant, preferably an ornamental plant, grass or a vegetable, comprising applying a fertilizer composition according to the invention in or to the soil in which the plant grows.

Description

Title: Fertilizer comprising protozoa.

The invention relates to agriculture and horticulture. In particular, it relates to organic fertilizers in a granular, powdered or pelleted form, and uses thereof.

Organic fertilizers are fertilizers derived from animal or plant matter. They can be naturally occurring such as manure and sludge or processed from waste materials such as hoofs, bones, feathers, cottonseeds, and soybeans. Organic fertilizers consist of relatively simple molecules such as amino acids and monosaccharides, and of more complex molecules such as proteins, collagen and polysaccharides. These organic molecules contain large amounts of carbon, nitrogen, phosphorous and potassium as well as other elements. When these organic materials are returned to the soil, they undergo decomposition. This is a biological process that includes the physical breakdown and biochemical transformation of the complex organic molecules into simpler inorganic molecules. The rate of decomposition of the organic materials provided by organic fertilizers is determined by several factors. For example the quality of the organic material, the soil (micro) organisms present and the physical environment (e.g. moisture and temperature).

The carbon-rich organic matter provided by the organic fertilizer serves as a food source for microorganisms and thereby stimulates microbial growth. As microorganisms break down the carbon-rich organic matter, excess nutrients are released into the soil in inorganic forms that can easily be taken up by plants. This release process is called mineralization.

Nitrogen is considered to be the main limiting plant nutrient. In nature, nitrogen can be present in a variety of forms, including organic forms (e.g. nucleic acids, amino acids), ammonium (NH4+), nitrite (NO2'), nitrate (NO3 ), nitric oxide (NO), and nitrous oxide (N2O) en nitrogen gas (N2). Plants generally absorb nitrogen as ammonium or nitrate. Ammonification is the process by which organically bound nitrogen is mineralized to ammonium. Nitrification is the process by which ammonium is oxidized to nitrite by bacteria in the genus Nitrosomonas. This nitrite is then rapidly oxidized to nitrate by bacteria in the Nitrobacter genus. Nitrate is a highly soluble nutrient that is easily absorbed by plant roots but is also easily lost due to leaching.

As a result of the relatively slow decomposition and mineralization processes, organic fertilizers continue to release nutrients over time, thereby feeding plants over the course of several months. Apart from their effects on biological soil components, organic fertilizer also affect several edaphic soil characteristics. Through the action of soil organisms, part of the organic material is converted to organic matter, which is known for its soilimproving characteristics. The organic matter causes soil particles to aggregate. As a result, pores of varying shapes and sizes arise which can be filled with water or air. The pores not only form habitats for aerobic and anaerobic bacteria, but also provide the plant roots with the oxygen that is required for respiration. The presence of pores also facilitates water infiltration in times of heavy rain. Apart from providing the plants with nutrients, organic fertilizers also improve plant growth indirectly since roots grow best in the crumbly soil that results from the aggregation of soil particles. The organic matter itself functions as a sponge that greatly increases the water-holding capacity of the soil. The organic matter also functions as a reservoir of nutrients which can be released into the soil over time. Organic fertilizers therefore not only stimulate plant and microbial growth directly but also indirectly by improving several soil characteristics.

The rate of decomposition of organic material is amongst other factors dependent on the carbon to nitrogen (C/N) ratio of the organic material. Organic material with a high C/N ratio can lead to the immobilization of nitrogen in microbial biomass. This reduces the amount of nitrogen available to plants. Some organic fertilizers are inoculated with bacteria and/or fungi that assist in the mineralization of the organic material, stimulate plant growth through the production of plant hormones, facilitate the uptake of nutrients and/or suppress pathogens. For example, WO2012047081 composition in the form of pelletized granules based on spores and mineral clays for its use in agriculture comprising: (a) a mixture of spores of endomycorrhizal fungi, (b) a mixture of mineral clay in a proportion of between 59% and 75% in weight of the composition and (c) a binder in a proportion of between 10 and 12% in weight of the composition. Preferred endomycorrhizal fungi include Glomus constrictum, Glomus fasciculatum, Glomus geosporum, Glomus intraradices and Glomus tortuosum, and mixtures thereof.

Unfortunately, the efficacy of fertilizers supplemented with bacteria is often limited by the low persistence of the bacteria in the soil.

The rhizosphere is a highly competitive environment and the introduced bacteria must not only compete with the indigenous microflora, but also resist predation from a variety of soil organisms.

Overall, it would be desirable to improve the survival chances of the introduced beneficial bacteria in the organic fertilizer and to improve the mineralization of the organic material provided by the organic fertilizer. Plant growth and plant health could be further enhanced by forcing the species composition of the native microflora to shift towards species that are beneficial to plants, e.g. nitrifying bacteria, plant hormone producing bacteria and/or species that produce metabolites that are active against soil pathogens.

It was found that at least some of the above goals can be met by further supplementing the fertilizers with protozoa which graze on (soil) bacteria resulting in the continuous remobilization of plant-essential nutrients as well as an improved survival or activity of the added beneficial bacteria once they enter the soil. Accordingly, the invention provides a fertilizer composition in a granular, powdered or pelleted form, comprising (i) an organic source of nitrogen, phosphorus and/or potassium, (ii) Plant-Growth Promoting Rhizobacteria (PGPR) in the form of spores or cysts and (iii) protozoa in the form of cysts. The addition of protozoa to organic fertilizers increases the efficacy of the fertilizer and promotes plant growth by stimulating the mineralization of organic material, improving the survival chances of the added PGPR, increasing the activity of the PGPR, and/or causing a shift in the species composition in the rhizosphere towards more beneficial microorganisms (e.g. nitrifying bacteria). GB1288122 relates to the decomposition of agricultural waste materials into constituents useful for animal or plant nutrition. Disclosed is a method of controlling the decomposition of organic materials containing polysaccharide constituents, comprising contacting the organic material in an inanimate environment with a symbiotic mixture of microflora capable of metabolizing cellulose and sufficient protozoa capable of feeding on both said cellulose-metabolizing microflora and putrefactive micro-organisms to maintain a stable population of said cellulose-metabolizing microflora.

However, the advantage of adding protozoa to an organic fertilizer in order to improve the efficacy of the fertilizer, to increase the survival chances or activity of the added beneficial bacteria and to enhance plant growth and/or health by changing the composition or activity of soil microorganisms, has not been taught or suggested in the art.

As will be understood, the best results are obtained if the selected PGPR and protozoa have a positive influence on each other, e.g. that they are compatible or even act synergistically. For example, the bacteria can defend itself against protozoan grazing; the protozoa is not capable of affecting the bacteria because the bacteria can for example form biofilms; or the protozoa does graze on the bacteria but this grazing increases the growth rate or activity of the bacteria.

Protozoa are aquatic organisms and need thin water films or water-filled pores to survive. In order to ensure that viable protozoa remain present in the organic fertilizer of the invention, they are added in their cyst stage. In nature many protozoa can transform from an actively grazing form (trophozoite) into metabolically inactive cysts when confronted with stress conditions such as starvation or changes in osmolarity. The cysts preserve viability of the protozoa until more favorable conditions occur and the cyst returns to its trophozoite form. Cysts can excyst after several decades and emerge as viable trophozoites.

Protozoa are unicellular eukaryotic microorganisms that range in size between 2 and 200 qm. Based on the morphology of their locomotion, they can be further grouped into amoebae, flagellates and ciliates. Protozoa are considered to be the most important predators of bacteria in soils, particularly in the rhizosphere where the microbial biomass is generally higher as a result of carbon-rich root exudates. Microorganisms in the rhizosphere compete for nutrients with plant roots. During microbial growth, nutrients are temporarily locked up in bacterial biomass. Protozoan grazing on these bacteria stimulates microbial mineralization and thus increases the availability of nutrients to plants. The addition of protozoa to organic fertilizers thus stimulates mineralization and makes the decomposition of organic material less dependent on the C/N ratio. This greatly improves the efficacy of the fertilizer.

Protozoa are selective grazers, favoring certain bacterial species over others. The phenomenon of grazing induced changes in microbial composition has been reported by several authors. For example, populations of Gram-negative bacteria often decrease as a result of protozoan grazing while Gram-positive bacteria benefit (Ronn et al. 2002). The cell wall of Gram-positive bacteria may be harder to digest, which may enable these bacteria to survive when they pass through protozoan cells. Protozoan grazing also often stimulates nitrifying bacteria, presumably because protozoa selectively graze on their faster-growing competitors (Griffiths 1989; Verhagen et al. 1995; Alphei et al. 1996). Others reported a stimulation of auxin producing bacteria in the rhizosphere which resulted in a highly branched root system (Bonkowski and Brandt 2002). Protozoa have also been reported to prefer to graze on senescent bacteria, thereby increasing the contribution of younger strains with a higher metabolic activity (Alphei et al. 1996). High grazing pressure also stimulates the contribution of grazing-resistant bacteria. Certain bacteria are able to produce anti-protozoan metabolites that can also be active against soil pathogens. Protozoa may therefore prefer to consume bacteria that do not produce these metabolites, thereby indirectly increasing the contribution of bacteria that can inhibit soil pathogens (Müller et al. 2013). Thus apart from their general effects on mineralization, protozoa can also be used to steer the composition of the microflora towards species that are beneficial to plants.

The beneficial bacteria in a fertilizer composition according to the invention can be any type of Plant-Growth Promoting Rhizobacteria (PGPR). PGPR are typically defined based on their functional activities as (a) biofertilizers (increasing the availability of nutrients to a plant), (b) phytostimulators (plant growth promotion, generally through the production plant hormones), (c) rhizoremediators (degrading organic pollutants) and/or (d) biopesticides (controlling diseases, mainly by the production of antibiotics and antifungal metabolites). Furthermore, a single PGPR will often reveal multiple modes of action. One or more distinct PGPR can be used.

Nitrogen is the most vital nutrient for plant growth and productivity. Although, there is about 78% N2 in the atmosphere, it is unavailable to most plants. The atmospheric N2 is converted into plant- utilizable forms through biological N2 fixation (BNF), which converts nitrogen to ammonia by nitrogen-fixing microorganisms using a complex enzyme system known as nitrogenase. Nitrogen-fixing microorganisms are generally categorized as (a) symbiotic N2 fixing bacteria including members of the family Rhizobiaceae which five in symbiosis with leguminous plants (e.g. Rhizobia) and non-leguminous trees (e.g. Frankia) and (b) non-symbiotic (free living, associative and endophytes) nitrogen fixing forms such as Cyanobacteria Azospirillum and Azotobacter.

Phosphate the second most important plant growth limiting nutrient and is abundantly available in many soils in both organic and inorganic forms. Despite of this large reservoir of phosphate in soils, the amount of forms available to plants is generally low. This low availability of phosphate to plants is because the majority of soil phosphate is found in insoluble forms, while the plants absorb it only in two soluble forms, the monobasic (H2PO4·) and the diabasic (HPO42') ions.

Phosphate-solubilizing bacteria (PSB) are considered as promising biofertilizers since they can supply plants with phosphate that would otherwise not be available. Bacterial genera like Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Microbacterium, Pseudomonas, Rhizobium and Serratia are reported as the most significant phosphate-solubilizing bacteria. Non-limiting examples of beneficial bacteria with biofertilizer potential for use in the present invention are fisted in table 1.

Apart from their roles as biofertilizers, PGPR can also play a role as biostimulants and/or bioprotectants. Species in for example the genera Azospirillum, Bacillus, Pseudomonas, and Rhizobium can produce plant hormones such as IAA, gibberelfine or cytokines as well as other substances such as 2,.3-butanediol that can stimulate plant development. Promotion of lateral root development and an increased uptake of nutrients as a result of auxin production by PGPR have often been reported.

As said, apart from direct plant growth-promoting effects, PGPR can also stimulate plant growth through the suppression of pathogens. PGPR can antagonize deleterious microorganisms through the secretion of lytic enzymes and antibiotics and through competition for nutrients or space. PGPR are also known to activate the immune response of plants, a phenomenon called ‘induced systemic resistance’ (IRS). The expression of IRS can involve several physiological mechanisms. For example, IRS can increase a plant’s tolerance to pathogens which suppresses the expression of symptoms. Other mechanisms include escape as a result of growth promotion and resistance through the reinforcement of cell walls or the induction of Pathogenesis-related (PR) proteins. Non-limiting examples of PGPR with potential as biostimulants or bioprotectants for use in organic fertilizers are listed in table 1.

The beneficial bacteria may be prepared using any suitable method known to the person skilled in the art, such as, solid state or liquid fermentation using a suitable carbon source. To ensure the stability of the fertilizer, the bacteria are added to the fertilizer in their cyst or spore forms. A fertilizer composition may comprise beneficial bacteria in an amounts of 10 exp3 to lOexplO spores or colony forming units per gram of composition. Preferably, it comprises 10 exp5 to lOexplO spores or colony forming units per gram of composition, more preferably a total of 10 exp6 to 10exp9.

Table 1: Effects of exemplary PGPR for use in the organic fertilizer

A fertilizer of the invention is characterized by the combined presence of beneficial bacteria and protozoa in the form of cysts. The protozoa are typically present in an amount of lOexpl to 10exp7 cysts per gram of composition. It will be appreciated by the skilled person that the earlier described stimulation of mineralization can be put into practice with any type of bacteria-eating protozoa. Preferably, the protozoa are species belonging to the phyla Cercozoa, Cihophora, Discoba, Discosea and Tubulinea. More preferably, the protozoa belong to those genera that are commonly found in soils in order to improve their survival chances. Non-hmiting examples of preferred genera are Acanthamoeba, Amoeba, Bodo, Cercomonas, Cercozoa, Chilodonella, Colpidium, Colpoda, Halteria, Hartmannella, Heteromita, Naegleria, Neocercomonas, Oikomonas, Spumella, Tetrahymena and Vorticella.

For example, one embodiment is specifically designed for plants that are nutrient-stressed or require a relatively high input of nutrients. The selected PGPR in this formulation is the phosphate-solubilizing Bacillus megaterium and the protozoa is a small raptorial Cercozoa. This protozoa can graze in even the smallest soil pores and can therefore stimulate the mineralization of organic matter and nitrification in a very broad manner.

Not any combination of PGPR and protozoa is equally suitable, since the selected PGPR is preferably not negatively affected by the selected protozoa. Preferred combinations are those combinations where (a) the bacteria can defend itself against protozoan grazing, (b) the protozoa is not capable of affecting the bacteria because the bacteria can for example form biofilms and/or (c) the protozoa does graze on the bacteria but this grazing increases the growth rate or metabolic activity of the bacteria.

For example, in one embodiment, the protozoa is Acanthamoeba castellanii and the bacteria is a gram-positive Bacillus species that is resistant to protozoan grazing. In another embodiment, the PGPR is a biofilm-producing species such as Serratia sp. and the protozoa is a surface feeder such as Chilodonella sp. In another embodiment, the PGPR is Serratia marcescens, a species that can produce toxic secondary metabolites such as prodigiosin and the protozoa is Tetrahymena sp., a species that can detect these toxic metabolites and will therefore avoid this species.

Non-limiting examples of preferred combinations of beneficial bacteria and protozoa are Serratia marcescens and Bodo saltans, Serratia marcescens and Acanthamoeba polyphaga, Serratia marcescens and Acanthamoeba castellanii, Serratia marcescens and Hartmannella vermiformis, Serratia marcescens and Tetrahymena sp., Serratiaplymuthica and Tetrahymena sp., Serratia sp. and Cercozoa sp., Bacillus subtilis and Acanthamoeba castellanii, Bacillus amyloliquefaciens and Acanthamoeba castellanii, Bacillus pumilus and Acanthamoeba castellanii, Bacillus megaterium and Acanthamoeba castellanii, Bacillus licheniformis and Acanthamoeba castellanii, Bacillus sp. and Cercozoa sp.,

Bacillus subtilis and Acanthamoeba polyphaga, Bacillus amyloliquefaciens and Acanthamoeba polyphaga, Bacillus amyloliquefaciens and Acanthamoeba sp., Bacillus pumilus and Acanthamoeba polyphaga, Bacillus megaterium and Acanthamoeba polyphaga, Bacillus licheniformis and Acanthamoeba polyphaga, Bacillus subtilis and Colpoda steinii, Bacillus amyloliquefaciens and Colpoda steinii, Bacillus megaterium and Colpoda steinii, Bacillus pumilus and Colpoda steinii, Bacillus licheniformis and Colpoda steinii, Bacillus licheniformis and Naegleria fowleri, Bacillus megaterium and Naegleria fowleri, Bacillus subtilis and Naegleria fowleri, Bacillus amyloliquefaciens and Naegleria gruberi, Bacillus subtilis and Naegleria gruberi, Bacillus licheniformis and Naegleria gruberi or any combination thereof.

Encystment of protozoa can be accomplished in various ways, for example by reducing the bacterial food source (e.g. Escherichia coli or Klebsiella pneumoniae) for protozoa that are grown in mono- or polyxenic cultures, or by depleting nutrients in the liquid growth medium for protozoa grown in axenic cultures (Neff et al. 1964). Protozoa can be grown in different types of commercially available growth medium but rapid and synchronous encystment is found in growth media that support rapid population growth (i.e. short generation times).

Encystation can also be induced by increasing the osmolarity of the growth medium through the addition of for example sodium chloride or glucose. The corresponding environmental condition behind this phenomenon is probably a loss of water from the soil due to evaporation. Once the encystment process is completed, the cysts can be harvested from the encystment medium and freeze-dried for preservation.

Accordingly, the invention also relates to a method for providing a fertilizer composition, comprising the steps of: a) mixing organic sources of nitrogen, phosphate and potassium in the desired ratio! b) providing a culture of PGPR and inducing cyst- or spore-forming, preferably through fermentation! c) providing a culture of protozoa and inducing encystation, preferably by removing the bacterial food source in monoxenic or polyxenic or by increasing the osmolarity of the growth medium or by transferring the protozoa from a growth medium to an encystation medium for axenic cultures! and d) preparing a granular, powdered or pelleted fertilizer from a mixture of the PGPR spores or cysts, the protozoan cysts and the organic components obtained in (a); or by applying the PGPR spores or cysts, and the protozoan cysts to granular, powdered or pelleted fertilizer prepared from the mixture of organic components obtained in (a).

Granules of an organic fertilizer can be produced by methods known in the art. For example, W02012/102641 describes a method for producing granulated organo-mineral fertilizers from organic waste materials. It involves mixing the organic waste materials, removing mechanical impurities, mixing with the addition of mineral components (NB. No mineral components need are added to organic fertihzers), grinding, decontaminating, homogenizing, granulating and drying.

According to the conventional fertilizer standards, the chemical makeup or analysis of fertilizers is expressed in percentages (by weight) of the essential primary nutrients nitrogen, phosphate and potassium. More specifically, when expressing the fertilizer formula, the first figure represents the percent of nitrogen expressed on the elemental basis as "total nitrogen" (N), the second figure represent the percent of phosphate, sometimes expressed on the oxide basis as "available phosphoric acid" (P2O5), and the third figure represents the percent of potassium, sometimes expressed on the oxide basis as "available potassium oxide" (K2O). This expression is otherwise known as N-P-K.

An aspect of the present invention allows fertilizer formulations to be customized with respect to levels of N-P-K to suite various plants or soil conditions. Listed in Table 2 are some of the many N-P-K variations that are possible within the scope of the present invention. By mixing different sources of organic materials, a wide range of fertilizers with varying N-P-K values can be formulated. Depending on the application, the amount of nitrogen, phosphate and potassium can range from 0 to 20%. In one embodiment, the fertilizer composition of the invention is of the formula NPK 7-6-6 . In another embodiment, it is of the formula 9-3-5.

Table 2 - N-P-K values of different raw organic materials that can be used for the production of organic fertilizers.

In one embodiment, raw materials, such as dried feather meal and meat meal are blended (in no specific order) and then conveyed into a granulator where they are pressed in the desired granule size. The desired size may range from a fine powder to granules ranging in size from about < 1 mm to approximately 1 cm. After pressing, an aqueous suspension of bacterial spores and cysts and protozoan cysts is sprayed on the resolving fertilizer. In another embodiment, the bacterial spores or cysts and the protozoan cysts are mixed with the organic components before they are granulated. A still further embodiment relates to a method for growing a plant, comprising applying to the soil in which the plant grows a (granular) fertilizer composition according to the invention. The amount and frequency of fertilizer to be applied will depend on various factors, e.g. type of plant, developmental stage, other growth conditions and the like. Typically, the amount of the fertilizer is effective to enhance growth such that fertilized plants exhibit an increase in growth, increased leaf area, improved flowering, an increase in yield, an increase in root length and/or root mass when compared to unfertilized plants. The suitable application rates vary according to the type of seed or soil, the type of crop plants, the amounts of the source of phosphorus and/or micronutrients present in the soil or added thereto, etc. A suitable rate can be found by simple trial and error experiments for each particular case. In one aspect, the fertilizer is added in an amount of 10 to 5000 kg per ha.

The invention is exemplified by the following non-limiting example.

Example 1: Formulation for a 7-6-6 granular fertilizer

Aerobic fermentation was carried out on the Gram-positive bacteria Bacillus amyloliquefaciens, a species that is known to exert positive effects in the rhizosphere. B. amyloliquefaciens was grown in an amino acid rich growth medium consisting of soy meal, skim milk powder, yeast extract, lactose and mineral salts in a 5,000 liter aerobic fermenter for 40 hours at 35 °C while continually agitated at 150 rpm and aerated at 35 m3^1. With the impoverishment of nutrients in the growth medium, the log phase was terminated which induced sporulation. The maximum cell density was l,5expl0 CFU/ml and the sporulation degree was almost 100%. The spores were separated from the culture medium with a separator (Westfalia). The resulting slurry was subsequently freeze dried at -30°C and dried in the vacuum. The dried product was subsequently milled to a mesh size of 630 qm resulting in a fine powder containing lexplO CFU/g. This powder was used to inoculate the formulation of the present example during the granulation step described below.

For the purpose of the present fertilizer, the naked amoebae Acanthamoeba castellanii was chosen because it was shown to greatly stimulate mineralization and plant hormone production in the rhizosphere. Thereby, the Gram-positive Bacillus amyloliquefaciens can produce bacteriocin-like substances that were previously shown to inhibit Acanthamoeba sp.

Trophozoites were grown axenically in proteose peptone-yeast extract-glucose (PYG) amended with 0,05 M CaCh, 0,4 M MgSCU, 0,25 M Na2HPC>4; 0,25 Μ KH2PO4, 0,005 M Fe(NH4)2( SC>4)2, Na Citrate and a 0,1 M glucose solution. The trophozoites were grown in a 5,000 liter fermenter at densities of 10exp5 cells/ml at 25 °C while continuously agitated at 40 rpm. To induce encystation, the osmolarity of the growth medium was increased with 0,3 M glucose. This increase in osmolarity caused cell division to stop and caused 85% of the trophozoites to form cysts within 40 hours. The cysts were harvested from the fermenter, freeze-died and milled to a mesh size of 700 ipn. The cysts were held until used in the granulation step.

The raw organic materials, the Bacillus spore powder and Acanthamoeba cysts were weighted and mixed according to the recipe given in table 3.. The mixture of raw materials was subsequently pressed, resulting in granules ranging in size from <1 mm to 5 mm. The product specifications of this fertilizer are listed in table 4.

Table 3: Recipe of the exemplary fertilizer composition

Table 4: product specifications of exemplary fertilizer.

REFERENCES

Alphei, J., Bonkowski, M. and Scheu, S. 1996. Protozoa, Nematoda and Lumbricidae in the rhizosphere of Hordelymus europaeus (Poaceae): faunal Interactions, response of microorganisms and effects on plant growth. Oecologia 106: 111-126.

Bonkowski, M. and Brandt, F. 2002. Do soil protozoa enhance plant growth by hormonal effects? Soil Biology &amp; Biochemistry 34: 1709-1715.

Griffiths, B.S. 1989. Enhanced nitrification in the presence of bacteriophagous protozoa. Soil Biology and Biochemistry. 21: 1045-1051. Müller, M.S., Scheu, S. and Jousset, A. 2013. Protozoa drive the dynamics of culturable biocontrol bacterial communities. PloS ONE 8: e66200.

Neff, R.J., Ray, S.A., Benton, W.F., Wilborn, M. 1964. Induction of synchronous encystment (differentiation) in Acanthamoeba sp. In: Methods in cell physiology. Vol. 1. Prescott, D.M. (Eds). Academic Press Inc., New York, The United States. Pp. 55-83.

Ronn, R., McCaig, A.E., Griffiths, B.S. and Prosser, J.I. 2002. Impact of protozoan grazing on bacterial community structure in soil microcosms. Applied and Environmental Microbiology 68: 6094-6105.

Verhagen, F.J.M., Laanbroek, H.J. and Woldendorp, J.W. 1995. Competition for ammonium between plant roots and nitrifying and heterotrophic bacteria and the effects of protozoan grazing. Plant and soil 170: 241-250.

Claims (14)

A fertilizer composition in a granular, powdered or pelletized form, comprising (i) an organic source of nitrogen, phosphorus and / or potassium, (ii) Plant Growth Stimulating Rhizobacteria (PGPR) in the form of spores or cysts and (iii protozoa in the form of cysts. The fertilizer composition of claim 1, wherein the selected PGPR and protozoa are compatible with one or more of the following aspects: (a) the bacteria can defend themselves against being protozoa grazed; (b) the protozoa are unable to influence the bacteria because the bacteria can, for example, form biofilms; (c) the protozoa graze on the bacteria but grazing increases the growth rate or activity of the bacteria. Fertilizer composition according to claim 1 or 2, wherein the PGPR belong to the genera Acinetobacter, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus, Beijerinckia, Bradyrhizobium, Burkholder ia, Enterobacter, Frankia, Gluconacetobacter, Klebsiella, Rhizobromium, Rhizobromium, Pizudium serium . A fertilizer composition according to any one of the preceding claims wherein the PGPR is present in an amount of 10xp3 to 10x1010 spores or colony forming units per gram of composition. The fertilizer composition according to claim 1, wherein protozoa increase the efficacy of the fertilizer and promote growth and / or health of the plant by (a) stimulating the mineralization of organic material, (b) improving the chances of survival of the PGPR, ( c) increasing the activity of the PGPR, and / or (d) causing a shift of the species composition in the rhizosphere toward more useful microorganisms. A fertilizer composition according to any one of the preceding claims, wherein the protozoa are selected from the phyla Cercozoa, Ciliophora, Discoba, Discoseeen and Tubulinea. A fertilizer composition according to any one of the preceding claims, wherein the protozoa belong to the genera Acanthamoeba, Amoeba, Bodo, Cercomonas, Cercozoa, Chilodonella, Colpidium, Colpoda, Halteria, Hartmannella, Heteromita, Naegleria, Neocercomonas, Oikomonort, Spumella, Spumella. A fertilizer composition according to any one of the preceding claims, wherein the protozoa are present in an amount from 10 x 10 to 10 x 7 cysts per gram of composition. 9. Fertilizer composition according to any one of the preceding claims, wherein the nitrogen, phosphate and / or potassium come from organic sources, preferably hoof meal, hair meal, meat meal, bone meal, cottonseed meal and / or soybean meal. The fertilizer composition of claim 9, wherein the amount of nitrogen, phosphate and / or potassium can vary between 0 to 20% depending on the application. A fertilizer composition according to any one of the preceding claims, wherein the fertilizer has a granular form, preferably with granules in the range of 0.01 mm to 5 mm. A method for providing a fertilizer according to any of claims 1 to 11, comprising the steps of: a) mixing organic sources of nitrogen, phosphate and potassium in the desired ratio; b) providing a PGPR culture and inducing cyst or spore formation, preferably by fermentation; c) providing a protozoa culture and inducing encapsulation in a cyst, preferably by removing the bacterial food source in a monoxenic or polyxenic culture or by increasing the osmolarity of the growth medium or by transferring the protozoa from a growth medium to a cytoforming medium for axenic cultures; and d) preparing a granular, powdered or pelletized fertilizer from a mixture of the PGPR spores or cysts, the protozoa cysts and the mixture of organic components obtained in (a); or by applying the PGPR spores or cysts, and the protozoa cysts to the granular, powdered or pelletized fertilizer prepared from the mixture obtained in step (a). A method for growing a plant, preferably an ornamental plant, grass or a vegetable, comprising applying a fertilizer composition according to any of claims 1 to 11 in or on the soil in which the plant grows. A method according to claim 13, wherein the fertilizer is added in or on the soil in an amount of 10 to 5000 kg per hectare.