WO2015199541A1

WO2015199541A1 - Fertilizer comprising bacteria and protozoa.

Info

Publication number: WO2015199541A1
Application number: PCT/NL2015/050465
Authority: WO
Inventors: Brenda LOZNIK; Pier Jouke Oosterkamp
Original assignee: Ecostyle B.V.
Priority date: 2014-06-26
Filing date: 2015-06-26
Publication date: 2015-12-30
Also published as: NL2013072B1

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) wherein the PGPR is a Bacillus species, preferably Bacillus subtilis, Bacillus amyloliquefaciens and/or Bacillus licheniformis; and encysted protozoa selected from the group comprising Neocercomonas sp., Cercomonas sp., Vannella sp., Sandona sp. and Bodomorpha sp., and combinations thereof, the invention also relates to a method for providing this fertilizer. 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 bacteria and protozoa.

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

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 that are essential for plant growth and development. When these organic materials are returned to the soil, they undergo

decomposition. This is predominantly a biological process that includes the physical breakdown and biochemical transformation of the complex organic molecules into smaller organic molecules and inorganic elements. 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 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), nitrous oxide (N2O) and nitrogen gas (N2). Plants generally absorb nitrogen in the form of 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 soil- improving 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. Decomposition of 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, WO2012/047081 discloses a 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. Soil, and in particular the small area of soil around the roots which is called '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 (micro)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. more nitrifying bacteria, plant growth -promoting bacteria and/or species that produce metabolites that are active against soil- borne pathogens.

It was found that at least some of the above goals can be met by further supplementing fertilizers comprising beneficial bacteria with specific 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. The best results were obtained when 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 themselves against protozoan grazing; the protozoa are not capable of affecting the bacteria because the bacteria can for example form biofilms; or the protozoa do graze on the bacteria but this grazing increases the growth rate or activity of the remaining bacteria. More in particular, the addition of selected species of protozoa cysts to an organic fertilizer comprising Bacillus species

surprisingly resulted in an increased plant growth (biomass) and

mineralization of organic material.

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, wherein the PGPR is a Bacillus species and (iii) protozoa in the form of cysts, wherein the protozoa are selected from the group comprising Neocercomonas sp., Cercomonas sp., Vannella sp., Sandona sp. and Bodomorpha sp., and combinations thereof. 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).

GB 1288122 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 the cellulose-metabolizing microflora and putrefactive micro-organisms to maintain a stable population of said cellulose-metabolizing microflora.

However, the advantage of adding one or more of the selected species of protozoa cysts to an organic fertilizer comprising Bacillus species 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.

WO2013/176777 relates to a bio-organo-phosphate fertilizer supplemented with "phosphorus solubilizing" and "plant growth regulating" micro-organisms, like algae, bacteria, protozoa, fungi. WO2013/176777 is silent about protozoa cysts, and also fails to teach which (combination of) genera/species of micro-organisms can be used.

AU2010202667 relates to a soil enhancing material for promoting the growth and/or development of a plant, the soil enhancing material comprising at least one viticulture material. The material may be

supplemented with composting micro-organisms, such as bacteria, fungi, algae and or protozoa. Nothing is mentioned about protozoa in the form of cysts. AU2010202667 is silent about the specific combination of bacteria / protozoa as disclosed in the present invention.

WO2014/043604 discloses a fertilizing composition comprising an organic source of N, P and/or K, agriculturally beneficial bacteria and protozoa in the form of cysts. Disclosed are long lists of exemplary bacteria and protozoa, among which Bacillus species and the genus Cercomonas. Preferred bacteria are Pseudomonas fluorescens and Sinorhizobium meliloti. Importantly, the specific combination of a Bacillus sp. and one or more of Neocercomonas sp., Cercomonas sp., Vannella sp., Sandona sp. and Bodomorpha sp. according to the present invention is not disclosed or suggested.

The beneficial bacteria in a fertilizer composition according to the invention can be any Bacillus species considered as 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.

Preferred Bacillus species for use in the present invention include B. amyloliquefaciens, B. atrophaeus, B. cereus, B. circulans, B. coagulans, B. licheniformis, B. luciferensis, B. megaterium, B. mucilaginosus, B.

mycoides, B. pasteurii, B. polymyxa, B. pumilus, B. sphaericus, B. subtilis and B. thuringiensis. Particularly preferred are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus licheniformis.

Combinations of one or more Bacillus species are also envisaged.

Non-limiting examples of further beneficial bacteria with biofertilizer potential for use in the present invention are listed 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, gibberelline 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' (ISR). The expression of ISR can involve several physiological mechanisms. For example, ISR 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 the form of cysts or spores. A fertilizer composition may comprise beneficial bacteria in an amounts of 10 exp l to lOexp lO, like 10 exp3 to lOexp lO, spores or colony forming units per gram of composition. Preferably, it comprises 10 exp5 to lOexp lO 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

Genus Bioprotecta Biofertilizer Biostimulant Exemplary species

nt

Acinetobacter X A. calcoaceticus

Arthrobacter X X A. globiformis and A. nicotianae

Azoarcus X A. communis and A. indigens

Azospirillum X X X A. amazonense, A. brasilense, A.

canadensis, A. doebereinerae, A. halopraeferens, A. irakense, A. largimobile, A. lipoferum, A. melinis and A. oryzae

Azotobacter X X X A. armeniacus, A. beijerinckii, A.

chroococcum, A. nigricans, A. paspali, and A. vinelandii

Bacillus X X X B. amyloliquefaciens, B.

atrophaeus, B. cereus, B.

circulans, B. coagulans, B.

licheniformis, B. luciferensis, B. megaterium, B. mucilaginosus, B. mycoides, B. pasteurii, B.

polymyxa, B. pumilus, B.

sphaericus, B. subtilis and B. thuringiensis

Beijerinckia X X B. derxii and B. mobilis

Bradyrhizobium X X B. elkanii and B. japonicum

Burkholderia B. brasilensis, B. cepacia, B.

kururiensis, B. unamae and B. vietnamiensis

Enterobacter E. aerogenes, E. asburiae and E.

cloacae

Frankia Frankia sp.

Gluconacetobacter G. diazotrophicus

Klebsiella K. aerogenes

Pseudomonas P. aeruginosa, P. cepacia, P.

fluorescens, P. lutea, P. mendocina, P. putida, P. stutzeri and P.

thivervalensis

Rhizobium Rhizobium sp.

Serratia S. liquefaciens, S. marcescens, S.

plymuthica and S. proteamaculans

Streptomyces S. anulatus, S. coelicolor, S.

griseoviridis, S. lydicus and S. pilosus

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 the form of cysts. 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 μιη. 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 (Miiller 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.

A fertilizer of the invention is characterized by the combined presence of beneficial bacteria at least comprising a Bacillus species, and protozoa in the form of cysts wherein the protozoa are selected from the group

comprising Neocercomonas sp., Cercomonas sp., Vannella sp., Sandona sp. and Bodomorpha sp., and combinations thereof. The protozoa are typically present in a total amount of lOexpl to 10exp7 cysts per gram of composition, more preferably a total of 10exp2 to 10exp4 cysts per gram of composition.

In one aspect, a species from the genus Neocercomonas is used, for example selected from Neocercomonas jutlandica, Neocercomonas sp. strain 10-3.4, strain 10-3.6, strain 10-4.1, strain 18-6E, strain 4-2.2, strain 7-3.6, strain 8-3.1, strain 9-3.7, strain 9-4.1, strain 9-6.2, strain C-43, strain C-56, strain C-59, strain C- 72,. strain C-84, strain C-85, strain CeS-2, strain CS-4, strain New Zealand 1- 7E, strain NY-1, and strain Panama53. In a further aspect, a species from the genus Cercomonas is used, for example one or more selected from the group consisting of Cercomonas lenta, Cercomonas agilis, Cercomonas alexieffi, Cercomonas ambigua, Cercomonas bodo, Cercomonas braziliensis, Cercomonas celer,

Cercomonas clavideferens, Cercomonas crassicauda, Cercomonas

dactyloptera, Cercomonas deformans, Cercomonas diparavarians,

Cercomonas edax, Cercomonas effusa, Cercomonas elliptica, Cercomonas fastiga, Cercomonas gigantica, ercomonas granulatus, Cercomonas hederae, Cercomonas hiberna, Cercomonas kiaerdammane, Cercomonas kolskia, Cercomonas laciniaegerens, Cercomonas laeva, Cercomonas lata,

Cercomonas lenta, Cercomonas longicauda, Cercomonas magna, Cercomonas media, Cercomonas metabolicus, Cercomonas mtoleri, Cercomonas mutans, Cercomonas nebulosa, Cercomonas paraglobosa, Cercomonas parambigua, Cercomonas paravarians, Cercomonas parincurva, Cercomonas parva, Cercomonas phylloplana, Cercomonas pigra, Cercomonas plasmodialis, Cercomonas primitiva, Cercomonas radiata, Cercomonas rapida,

Cercomonas ricae, Cercomonas rotunda, Cercomonas simplex, Cercomonas sphagnicola, Cercomonas vibrans, Cercomonas vacuolata, Cercomonas wylezichi, Cercomonas zhukovi, Cercomonas sp. ATCC 50317, Cercomonas sp. ATCC 50318, Cercomonas sp. ATCC 50319. In a specific aspect, the fertilizer composition comprises Cercomonas lenta.

In a still further aspect, a species from the genus Vannella is used, for example one or more selected from the group consisting of Vannella aberdonica, Vannella anglica, Vannella aberdonica, Vannella arabica, Vannella bursella, Vannella cf. miroides, Vannella danica, Vannella devonica, Vannella ebro, Vannella epipetala, Vannella lata, Vannella miroides, Vannella persistens, Vannella platypodia, Vannella plurinucleolus, Vannella septentrionalis, Vannella simplex, Vannella sp. 15i, Vannella sp. CCAP 1589/17, Vannella sp. CRIB-39, Vannella sp. ED-AS, Vannella sp. ED40, Vannella sp. Geneva, Vannella sp. S002, Vannella sp. strain 4354/1, Vannella sp. strain 4362V 711, Vannella sp. strain 4432/1, Vannella sp.

strain CAZ6/I, Vannella sp. strain CH88/I, Vannella sp. strain IS013/I,

Vannella sp. strain RSL/I, and Vannella sp. strain S2M2/I, Vannella sp.

ATCC 50922, Vannella sp. ATCC 30945, Vannella sp. ATCC 5PRA-37, Vannella sp. ATCC 50989, Vannella sp. ATCC 30947, Vannella sp. strain

1589/18, Vannella sp. strain 1589/21.

Exemplary Bodomorpha species for use in the present invention include Bodomorpha minima, Bodomorpha prolixa, Bodomorpha sp.

HFCC106, Bodomorpha sp. HFCC55, Bodomorpha sp. HFCC57,

Bodomorpha sp. HFCC92, Bodomorpha sp. Panamal05, Bodomorpha sp.

Panamall5, and combinations thereof.

Examplary Sandona species for use in the present invention include Sandona afra, Sandona aestiva, Sandona aporians, Sandona campae, Sandona dimutans, Sandona dismilis, Sandona erratica, Sandona limna, Sandona mutans, Sandona pentamutans, Sandona similis, Sandona tetramutans, Sandona tetrasimilis, Sandona ubiquita, and combinations thereof.

In a preferred embodiment, the fertilizer comprises at least a Neocercomonas sp. and/or a Cercomonas sp., optionally in combination with a Vannella sp.

Non-limiting examples of preferred combinations of beneficial bacteria and protozoa are Bacillus sp. and Cercozoa, Bacillus subtilis and Cercomonas longicauda, Bacillus amyloliquefaciens and Cercomonas longicauda,

Bacillus subtilis and Cercomonas lenta, Bacillus amyloliquefaciens and

Cercomonas lenta, Bacillus subtilis and Cercomonas diparavarians, Bacillus subtilis and Cercomonas sp., Bacillus subtilis, Bacillus amyloliquefaciens and Cercomonas lenta, Bacillus subtilis, Bacillus amyloliquefaciens and Cercomonas longicauda, Bacillus subtilis and Neocercomonas jutlandica, Bacillus amyloliquefaciens and Neocercomonas jutlandica, Bacillus subtilis with Bacillus amyloliquefaciens and Neocercomonas jutlandica, Bacillus subtilis and Neocercomonas sp., Bacillus subtilis, Bacillus

amyloliquefaciens, Cercomonas lenta and Neocercomonas sp., Bacillus subtilis, Bacillus amyloliquefaciens, Cercomonas longicauda and

Neocercomonas sp., Bacillus subtilis, Cercomonas sp. and Vannella sp.,

Bacillus subtilis, Neocercomonas sp. and Vannella sp., Bacillus subtilis and Vannella persistens, Bacillus amyloliquefaciens and Vannella sp., Bacillus subtilis and Vannella sp., Bacillus subtilis, Bacillus amyloliquefaciens, Cercomonas lenta and Vannella sp., Bacillus subtilis, Bacillus

amyloliquefaciens, Neocercomonas sp. and Vannella sp., Bacillus subtilis and Bodomorpha minima, Bacillus amyloliquefaciens and Bodomorpha minima, Bacillus subtilis, Cercomonas lenta and Bodomorpha sp., Bacillus subtilis and Sandona aporians, Bacillus amyloliquefaciens and Sandona aporians, Bacillus subtilis, Bacillus amyloliquefaciens and Sandona aporians, Bacillus subtilis, Cercomonas lenta and Sandona aporians, Bacillus subtilis, Vannella sp. and Sandona aporians, Bacillus

amyloliquefaciens, Cercomonas lenta, Vannella sp. and Sandona aporians,

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 a Bacillus species and inducing cyst- or spore -formation , preferably through fermentation;

c) providing a culture of one or more of the selected 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 Bacillus spores or cysts, the protozoan cysts and the organic components obtained in (a); or by applying the Bacillus 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, WO2012/102641 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 are added to organic fertilizers), 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. In yet another embodiment it is of the formula 7-2-4.

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

Raw material N-P-K value

Hair meal 14-0-0

Feather meal 13-0-0

Meat meal 8-4-0

Bone meal 6-10-0

Cottonseed meal 6-10-0

Soybean meal 7-2- 1

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 in a powdered form with the organic components before they are granulated.

A fertilizer composition may include further useful ingredients, such as a stabilizer, bulking agent, and/or additional micro-organisms. In one embodiment, the composition comprises, in addition to the selected bacteria and protozoa, one or more selected from the group of algae, fungi and actinomycetes. Preferred endomycorrhizal fungi include Glomus constrictum, Glomus fasciculatum, Glomus geosporum, Glomus intraradices, Glomus tortuosum, and mixtures thereof.

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 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. Also provided herein is a method for enhancing the mineralization process in a soil, comprising applying a sufficient amount of fertilizer composition of the invention. Soil is made up of many components. A significant percentage of most soil is clay. Organic matter, while a small percentage of most soil, is also important for several reasons. Both of these soil fractions have a large number of negative charges on their surface, thus they attract cation elements. At the same time, they also repel anion nutrients. The cation exchange capacity (CEC) value of soil, as reported by nearly all soil testing laboratories, is a calculated value that is an estimate of the soils ability to attract, retain, and exchange cation elements, the total capacity of a soil to hold exchangeable cations. The CEC is an inherent soil characteristic and it influences the soil's ability to hold onto essential nutrients and provides a buffer against soil acidification. Soils with a higher clay fraction tend to have a higher CEC. Organic matter has a very high CEC. Sandy soils rely heavily on the high CEC of organic matter for the retention of nutrients in the topsoil. The CEC is typically reported in millequivalents per 100 grams of soil (meq/lOOg ). Larger CEC values indicate that a soil has a greater capacity to hold cations.

It was surprisingly found by the present inventors that certain specific combinations of bacteria and protozoa had good results when applied to a poor sandy soil, whereas other combinations were particularly effective in richer sandy soils. As used herein, the term "poor" or "soil" refers to both the nutrient supply and microbial soil life. For example, a poor soil can have an organic matter of up to about 2%, preferably up to about 1.5%; a CEC in the range of 1- 10 mmol/kg soil, and/or a soil-derived nitrogen content of up to 7 mg/kg soil. In contrast, a 'rich' soil can have an organic matter of at least about 4%, preferably at least 6%; a CEC in the range of 11-50, like at least 40 mmol/kg soil, and/or a soil-derived nitrogen content of at least 20 mg N /kg soil, preferably at least 30 mg N/kg soil.

In one embodiment, the invention provides 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, wherein the soil is a poor soil and wherein the fertilizer comprises at least a Vannella sp. For example, a fertilizer composition comprising B. subtilis in combination with a Vannella species or a Sandona species is used for growing a grass (e.g. Lolium perenne) on a poor sandy soil. In another embodiment, the soil is a rich soil and the fertilizer comprises a Neocercomonas sp. or Cercomonas sp. For example, a fertilizer composition comprising B. amyloliquefaciens and a Cercomonas species is used for growing a grass (e.g. Lolium perenne) on a rich sandy soil.

LEGEND TO THE FIGURES

Figure 1: Relative growth of the 4 selected protozoa species (SP1, SP2, SP3 and SP4) on a monoculture of 6 different species of Bacillus species that are commonly found in soil. Light colors indicate slow growth rates, whereas darker colors indicate faster growth.

Figure 2: Effect of fertilizers supplemented with or without selected protozoa on shoot dry weight of Lolium perenne (mean ± standard error) measured 6 weeks after applying the fertilizers on a poor sandy soil. The horizontal black line indicates the shoot dry weight of the unfertilized and unsupplemented control treatment. Treatments that are significantly different from the unsupplemented fertilizer (NOP) at a p-value < 0,05 are indicated with a ' · ', whereas differences at a p-value of < 0, 1 are shown with a

Figure 3: Effect of fertilizers supplemented with or without selected protozoa on root dry weight of Lolium perenne (mean ± standard error) measured 6 weeks after applying the fertilizers on a poor sandy soil. The horizontal black line indicates the root dry weight of the unfertilized and unsupplemented control treatment. Treatments that are significantly different from the unsupplemented fertilizer (NOP) at a p-value < 0,05 are indicated with a ' · ', whereas differences at a p-value of < 0, 1 are shown with a '°'. Figure 4: Effect of fertilizers supplemented with or without protozoa on clippings dry weight oi Lolium perenne (mean ± standard error) measured 3 weeks after applying the fertilizers on a rich sandy soil. The horizontal black line indicates the clippings dry weight of the unfertilized and unsupplemented control treatment. Treatments that are significantly different from the unsupplemented fertilizer (NOP) at a p-value < 0,05 are indicated with a ' · ', whereas differences at a p-value of < 0, 1 are shown with a '°'.

Figure 5: Effect of fertilizers supplemented with or without protozoa on root dry weight of Lolium perenne (mean ± standard error) measured 6 weeks after applying the fertilizers on a rich sandy soil. The horizontal black line indicates the root dry weight of the unfertilized and unsupplemented control treatment. Treatments that are significantly different from the

unsupplemented fertilizer (NOP) at a p-value < 0,05 are indicated with a ' · ', whereas differences at a p-value of < 0, 1 are shown with a

Figure 6: Relationship between fertilizers supplemented with different dosages of SP1 (in log 10 cysts per gram of fertilizer) and the number of Bacillus bacteria on the roots oiLolium perene (in log 10 CFU Bacillus per gram of root dry weight). R² indicates the degree of correlation between the rate of SP1 in the fertilizer and the number of Bacillus bacteria on the root system.

The invention is exemplified by the following non-limiting examples. 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/h^-1. With the impoverishment of nutrients in the growth medium, the log phase was terminated which induced sporulation. The maximum cell density was l,5exp l0 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 um resulting in a fine powder containing lexp lO 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 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) supplemented with 0,05 M CaC , 0,4 M MgS0₄, 0,25 M Na₂HP0₄, 0,25 M KH₂P0₄, 0,005 M Fe(NH₄)₂( S0₄)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 um. 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

Raw materials Amount (kg)

Dried hair meal 600,0

Dried meat meal 506,2

Dried bone meal 382,3

Vinasse potassium 288,4

Meal of oil containing seeds 222,3

Bacillus amyloliquefaciens spores 0,5

Acanthamoeba castellanii cysts 0,3

Total 2.000,0

Table 4: product specifications of exemplary fertilizer.

Product specifications

N-P-K rating 7-6-6

% Organic matter 62 %

Bacillus amyloliquefaciens spore count 2,4exp6 CFU / gram

Acanthamoeba castellanii cyst count l,3exp3 cysts / gram

Granule diameter 0,01 - 5 mm

Recommended use rate for ornamentals 1 kg / 10m²

Example 2: Preparation of exemplary fertilizers

The following fertilizers were prepared for use in the trials described below. The basic organic fertilizer used for trials is commercially available and has a N-P-K of 9-3-5. Four species of protozoa were isolated from field soils and identified using a combination of 18S barcoding and morphological determination. This resulted in the identification of the following species: Cercomonas sp. (SP1), Vannella sp. (SP2), Sandona sp. (SP3) and

Platyamoeba sp. (SP4). All protozoa were grown in 1 L Erlenmeyer flasks in a diluted phosphate buffer (Page's Amoeba Saline) supplemented with an E. coli suspension. Cysts were formed once the protozoa ran out of food. The cysts were sieved from the growth medium and stored at 4°C. The fertilizer was supplemented with three different dosages of dried cysts (i.e. 10exp2, 10exp3 and 10exp4 cysts / gram of fertilizer) resulting in a total of 12 different fertilizer formulations (4 protozoa species x 3 cysts dosages). The specifications of the fertilizers used for the trials are described in table 5. Table 5: Specifications of fertilizers used for trials

Product specifications

Basic fertilizer

N-P-K rating 9-3-5

% Organic matter 60 %

Bacillus sp. spore count l,0exp6 CFU / gram

Glomus sp. propagule count 14 propagules / gram

Granule diameter 0,01 - 5 mm

Recommended use rate for lawns 1 kg / 10m²

Protozoa additions

Composition Protozoa species Cysts / gram of fertilizer

1 NOP (No Protozoa) -

2 SP1 - Cercomonas sp. 10exp2

3 SP1 - Cercomonas sp. 10exp3

4 SP1 - Cercomonas sp. 10exp4

5 SP2 - Vannella sp. 10exp2

6 SP2 - Vannella sp. 10exp3

7 SP2 - Vannella sp. 10exp4

8 SP3 - Sandona sp. 10exp2

9 SP3 - Sandona sp. 10exp3

10 SP3 - Sandona sp. 10exp4

11 SP4 - Platy 'amoeba sp. 10exp2

12 SP4 - Platyamoeba sp. 10exp3

13 SP4 - Platyamoeba sp. 10exp4 Example 3

The four species of protozoa were grown on a monoculture of 6 different Bacillus species that are commonly found in soil to determine their individual feeding preferences and feeding rates. The results of this experiment are shown in figure 1.

Figure 1 illustrates that the protozoa differ in their individual feeding preferences. For example, SP4 can grow rapidly on all 6 of the tested Bacillus species, indicating that this protozoa is not affected by defense system that Bacillus bacteria are known to have. This makes SP4 on forehand less suitable for the present invention. SP1, SP2 and SP3 are much more selective in their feeding preferences and thus more preferred for use in a fertilizer of the present invention.

Example 4

The purpose of this trial was to determine the effects of the fertilizer compositions described in table 5 on the growth oiLolium perenne, Lolium perenne (common name perennial rye-grass or English ryegrass or winter ryegrass), is a grass from the family Poaceae. A sandy soil (97% sand) was collected from the northeast of The Netherlands. The soil had pH 3,7 which was raised to pH 5,5 by liming. The organic matter content of the soil was 1,4%, the CEC was no more than 4 mmol/kg soil and soil life derived nitrogen was 5 mg/kg soil. The soil could therefore be characterized as poor in terms of both nutrient supply and soil life.

The soil was transferred into plastic containers (64 cm²) which were placed in a greenhouse where temperatures between 15°C and 18°C were maintained for the duration of the trial. There were 5 replica's for each fertilizer composition. Lolium perenne was sown at a rate of 20 g/m². The grass was allowed to grow for 3 weeks before it was cut back to a length of 3 cm to ensure that the starting conditions for each container were the same before the start of the trial. After these 3 weeks, the fertilizer compositions were applied at a rate of 100 g/m².

Three weeks after applying the fertilizers, the grass was cut back to a length of 3 cm. The grass clippings were dried overnight at temperatures of 80°C before being weighed. The remaining grass was allowed to grow for a final 3 weeks (i.e. 6 weeks after applying the fertilizer compositions) before the above ground parts (i.e. shoots) and below ground parts (i.e. roots) were separated from each other and dried and weighed as described above.

Results

Whereas the lowest dosage of SP1 (i.e. 10exp2 cysts per gram of fertilizer) resulted in a significantly increased clippings dry weight, none of the other protozoa and/or cyst dosages increased the clipping dry weight in a significantly (data not shown). The lack of results may very well be caused by overgrazing of the microbially poor soil. Three weeks later, this changed dramatically. SP2 was shown to increase shoot dry weight with more than 80% in comparison to unsupplemented fertilizers (table 6 and figure 2). The increased shoot biomass is likely the result of the previously described stimulation of mineralization caused by protozoan grazing on bacteria. None of the other protozoa (with the exception of the lowest rate of SP3) had a significant effect on clippings dry weight. Table 6: Effect of fertilizers supplemented with selected protozoa on Lolium perenne shoot dry weight (mean ± standard error) measured 6 weeks after applying the fertilizer on a poor sandy soil. The relative change compares the shoot dry weight of protozoa supplemented fertilizers with that of unsupplemented fertilizers (NOP). P-values of a MANOVA are shown in the last column.

Interestingly, apart from an increase in shoot biomass, SP2 was also shown to increase root biomass (table 7 and figure 3).

Table 7: Effect of fertilizers supplemented with selected protozoa on Lolium perenne root dry weight (mean ± standard error) measured 6 weeks after applying the fertilizer on a poor sandy soil. The relative change compares the root dry weight of protozoa supplemented fertilizers with that of unsupplemented fertilizers (NOP). P-values of a MANOVA are shown in the last column. Relative

Cysts / g

Protozoa DW (g) Change P

fertilizer mean ± s.e.

(%)

NOP - 0,061 ± 0,010 - -

SP1 10exp2 0,057 ± 0,010 -6,7 ,889

SP1 10exp3 0,096 ± 0,009 57,2 ,214

SP1 10exp4 0,084 ± 0,020 36,5 ,445

SP2 10exp2 0, 114 ± 0,020 86,5 ,063

SP2 10exp3 0,083 ± 0,007 34,9 ,465

SP2 10exp4 0, 112 ± 0,023 82,9 ,074

SP3 10exp2 0, 122 ± 0,023 98,4 ,059

SP3 10exp3 0,057 ± 0,015 -6,7 ,882

SP3 10exp4 0,063 ± 0,004 2,7 ,955

SP4 10exp2 0,062 ± 0,027 0,4 ,992

SP4 10exp3 0,076 ± 0,024 23,3 ,610

SP4 10exp4 0,069 ± 0,011 12,8 ,778

Elemental analysis of the shoots further substantiated the improved mineralization hypothesis as the shoots of fertilizers treated with SP2 contained significantly greater concentrations of calcium, magnesium and potassium (table 8) which could of course be taken up through the increased root system of the plant. No differences were found for nitrogen, phosphorus and sulfur.

Interestingly, there appears to be a certain optimum in the rate of SP2 to be used in the fertilizer as the highest rate of SP2 seemed to lower the amount of calcium, magnesium and potassium in the shoots. When too many protozoa are introduced in the soil, they likely overgraze on the native microflora resulting in reduced mineralization and therefore reduced plant growth. Different species of protozoa have a different optimum making them more or less interesting to be used in the present invention. Table 8: Effects of fertilizers supplemented with different rates of protozoa SP2 on the amount of calcium, magnesium and potassium ^g / mg dry weight) in the shoots of Lolium perenne. Measurements were taken 6 weeks after applying the fertilizers on a poor sandy soil. The relative change compares the element values of the protozoa supplemented fertilizers with those of unsupplemented fertilizers (NOP). P-values of a MANOVA are given.

Example 5:

A trial similar to that of Example 4 was performed in a second sandy soil that contained 6,5% organic matter and had a CEC of 58 mmol /kg soil. The amount of nitrogen derived from soil life was 34 mg N/kg soil which makes the soil more fertile than the soil used in Example 4. The pH of the soil was initially 4,4 but was raised to 6,5 by liming.

Where in the poor soil no significant increases in clippings dry weight could be found, this was very different in the rich soil. In the rich soil, SP1 significantly increased the dry weight of the clippings with up to 90% when compared to the unsupplemented fertilizer (NOP). See table 9 and figure 4. It is thought that microbially rich soils are less sensitive to overgrazing, resulting in a more rapid stimulation of the mineralization process. Table 9: Effect of fertilizers supplemented with selected protozoa on Lolium perenne clippings dry weight (mean ± standard error) measured 3 weeks after applying the fertilizer on a rich sandy soil. The relative change compares the clippings dry weight of protozoa supplemented fertilizers with that of unsupplemented fertilizers (NOP). P-values of a MANOVA are shown in the last column.

SP1 was also shown to significantly increase the dry weight of the roots (table 10 and figure 5). This increase in root biomass can be the result of the improvement of the minerahzation process resulting in a greater supply of nutrients to the plant. Another explanation is that SP1 improved the establishment and/or survival of the Bacillus in the fertilizer and/or the native Bacilli in the soil. Bacillus bacteria are known to produce a range of metabolites that stimulate root development in plants. SP1 was shown to increase the number of Bacillus bacteria on the root system of Lolium perenne (figure 6). Again, there appears to be an optimum in the rate of protozoa to be used since the number of Bacilli on the roots were reduced when fertilizers were supplemented with the highest rate of SPl (i.e. 10exp4 cysts per gram of fertilizer).

Table 10: Effect of fertilizers supplemented with selected protozoa on Lolium perenne root dry weight (mean ± standard error) measured 6 weeks after applying the fertilizer on a rich sandy soil. The relative change compares the root dry weight of protozoa-supplemented fertilizers with that of unsupplemented fertilizers (NOP). P-values of a MANOVA are shown in the last column.

Relative

Cysts / g

Protozoa DW (g) Change P

fertilizer mean ± s.e.

(%)

NOP - 0, 112 ± 0,014 - -

SPl 10exp2 0, 132 ± 0,013 18,5 ,413

SPl 10exp3 0, 172 ± 0,033 53,7 ,020

SPl 10exp4 0, 180 ± 0,015 60,6 ,013

SP2 10exp2 0,099 ± 0,011 -11,4 ,613

SP2 10exp3 0, 105 ± 0,008 -6,4 ,776

SP2 10exp4 0,090 ± 0,008 -19,8 ,381

SP3 10exp2 0,043 ± 0,010 -61,5 ,008

SP3 10exp3 0, 103 ± 0,018 -8,0 ,722

SP3 10exp4 0,092 ± 0,024 -18, 1 ,447

SP4 10exp2 0, 127 ± 0,019 13,9 ,560

SP4 10exp3 0,087 ± 0,012 -21,8 ,335

SP4 10exp4 0,068 ± 0,021 -39,5 ,084 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 & Biochemistry 34: 1709- 1715.

Griffiths, B.S. 1989. Enhanced nitrification in the presence of bacteriophagous protozoa. Soil Biology and Biochemistry. 21: 1045-1051. Miiller, 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

1. 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, wherein the PGPR is a Bacillus species, preferably Bacillus subtilis, Bacillus amyloliquefaciens and/or Bacillus licheniformis; and (iii) protozoa in the form of cysts, wherein the protozoa are selected from the group comprising Neocercomonas sp., Cercomonas sp., Vannella sp., Sanalona sp. and Bodomorpha sp., and combinations thereof.

2. Fertilizer composition according to claim 1, comprising at least a Neocercomonas sp. and/or a Cercomonas sp., preferably in combination with a Vannella sp. and/or a Bodomorpha sp. and/or a Sandona sp.

3. Fertilizer composition according to claim 1 or 2, further comprising one or more PGPR that belongs to the genera Acinetobacter, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Beijerinckia, Brady rhizobium,

Burkholderia, Enterobacter, Frankia, Gluconacetobacter, Klebsiella,

Pseudomonas, Rhizobium, Serratia or Streptomyces.

4. Fertilizer composition according to any of the preceding claims wherein the Bacillus species are present in a total amount of 10exp3 to lOexp lO spores or colony forming units per gram of composition.

5. Fertilizer composition according to claim 4, wherein the Bacillus species are present in an amount of 10exp4 to 10exp8 spores or colony forming units per gram of composition.

6. Fertilizer composition according to any of the preceding claims, further comprising protozoa that belong to the genera Acanthamoeba, Amoeba, Bodo, Chilodonella, Colpidium, Colpoda, Halteria, Hartmannella, Heteromita, Naegleria, Oikomonas, Spumella, Tetrahymena and/or

Vorticella.

7. Fertilizer composition according to any of the preceding claims, wherein the protozoa are present in a total amount of lOexp l to 10exp7 cysts per gram of composition.

8. Fertilizer composition according to claim 7, wherein the protozoa are present in a total amount of lOexp l to 10exp4 cysts per gram of composition.

9. Fertilizer composition according to claim 7 or 8, comprising 10exp2 to 10exp3 Neocercomonas sp. cysts, and/or 10exp2 to 10exp3 Cercomonas cysts, and/or 10exp2 to 10exp3 Vannella sp. cysts per gram of composition.

10. Fertilizer composition to any of the preceding claims, comprising 10exp3 to lOexp lO spores or colony forming units of a Bacillus sp., lOexp l to 10exp4 Cercomonas sp. cysts, and lOexp l to 10exp4 Vannella sp. cysts per gram of composition.

11. Fertilizer composition to any of the preceding claims, wherein the nitrogen, phosphate and/or potassium are derived from organic sources, preferably hoof meal, hair meal, feather meal, meat meal, bone meal, cottonseed meal and/or soybean meal.

12. Fertilizer composition according to claim 11, wherein the amount of nitrogen, phosphate and potassium can range from 0 to 20% depending on the application.

13. Fertilizer composition according to any of the preceding claims, wherein the fertilizer has a granular form, preferably with granules ranging in size from 0,01 mm to 5 mm.

14. A method for providing a fertilizer according to any of claims 1 to 13, comprising the steps of:

b) providing a culture of said PGPR and inducing cyst- or spore- formation, preferably through fermentation;

c) providing a culture of said 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 mixture of 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 obtained in step (a).

15. A method for growing a plant, preferably an ornamental plant, grass or a vegetable, comprising applying a fertilizer composition according to any one of claims 1 to 13 in or to the soil in which the plant grows.

16. Method according to claim 15, wherein the fertilizer is applied in or to the soil added in an amount of 10 to 5000 kg pro hectare.

17. Method according to claim 15 or 16, wherein the soil is a poor sandy soil and wherein the fertihzer comprises a Sandona and/or a

Vannella sp.

18. Method according to claim 15 or 16, wherein the soil is a rich sandy soil and wherein the fertilizer comprises a Neocercomonas sp. and/ or a Cercomonas sp.