WO2014042517A2 - Biofertilizer - Google Patents

Abstract

The present invention relates to a biofertilizer comprising at least one bacterial strain selected from the group comprisingPseadomonas aeruginosa strain AGKT 1,Serratia marcescens strain AGKT4 andBacillus amyloliquefaciens, and optionally also a suitable culture medium. The biofertilizer may further comprise a carrier, which is selected from cocoa pod husk, cocoa bean shell and peat moss. The biofertilizer may be used in combination with an NKP fertilizer.

Description

Biofertilizer

Field of Art The present invention relates to biofertilizers and their optimum composition. Background Art

Nutrients such as Nitrogen (N), Phosphorus (P) and Potassium (K) are essential and they should be adequately supplied for the growth of plants. In plants, P is essential for photosynthesis and, therefore, the assimilation of carbohydrates, such as sugars. In its many compounds, phosphorus has roles in cell division, in stimulation of early root growth, in hastening plant maturity, in energy transformations within the cells and in fruiting and seed production. In all types of farming system in Malaysia, mineral fertilizers account for more than 90 percent of the fertilizers used (Sabri, M.A. (2009/ Evolution of fertilizer use by crops in Malaysia: Recent trends and prospects, Paper presented at IFA Crossroad Asia- Pacific in Kota Kinabalu, Sabah, Malaysia. Dec 2009). Large amounts of straight, mixture and compound fertilizers for the sources of N, P and K are commonly used. However, the prolonged use of mineral fertilizers, (especially N), can result in groundwater contamination. Therefore, these problems can be tackled using biofertilizer as alternative fertilizer replacement for the reduction use of mineral fertilizers. Biofertilizer is a low cost technology, eco-friendly and harmless to environment with the ability of supplementing nutrients Many definitions and interpretations of the term biofertilizer exist (Vessey, J.K. (2003). Plant growth promoting rhizobacteria as biofertilizers. Plant and Soil. 255: 57 1-586). In this chapter, a biofertilizer is a product that consisting of selected and different types of living microorganism, which are known to improve plant growth through the supply of available plant nutrients through biological processes (Hegde, D.M., Dwiv, B.S. and Sudhakara, S.N. ( 1999). Biofertilizers for cereal production in India— a review. Indian Journal of Agricultural Science. 69: 73-83; Vessey, 2003). Development of biofertilizer requires carrier material to be mixed with microorganisms. Such carriers being used are peat (Albareda, M., Rodriguez-Navarro. D.N.. Camacho. M. and Temprano, F.J. (2008). Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations. Soil Biology and Biochemistry. 40: 2771-2779), lignite (Stella, D. and Sivasakthivelan, P. (2009). Effect of different organic amendments additions into Azosprillum bioinoculant with lignite as carrier material. Botany Research International. 2(4): 229-232) and bagasse (Albareda et al, 2008), soil and sand, compost, soil and aggregate of soil, charcoal and other agriculture waste (Jauhri, .S, Gupta, M. and Sadasivam, K.V. ( 1989). Agro-industrial wastes as carriers for bacterial innoculants. Biological Wastes. 27: 81 -86). So far, reports on the research on the use of cocoa pod husk as carrier material in development of biofertilizer are not available. At the moment, pod husks are waste product of the cocoa industry, and present a serious disposal problem. They are sources of disease inoculums when used as mulch inside the plantation. It was known that Pseudomonas and Bacillus are widely used in organic production system and also important phosphorus-solubilizing microorganisms (PSB), resulting in improved crop growth and yield (Doebereiner, J. and Pedrosa, F.P. ( 1987). Nitrogen-Fixing Bacteria in Nonlegumino s Crop Plants: Science Tech Publishers (Madison, Wis. and Berlin and New York)). Research by Wu et al. (W , S.C., CaO, Z.H., Cheung, K.C. and Wong, M.H. (2005). Effects of biofertilizer containing N-fixer, P and solubilizers and AM fungi: A green house trial. Geoderma. 125: 155-166) identified that biofertilizer was an alternative to chemical fertilizer to increase soil fertility and crop production in sustainable farming. Such biofertilizers containing an arbuscular mycorrhizal fungus {Glomus mosseae or Glomus intraradices) with or without N-fixer {Azotobacter chroococcum), P sokibilizer {Bacillus megaterium) and K solubilizer {Bacillus mucilaginous) was experimentally proven to significantly increase the growth of Zea mays.

The aim of the present invention is to select bacteria isolates that are capable of soliibilizing P in a liquid medium at the amount of at least 100 μg/mL, and to develop an optimum biofertilizer composition for these bacteria.

Disclosure of the Invention

The object of the present invention is a biofertilizer comprising at least one bacterial strain selected from the group comprising Pseudomonas aeruginosa strain AGKT 1 , Serratia marcescens strain AG T4 and Bacillus amyloliquefaciens. In a preferred embodiment, the bacteria are grown and used in a culture medium comprising peptone, KC1, MgS0₄.7H₂0, MnS0₄.H₂0, FeS0₄.7H₂0, NaCl and a sugar selected from glucose and fructose, and having a pH between 6 and 10.

A particularly suitable culture medium for Pseuclomonas aeruginosa strain AG T 1 comprises glucose 0.5 g, peptone 0.325 % (w/v), KC1 0.02 g/L, MgS0₄.7H₂0 0.1 g, MnS0₄.H₂0 0.002, FeSO₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L, pH 9.0. Inoculation of the bacterial inoculum is preferably at 2.750% (v/v).

A particularly suitable culture medium for Serratia marcescens strain AGKT4 comprises fructose 60 g, peptone 6 g, KC1 0.02 g, MgS0₄.7H₂0 0.1 g ,MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L, pH 7. Inoculation of the bacterial inoculum is preferably at 1 .875% (v/v).

A particularly suitable culture medium for Bacillus amyloliquefaciens comprises fructose 60.0 g, peptone 6.0 g, MgSO₄.7H₂0 0.10 g, KC1 0.2 g, MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCl 0.2 g in 1.0 L distilled water, pH 8.0. Inoculation of the bacterial inoculum is preferably at 2.750% (v/v).

In a preferred embodiment, the biofertilizer further contains a carrier, which is selected from cocoa pod husk, cocoa bean shell and peat moss, more preferably the carrier is cocoa bean shell. The biofertilizer in both dry and liquid forms may be stored at temperatures of 10 °C to 30 °C. In these conditions, the biofertilizer compositions were capable of maintaining the number of cfu at the levels sufficient for the use in the application. The number of cfu were above 5 logio cfu/g for dry formulation and 5 logio /mL for liquid formulation after 6-month storage. Generally, the dry formulations displayed superioriority over the liquid formulations in terms of the survivability of bioinoculant. Also, the dry formulations exhibit the desirable characteristic of pH above 5 and near to the neutral pH. The moisture content of above 37% in all the formulations at both temperatures were satisfactory. The liquid formulations were also satisfactory as the number of cfu were in the suitable range for a formulation.

In a preferred embodiment, the biofertilizer can be administered in combination with an N P fertilizer.

The biofertilizer may be used in the agriculture, in particular for crop and soil improvement, as biofungicide or plant growth regulator . Examples of carrying out the Invention

Example 1 : Isolation and characterization of bacteria

Isolation of bacteria: Bacterial isolates were isolated from soil with dumped palm oil shell, rhizosphere soil of cocoa, soil under dumped cocoa pods, dumped cocoa pods, liquid sample from cocoa fermentation box, healthy cocoa pods, rhizosphere soils of grass, corn and groundnut. All sources of bacteria samples were taken from Tawau, Sabah. All samples of 10 g each was put in 90 mL of Nutrient Broth in a 200 mL conical flask shaken for 24 to 48 h. Solution of soil or sample of 1 mL was diluted in a serial dilution technique. Exactly, an aliquot of 0.1 mL was put on the nutrient agar (NA) for isolation of bacteria. The nutrient agar plates were incubated at 28 °C for one to six days. Isolated, predominant, morphological distinct colonies were selected, purified by repeated culturing and maintained on NA slants. The Bacillus amyloliquefaciens was isolated from rhizosphere soil of cocoa at 25 °C at the depth of sampling point 0-15 cm. The AGKT 1 bacterial strain was isolated from soil under dumped oil palm shell at 30 °C at the depth of sampling point 0-1 5 cm. The AGKT4 bacterial strain was isolated from rhizosphere soil of groundnut at 30 °C at the depth of sampling point 0- 15 cm. Screening for PSB Using Plate Assay: The NBRIY medium as described by Nautiyai (1999) which comprised Ca₃(P0₄)₂ 5 g, glucose 10 g, MgSO₄.7H₂0 0.10 g, KC1 0.2 g, (NH₄)₂S0₄ 0.5 g, MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g, NaCl 0.2 g, bromophenol blue 0.03 g, Bacto agar 20 g and distilled water to 1 L, pH 7 was used. Aseptically, the bacterial inocula were prepared by inoculating a loopful of bacterial stock cultures from NA slants into 10 mL of sterile LB broth in a universal bottle. The bottles were incubated for 18 h at 28 °C in an oven. After 18 h, a loopful of inoculum from each bacterial inoculum was aseptically streaked on the plate assay. The plates were incubated at 28 °C in an incubator for one to four days. Observation on the growth of bacteria, clearing zone surrounding the colonies of bacteria as well as the change of bromophenol blue color was recorded as potential PSB.

Table 1. The growth of bacterial isolates on phosphate solubilizing medium with bromophenol blue

Growth Clearing zone

+ : slow growth observed (1^1 days at 28 "C) + : slightly observed

++ : moderate growth (1-4 days at 28 °C) ++ : clearly observed

+++ : heavy growth (1-4 days at 28 "C)

Mostly, these bacterial isolates exhibited slight clearing zones and intense blue color of colony. The clearing zone which was observed surrounding the colony of bacteria was due to the solubilization of insoluble phosphate. The intensification of blue color on the bacterial colonies was due to the change of bromophenol blue indicator in the acidity or at low pH. The low pH might be due to the production of organic acids in the medium. Screening of PSB in Liquid Culture Medium: A total number of 22 bacterial isolates were used in the screening for the ability of these bacteria to solubilize phosphate in a liquid culture medium. The NBRIY medium (Nautiyal, 1999) was used which comprised Ca₃(PO₄)₂ 5 g, glucose 10 g, MgSO₄.7H₂0 0.10 g, KC1 0.2 g, (NH₄)₂SO₄ 0.5 g, MnS0₄.H₂0 0.002 g, FeS0 .7H₂0 0.002_g/ NaCl 0.2 g, 1.0 L distilled water, pH 7.0. The inoculum of each bacterial isolate was prepared by inoculating a loopful of bacterial stock culture into the LB broth and incubated at 28 °C in an incubator for 18 h. One millilitre of each bacterial inoculum which had an optical density at 600 nm of 0.8 (« 10¹² cfu) was inoculated into a 50 mL medium in a 100 mL conical flask. The flasks were incubated for eight days at 28 °C and agitated at 70 rpm on an orbital shaker (Lab-Line Orbit Shaker, Lab-Line Instrument, Inc.).

Isolate of AGKT1 could solubilize phosphate at the amount of 50.2 ng/mL, 121.2 Hg/mL, 128.1 μg/mL, 129.1 μg/mL, 145.8 μ /mL, 138.7 μg/mL, 133.2 μg/mL and 103.2 μ / mL at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively.

Isolate of AGKT4 could also solubilize phosphate at the amount of 99.0 μg/ mL, 125.1 μg/mL, 94.7 μg/mL, 163.6 μg/mL, 122.4 μg/mL, 128.0 μg/mL, 144.9 μg/mL and 108.6 μg/ m at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively.

Bacillus amyloliquefaciens could solubilize phosphate at the amount of 103.4 μg/ mL, 121.1 μg/mL, 142.8 μg/mL, 123.8 μg/mL, 143.9 μg/mL, 129.4 μg/mL, 123.7 μg/mL, and 127.1 μg/ m at at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively. Ability of Selected PSB to Solubilize Rock Phosphates: The bacterial isolates, AGKT1, AGKT4 and Bacillus amyloliquefaciens with high P solubilization were used to test the solubilization of P from various poorly soluble rock phosphates using the NBRIY medium. Christmas Island Rock Phosphate (CIRP, 32.0% P₂0₅), North Carolina Rock Phosphate (NCRP, 30.2% P₂0₅ ), China Rock Phosphate (CRP, 34.0% P₂0₅) and Fused Calcium Magnesium Phosphate (FMP, 22.0% P₂O5) at the amount of equivalent to 50 mg P₂Os was used in replacement of Ca₃(PO4)₂. The control was used using NBRIY medium containing all the rock phosphate sources without bacterial inoculation. The inoculum of each bacterial isolate was prepared by inoculating a loopful of bacterial stock culture into the LB broth and inctibated at 28 °C in an incubator for 18 h. One millilitre of each bacterial inoculum which had an optical density at 600 nm of 0.8 (^« 10¹² cfu) was inoculated into a 50 mL medium in a 100 mL conical flask. The flasks were incubated for four days at 28 °C and agitated at 70 rpm on an orbital shaker (Lab-Line Orbit Shaker, Lab-Line Instrument, Inc.). After 72 h of incubation, 5 mL of aliquot from inoculated medium was pipetted and centrifuged at 26,920 x g for 10 min using a Contifuge Stratos (Heraeus Instruments, Germany) refrigerated centrifuge at 10 °C. The clear supernatant was taken for the determination of P solubilization by bacterial isolates. To a 3 mL of the supernatant, 1.25 mL Barton's reagent was added and the volume was made up to 50 mL with distilled water. After 10 min, the solution with yellowish color was read using a Spec ronic 20 Genesys Spectrophotometer at 430 nm wavelength.

A standard curve was prepared by dissolving 0.2195 g KH₂PO₄ in water and the solution made up to 1 L (1 mL=59 ppm P or 1 mL=59 μ /mL P). Further dilution of 10 mL in to 250 mL was made to give 1 mL = 2 ppm. Aliquots of 2, 3, 3, 5, 6, 8, 10, 15 and 20 mL of the 2 ppm stock solution were taken in 50 mL volumetric flasks, 1.25 mL of Barton reagent added and the volume made up to 50 mL mark with distilled water. A standard graph was then prepared from which P values for experimental samples were calculated. The degree of ability of the bacterial isolates to solubilize insoluble phosphate on various poorly rock phosphates was diverse. For CRP, it showed that sequence of the ability of bacterial isolates to solubilize phosphate was AG T4>BA>AGKT1 while for CIRP, NCRP and FMP, the sequences were BA>AGKT1>AGKT4, BA>AGKT4>AGKT1 and AGKT1>BA>AGKT4_/ respectively. In all cases, the ability of bacterial isolates to solubilize the rock phosphates was due to the reduction of pH in the medium. The sequences of pH reduction in the medium for CRP, CIRP, NCRP and FMP were AGKT4<BA<AGKT1, AGKT4<BA<AGKT1, AGKT4<BA<AGKT1 and AGKT4<BA<AGKT1, respectively.

The bacterial isolates were further subjected to morphological observations, Gram staining and spore staining by Schaeffer-Fulton method. Table 2. Colony morphology of AGKT1 and AGKT4 isolates

Table 3. Cell Morphology and Gram Reaction of Isolates

Isolates Cell Morphology Gram Reaction Spore

AGKT1 Rod Negative Absent

AGKT4 Rod Negative Absent 16s rDNA sequencing : Further, the bacteria were identified using 16s rDNA sequencing. Two bacterial isolates from AGKTl and AGK.T4 were used for genomic DNA extraction to identify the isolates. The genomic extraction was done according to DNeasy Blood and Tissue Kit (QIAGEN) manual book. An approximately 1.5 kbp band was obtained.

Polymerase Chain Reaction (PCR): Universal primers were used for amplifying bacterial 16S rDNA. First, 2 μΙ ( 1 μg) DNA template and 1 μΐ each of 16s rRNA forward primer fD l (5'- CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1) and 16s rRNA reverse primer rD l(5'-CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT- 3', SEQ ID NO. 2) were added into μa ime PCR premix tubes (Ί-taq). Genomic DNA of each bacterial isolates from AGKTl and AGKT4 was added into each tube. Distilled water was added into the tubes to give a total volume of 20 μΐ. The reaction mixture was then subjected to PGR reactions containing the above forward and reverse primers using a PCR machine. The reaction mixture was initially heated for 4 min at 94 °C, followed by 30 PCR cycles (94 °C 1 min, 58 °C 1 min and 72 °C 1 min); then, one cycle of 7 min at 72 °C and held at 4 °C. The primers of AGKT l and AGKT4 were amplified. The PCR products of 1442 kbp and 1445 kbp were obtained from AGKT l and AGKT4, respectively. The amplified products were examined by electrophoresis and extracted by using gel extraction kit (QIAquick) according to the manufacturer's instructions. The DNA purification was done according to QIAquick Gel Extraction Kit manual book.

Sequencing and Blasting: The extracted products were sent to First BASE Laboratories Sdn Bhd, Malaysia for sequencing. A homology search was conducted using a Basic Local Alignment Search Tool (BLAST) from the website address of http://www.ncbi.nlm.nih.gov/BLAST/.

Table 4. 16s rDNA Sequence for Isolates AGKT1

Designated Sequence (SEQ ID NO. 3)

name

AGKT1 GTCATTGTAGGACGTACGCATGCAGTCGAGCGGATGAAGGGAGCTTGCTCCTGGATTCAGCGGCGGA

CGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACC GCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGA

1442 kbp

TTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAG TCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGG GCGAAAGCCTGATCCAGCCATGCCCGCGTGTTGAAAGAAAGGTCTTCGGATTGTAAAAGCCCTTTAA GTTGGGAAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAAAATAAGCACCGGC TAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAA GCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAA ACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATA GGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGG GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCT TGAGATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCCGCCTGGGGGAAGTATCGGCCGCAAGGTT AAAACTCAAATGAATTTGACGGGGCCCCGCACAAGGCCGGTGGAGCATGTGGTTTAATTCGAAGCAA CGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAA CTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAAC GAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAA ACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTAC AATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCG GATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGG TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAG CTAGTCTAACCGCAAGGGGACGTACCAGCAGATGA

Table 5. 16s rDNA Sequence for Isolates AGKT4

Example 2: Preparation of the liquid composition of the bacteria

The optimized medium for Pseudomonas aeruginosa strain AGKT I comprised glucose 0.5 g, peptone 0.325 % (w/v), KCl 0.02 g/L, MgS0₄.7H₂0 0. 1 g, MnS0₄.H₂0 0.002, FeSO₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L distilled water, pH 9.0. Inoculation of the bacterial inoculum was at 2.750% (v/v).

The optimized medium for Serratia marcescens strain AGKT4 comprised fructose 60 g, peptone 6 g, KCl 0.02 g, MgS0₄.7H₂0 0.1 g ,MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L distilled water, pH 7. Inoculation of the bacterial inoculum was at 1.875% (v/v). The optimized medium for Bacillus amyloliquefaciens comprised fructose 60.0 g, peptone 6.0 g, MgSO₄.7H₂0 0.10 g, KC1 0.2 g, MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCI 0.2 g in 1.0 L distilled water, pH 8.0. Inoculation of the bacterial inoculum was at 2.750% (v/v).

Example 3 : Preparation of the solid composition of the bacteria

The cocoa pods husks were dried in an oven at 70 °C for 7 to 14 days or sun dried for a few weeks. All dried cocoa pod husks, cocoa bean shell and peat moss were ground using a China Made Disk Mill Model FFC-45A manufactured by Qingdao (Dahua Double Circle Machinery, Co. Ltd., China) to pass through a 1 mm sieve (Testing Sieve, SANPO, Tokyo, Japan). Two hundred and ten grams of each carrier materials powder was packed in low density polyethylene plastic bags of 15 cm x 23 cm (0.05 mm gauge), sealed using a hot plastic sealer ( Hind, Malaysia) and sterilized at 121 °C and 15 psi for 3 h (1 h intervals).

The carrier materials were then mixed with the liquid compositions.

Example 4: Effect of the biofertilizer on cocoa seedlings Preparation of Soil for Potting and Seedlings: Non-sterile of low nutrient soil from the top of 15 cm of a soil from the Table Series was collected from QL Farms Sdn Bhd, Quoin Hill,

Tawau, Sabah. This soil is a member of the Table Family which is very fine, oxidic, isohyperthermic, brown Tipik Teinpalemoks. It typifies this family and is developed over basalts (basic igneous rocks). Soils of the Table Series are characterized by their deep heavy clay textured oxic horizons with dark yellowish brown color and an ECEC that is more than 1.5 cmol (+) kg^"' clay in all horizons between 25 to 100 cm depth. Structures are weak, medium to fine subangular blocky and consistence is friable. The soil was dried, sieved (2 mm), and used in a pot study. The bulked soil sample was also analyzed for nutrient contents prior to the experiment. Eight kilograms of soil were put into clean plastic pots, which were thoroughly cleaned by rubbing using a clean gauge cloth, which dipped in 70% ethanol. The pots were arranged in the experimental design layout. Cocoa seeds from open-pollinated seed garden from UIT 1 x NA33 were surfaced sterilized for 1 5 min using 70% ethanol. Then, the seeds were put into a double layer of sterilized moist gunnysacks for germination. After two days, three germinated seeds were transferred into the pots. The pots were watered at field capacity during the course of experiment. After one month of sowing, all seedlings were thinned out and one healthy and uniform seedling was retained in the pots. Experimental Design and Treatments: A randomized complete block design (RCBD) consisted of 15 treatments with 4 replications was used as experimental design (Figure 6. 1 and Appendice A. 6.1). Each treatment consisted of 6 seedlings in all blocks. The treatments were designated as T l , T2, T3, T4, T5, T6, T7, T8, T9, T10, T i l , T 12, T 13, T14 and T15. The treatments were the combination of the bacterial formulations and fertilizers (Urea as Nitrogen source and KC1 as potassium source and Ca₃(PC>4)₂ ) with exception of treatments T l , T2 and T3. The details of the treatments are shown in Table 6.1. Application of N was made using Urea, P using Ca₃(P0₄)₂ and K using C1 at the rate of 1.38 g N/plant, 0.56 g P/plant and 0.45 g K/plant, respectively. Fertilizers of N and K were applied at fortnightly intervals starting from 30 DAS up to the fifth month, while P was applied after 30 DAS as the only one-application. Treatment Tl was served as the control without any application of fertilizer. Treatment T2 was the standard nursery fertilizer application using NPK blue 12: 12: 12+ TE which was applied at the rate of 10 g/plant split to fortnightly intervals as recommended by Teoh ( 1980). All inorganic fertilizers were applied to the seedlings near the root zone of cocoa seedlings.

Table 6: The details of treatments

CPH- Cocoa pod husk, CBS-cocoa bean shell; Ml, M2,M3 - liquid media for growing the respective bacteria ;

NKP [Urea, KC1 and Ca₃(P0₄)₂]

N as Urea at 1.38 g N/ plant, K as KC1 at 0.45 g K/plant and P as Ca₃(P0₄)₂ at 0.56 g P/plant (N and K applied at fortnightly intervals starting from 30 DAS up to 5-months old seedlings and P is applied 30 DAS, once).

T2 applied at fortnightly intervals at the rate of recommendation by Teoh

(1980).

Treatments T4-T12, applied at 37.5 g/ plants/ once at the adjacent of root zone in a circle furrow of 2 cm in depth and 5 cm apart from the basal stem.

Treatments T13-T15, applied at 25 mL/ plant /month at the adjacent of the root zone 5 cm apart from the basal stem.

Treatment T3 consisted of fertilizer from N and K source at the same rate as in treatments, T4 to T15. Treatments of T4 to T12, dry bioinoculant formulations were applied at 37.5 g/ plant once («10¹⁴ cfu/ g), near the root zones of cocoa seedlings in a circular furrow of 2 cm in depth and 5 cm apart from the basal stem of cocoa seedling. The furrow was then covered with the soil. Treatments of T13, T14 and T15, which were liquid bioinoculant formulations, applied at 25 mL/ plant (^IO¹⁴ cfu/mL) by dispensing into the pots using a pipetor at 30-day interval. The liquid formulation was applied near the root zones of cocoa seedlings, 5 cm apart from the basal stem. All treatments were commenced after one month-old of cocoa seedlings. All pots were also amended with lime using CaCO₃ at the rate of 6 g/ pot as top dressing to raise the pH of soil. Filtered deionized water was used as watering the seedlings in the pots at the field capacity (22%) using ELGA deionized cartridge connected to the water supply. Regular maintenance of pest and disease was carried out during the course of the experiment.

The growth parameters were measured. The plant height was measured from the cotyledon scar to the young shoot of cocoa seedlings using a measuring tape. The diameter of plant was measured using a digital clipper below the cotyledon scar of cocoa seedlings at the end of experiment. The number of leaves of cocoa seedlings was counted at the end of experiment. Only the recently matured leaves and matured leaves were counted. The results showed that the treatments had significant effect on the growth parameters of cocoa seedlings. Most of the treatments using phosphate-solubilizing bacteria in the formulation either in dry formulations or liquid formulation increased significantly the stem diameter, plant height and the number of leaves as compared to Tl (control). Formulation in T7 (NKP + Pseudomonas aeruginosa strain AGKT1 + cocoa bean shell) and T8 (NKP + Serratia marcescens strain AGKT4 + cocoa bean shell) had the greatest effect on all growth parameters followed by T4 (NKP + Pseudomonas aeruginosa + CPH), T5 (NKP + Serratia marcescens + CPH),T9 (NKP + Bacillus amyloliqiiefaciens + peat moss) and T6 (NKP + Bacillus amyloliqiiefaciens + CPH). The results revealed that the formulation using Serratia marcescens strain AGKT4, Pseudomonas aeruginosa strain AGKT1 and Bacillus amyloliqiiefaciens with carrier materials either cocoa bean shell and cocoa pod husk and peat as well as liquid formulation was the best formulation for the growth of cocoa seedlings. Significant increase in stem diameter, plant height and number of leaves was observed with these bacterial formulations as compared to Tl. However, the inoculated treatments (T4-T15) had no significant difference on the growth of cocoa seedlings as compared to the treatments of T2 (Standard NPK Blue 12:12:17:2 + TE) and T3 (NPK). The results indicated that these treatments were comparable to the standard fertilizer application using NPK Blue.

Table 7. The effects of treatments on stem diameter, height and number of leaves for 5-month old seedlings.

Means followed by the same letter in the same column are not significantly different according to the least significant difference (LSD) test (p<0.05). The p-value is for the analysis of variance (ANOVA). Each value is the mean of four replicates. In most cases, the stem diameter significantly increased in the treatments with inoculated PSB in all dry formulations (T5, T6, T8, T9 and T10) as compared to the control (Tl). The results revealed that these formulations tising carrier materials from cocoa pod husk powder, cocoa bean shell powder and peat moss seemed to be better as compared to the liquid formulations T13-Pseiidomonas aeruginosa strain AGKTl; T14-Serratia marcescens strain AGKT4 ; Tl5-Bacilhis amyloliquefaciens). Height of plant significantly increased in the treatments of T4, T5, T8, T9 and Til as compared to T3 (uninoculated bacteria). In addition, other treatments with inoculated PSB in the formulation with the treatments of T6, T7, T10, T12, T13, T14 and T15 also showed an increase in stem diameter, despite no significant differences. The treatments also had the significant effect on the number of leaves. Obviously, treatments T7 and T8 showed the greatest effect on the number of leaves as compared to the other treatments, including control (Tl), standard fertilizer application (T2) and straight fertilizer NPK (T3). Significant increase on the number of leaves indicated that these two treatments, which derived from Pseudomonas aeruginosa strain AGKTl and Serratia marcescens strain AGKT4 with cocoa bean shell as carrier material seemed to be the best formulations. At overall performance of all formulations, it seemed that dry formulations using carrier materials from cocoa bean shell, cocoa pod shell and peat moss s carrier materials for Pseudomonas aeruginosa strain AGKTl, Serratia marcescens strain AGKT4 and Bacillus amyloliquefaciens had a greater efficiency on the growth parameter of 5-month old cocoa seedlings. Fresh weight of plant components was determined: The plants were harvested at twenty weeks (5 months) of planting. The root, stem and leaves were separated and put into a handmade brown translucent paper (brown onionskin) envelope. Then the root, stem and leaves were weighed. The weight of fresh root was divided by the weight of fresh shoot (the total weight of fresh leaves and stem) to get fresh root:shoot ratio. Table 8 shows that the treatments had a significant effect on the fresh weight of plant components. Treatments of T4, T5, T6, T7 and T8 resulted of significant increase of fresh weight of leaf, stem, root, total fresh of plant as well as shoot as compared to the other treatments. Most of these treatments had low ratio of root: shoot indicating that the effect of treatments was greater on the growth of the upper part of plant than the root growth. This case might explain that most nutrient uptake was on the upper part of vegetative growth, stem and leaves. These treatments were better than the standard nursery fertilizer application.

Table 8. The effects of treatments on the fresh weight of leaf, stem, root, total weight, shoot and the ratio of roofcshoot of 5-month old seedlings.

Means followed by the same letter in the same column are not significantly different according to the least significant difference (LSD) test (p<0.05). The p-value is for the analysis of variance (ANOVA). Each value is the mean of four replicates.

Dry formulation using PSB from Pseudomonas aeruginosa strain AGKT 1 , Serratia marcescens strain AG T4 and Bacillus amyloliqiiefaciens in all carrier materials from cocoa pod husk, cocoa bean shell and peat moss (T4,T5,T6, T7,T8, T9,T 10,T 1 l and T 12) also resulted in greater effectiveness on fresh weight of plant components as compared to liquid formulations (T13, T 14 and T 15). Nevertheless, in liquid formulations, all PSB also showed significant different as compared to the control (Tl), standard fertilizer application (T2), and straight fertilizer NKP (T3-without PSB). Dry weight of plant components was determined: The plants were harvested at twenty weeks of planting. The root, stem and leaves were separated and put into a handmade brown translucent paper (brown onionskin) envelop and oven dried separately at 70 ° C for about 5 days for a constant weight. Then the root, stem and leaves were weighed. The weight of dry root was divided by the weight of dry shoot (the total weight of dry leaves and stem) to get dry root:shoot ratio.

The effect of treatments on dry weight of plant components is shown in Table 9. The results showed that the treatments had the significant effect on the dry weight of plant components. The dry weight of leaves increased significantly in the treatment of T7 followed by T9, T4, T5, T8 and T6 as compared to the treatment of T l (control). For the dry matter weight of stem it showed that treatment of T8 had the highest significant effect as compared to the control, normal fertilizer practice, and other treatments. It indicated that the formulation in T8 exerted the best effect to the stem growth in cocoa seedlings. This effect might be attributed by the availability of nutrients to the plants. However, dry matter weight of roots was not significantly affected by the treatments.

Table 9. The effects of treatments on the dry weight of leaf, stem, root, total weight, shoot and the ratio of root: shoot

of 5-month old seedlings.

Means followed by the same letter in the same column are not significantly different according to the least significant difference (LSD) test (p<0.05). The p-value is for the analysis of variance (ANOVA). Each value is the mean of four replicates.

However, there was a trend of increasing dry matter weight in the formulations used particularly in the dry formulations. For total dry weight of plants, there were significant differences among the treatments. The significant increase in total dry matter weight was observed in the treatments of T4, T5, T7 T8 and T9 as compared to the other treatments, indicating that the formulations of bioinoculant using three PSB from Pseudomonas aeruginosa strain AGKT 1 and Serratia marcescens strain AG T4 and Bacillus amyloliquefaciens using cocoa pod husk and cocoa bean shell were applicable and effective. Formulation of bioinoculant using all PSB in cocoa bean shell as dry formulations exhibited greater effective formulation (T7, T8 and T9) as compare to other treatments.

Claims

1. A biofertilizer comprising at least one bacterial strain selected from the group comprising Pseudomonas aeruginosa strain AGKT 1 , Serratia marcescens strain AGKT4 and Bacillus amyloliquefaciens.

2. The biofertilizer according to claim 1 , wherein the bacteria are grown and used in a culture medium comprising peptone, KCl, MgS0₄.7H₂0, MnS0₄.H₂0, FeS0₄.7H₂0, NaCl and a sugar selected from glucose and fructose, and having a pH between 6 and 10.

3. The biofertilizer according to claim 2, wherein the bacterial strain is Pseudomonas aeruginosa strain AGKT 1 and the culture medium comprises glucose 0.5 g, peptone 0.325 % (w/v), KCl 0.02 g/L, MgS0₄.7H₂0 0. 1 g, MnS0₄.H₂0 0.002, FeSO₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L, pH 9.0.

4. The biofertilizer according to claim 2, wherein the bacterial strain is Serratia marcescens strain AGKT4 and the culture medium comprises fructose 60 g, peptone 6 g, KCl 0.02 g, MgS0₄.7H₂0 0.1 g ,MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCl 0.2 g in 1 L, pH 7.

5. The biofertilizer according to claim 2, wherein the bacterial strain is Bacillus amyloliquefaciens and the culture medium comprises fructose 60.0 g, peptone 6.0 g, MgSO₄.7H₂0 0. 10 g, KCl 0.2 g, MnS0₄.H₂0 0.002 g, FeS0₄.7H₂0 0.002 g and NaCl 0.2 g in 1 .0 L distilled water, pH 8.0.

6. The biofertilizer according to any of the preceding claims, further comprising a carrier, which is selected from cocoa pod husk, cocoa bean shell and peat moss, more preferably the carrier is cocoa bean shell.

7. The biofertilizer according to any of the preceding claims, further comprising an NKP fertilizer.

8. Use of the biofertilizer according to any of the preceding claims in the agriculture, in particular for crop and soil improvement, as biofungicide or as plant growth regulator .