Indi an lournal of Biotechnology Vol. I , April 2002, pp 180-187

Efficacy of a Rock Phosphate Based Soil Implant Formulation of Phosphobacteria in Soybean (Glycine max Men-ill)

G Viveganandan and K S Jauhri*

Divi sion of Microbiology , Indian Agricultural Research Institute, New Delhi 11 00 12, India

Received 3 March 2001; revised 9 January 2002

For improving the effectiveness of low-grade rock phosphate, a granular formulation was developed by immo­bilizing phosphate solubilizing bacteria (PSB) impregnated rock phosphate in calcium alginate. This process ensured requisite standards of PSB viability in rock phosphate. The formulation was compared with powdered soil and seed formulations 1'01' its efficacy in soybean; maximum weight of nodule, dry matter and grain yield, Nand P uptake of shoot and grain were recorded with granular preparation. The granular formulation can directly be applied in soil and is environmentally safe.

Keywords: rock phosphate, phosphobacteria, formulation, efficacy, soybean

Introduction Soils in general, are poor in phosphorus in Indi a.

The efficiency of phosphatic fertilizers is also low and se ldom increases more than 20% due to hi gh rates of P fixation in so il (Kanwar et aI, 1982). The NPK con­sumption ratio has deteriorated from 5.9:2.4: 1 in 1991 to 8.5:3. 1: 1 in 1999 because of the sharp increase in the prices of phosphatic fertilizers (FAr, 1999). The high processing cost of phosphatic fertilizers has made it more necessary to find al ternative and cheaper means of P-fertilization.

At present, low-grade rock phosphate is used as a cheap alternative source of phosphorus for cultivation of crops but its utili zati on effic iency is low. Of the several methods (Narayanasamy & Biswas, 1998), seed bacterization with phosphobacteria has been pre­fen"ed for improving effic iency of rock phosphate ap­pli ed to soi l for crop production. However, maximum potential has not been reali zed because of the spatial differences between bacteria and rock phosphate. To accomp li sh thi s, attempts were made to develop for­mulations, which can ensure close contact of phos­phobacteri um with the rock phosphate. The efficacy of the new form ul ation was tested in soybean.

Materials and Methods Phospho bacterial Cultures

Phosphate solu bi li zi ng bacteria CPSB), Pseudomo-

" Author for correspondence: Tel: 578511 2, 5787649; Fax: 011-9 1-5751 7 19, 5766420 E ma il: ksj 3uilri @yailoo.co. ill

nas striata (P-27) and Bacillus polymyxa CH-5), were obtained from the Divi sion of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi. Pikovskaya's medium (Pikovskaya, 1948) was used for growth and maintenance of PSB .

Rock Phosphate and Additives

The low grade rock phosphate was obtained from Mussoorie Phosphorite Project, l-AB, Tagore Marg, Dehra OLIn UP. The additive materials, vermi compost and farmyard manure (FYM), were obtai ned from Snowview M L1 shroom Laboratory,Gandhi Ashram, Narela, Delhi , and the Division of Agronomy, JART, New Delhi , respectively. The rock phosphate and ad­ditives were sun-dried and powdered to pass through a 100-mesh sieve. The general characteristi cs of rock phosphate and additives are given in Table I .

Preparation of Formulation

Rock phosphate was mixed with FYM and vermi ­compost, separately to achieve 0, 10, 20, 30, 40, 50 and 100% concentrations. From the mixtures , 100 g each was dispensed into autoclavable polypropylene bags and sterili zed at 15 p.s .i. for 4 hrs. After cooling, the bags were inoculated separately with 10 ml of 48 hrs o ld culture broth of P. striata (P-27) and B. polymyxa (H-5) containing 15x 109 and 13x 109

CFU-I/ml , respectively. The fi nal moisture content of each sample was adjusted to 1/3 of its water holding capacity , and the packets were sealed. The contents

VIVEGANANDAN & JAUHRI: ROCK PHOSPHATE BASED SOIL IMPLANT FORMULATION 181

Table I - Characteris tics of rock phosphate and additives

Rock Additives J2hosQhate

(Mussoorie) Vermi- FYM compost

Water holding capac ity (%) 3 1.58 108.86 93.91 pH 6.81 6.70 7.20 Organi c carbon (%) 1.63 25.01 28.26 0.5 M NaHCO) ex tr. P 1.31 3 10 280.00 (Ilg/g) Citrate soluble P20 S (%) 2.25 ND ND Citrate in soluble P20 5 (%) 16.02 ND ND Total P20 5 (%) 18.27 0.91 0.40

ND: Not determined

were mixed thoroughly by massaging the packets from out side.

Granular inoculant was prepared by immobilizing PSB impregnated rock phosphate in calcium alginate. Rock phosphate amended with 30% additive and Pik­ovskaya's broth containing 4% sodium alginate were sterilized. A 48 hrs old culture of PSB was mixed thoroughly with equal volume of sterile, 4% sodium alginate solution to obtain homogeneous slurry of PSB in 2% sodium alginate. Rock phosphate-additive mixture (l00 g) was then mixed with 100 ml of the sIUlTY. The resulting slurries were pressed through a vibrating screen (pore size, 2 mm) and the emerging drops were allowed to gelate in 0.1 M calcium chlo­ride solution . On gelation, each drop of slurry formed a spherical bead, which was allowed to stand in cal­cium chloride solution for 30 min. Beads were washed with several changes of sterile distilled water, spread on a blotting paper and kept for 6 hrs in a laminar chamber. One hundred grams of air-dried beads (about 25% moisture) were dispensed into ster­ile polypropylene bags and sealed aseptically. The inoculant packets were stored at ambient temperature (26.0o-35 .6°C) .

Assessment of Inoculant Quality The colony forming units (CPU) of PSB were

enumerated by plating on Pikovskaya's agar. The moisture content of the inoculant was determined by drying the samples to a constant weight at 105°C in a hot-air oven and cooling in a desiccator for 6 hrs. The NaHC03 extractable-P in the inoculants was deter­mined by Olsen's method (Olsen et al, 1954).

Pot Culture Experiment The performance of the inoculant was evaluated in

a pot culture experiment conducted during kharif in

1999 at IARI, New Delhi . The soil used was sandy­loam with moderate organic carbon (0.63 %) and ni­trogen (0.07%). The total phosphorus (0 .04%) and NaHC03 extractable-P (8 .9 kg ha- I

) contents were low. The pH, EC and cation exchange capacity (CEC) of the soil were 8.3, 0.24 dS/m and 16.00 cmol (p+) kg-I, respectively .

The rock phosphate-vermicompost based PSB for­mulations were used as soil inoculants as well as a source of the required level of phosphorus (@ 60 kg P20S ha- I) in soil. In case of seed formulation, PSB were applied on seed and the required quantity of plain rock phosphate (60 kg P20 S ha-I) was applied to soil. Seeds of soybean var. Pusa-22 were surface ster­ilized and treated with Rhizobium japonicUln (SB-102) for all the treatments . The granulated soil­implant inoculants of PSB were placed 3 cm below seed while the powdered formulation was incorpo­rated in the topsoil of the pot. All the treatments were replicated six times. Three replicates of each treat­ment were used for observations on nodulation, dry matter yield, Nand P uptake of shoot. The remaining three replicates were maintained till the harvest of the crop for recording the grain yield and Nand P uptake of seed.

Determination of Dry Weight of Nodules and Shoot Roots were washed thoroughly to remove the ad­

hering soil particles and nodules were removed. Shoots were cut off from the roots and dried to con­stant weight at 65°C.

Grain Yield After maturity, pods removed from plants were

sun-dried for 3 days. The seeds obtained after shelling of pods were weighed_

Determination of Total Nitrogen and Total Phos­phorus

The total nitrogen and phosphorus contents of shoot and seed were determined by Kjeldahl's and Va­nadomolybdo-phosphoric yellow complex methods , respectively (Jackson, 1973).

Statistical Analysis The data were analyzed by using standard methods

for correlation, linear correlation and regression and analysis of variance (Panse & Sukhatme, 1985).

Results and Discussion Any carrier selected for inoculant production of

PSB must be able to support desirable number of

182 INDIAN J BIOTECHNOL, APRIL 2002

viable cells over a period of time. The organic matter content and moisture holding capacity of the rock phosphate were lower than the standard carrier for optimal growth and multiplication of PSB . It was, therefore, necessary to overcome these limitations for improved survival of PSB in the carrier. Addition of organic matter improves aeration and moisture re­taining capacity of soil (Buckman & Brady, 1974). The suitability of vermicompost and FYM for im­proving survival of PSB in rock phosphate was, there­fore, examined.

The viability of PSB in rock phosphate was better . with vermicompost than FYM (Fig. 1). Vermicom­post, a well known soil conditioner, is rich in plant nutrients and improves fertility of soil by supplying humus, increases water retention capacity and reduces the leaching of nutrients by slowing down their re­lease (Hartenstein, 1986). These attributes of vermi­compost appear to have supported growth and ensured better survival of PSB in rock phosphate. While in­crease in the additive concentration corresponded with the viability of PSB in rock phosph ate, the de­cline in cell number over time was a common obser­vation; this was irrespective of the additive used (Fig. 1).

Amendment of rock phosphate with vermicompost is an important consideration. Since increase in vi­ab ility of PSB was not significant with more than 30% vermicompost in rock phosphate, this ratio was maintained while devising a suitable formulation of PSB. The availability of P improved with additive concentration but declined linearly with time during storage (Fig. 2) . Various organic acids, carbonic acid and chelating substances are produced during the de­composition of organic matter, which help in liberat­ing phosphorus from rock phosphate (Chien, 1979).

The fertili zer li se efficiency to a greater extent de­pends on the type of formulation and method of appli­cation. Although particle size is an important variable in rate at which P is released, transported and replen­ished in soil system, concentrated pellets/granules of fer tili zers have been found more effective than finely powdered fertilizers when applied in the root zone of a crop. Efforts were, therefore, made to develop a granular formul ation , which would ensure pro longed survival of PSB and release of phosphorus from rock phosphate in soil. An alginate entrapped formulation is most suitab le for thi s purpose since the entrapment of PSB in alginate gel is a one step process and gives a gentle environment to the entrapped microorgan­isms (Smidsrod & Skjak Braek, 1990). Its easy avail-

ability makes it further attractive for commercial use. The final product is light and poses no threat to the environment because of its high biodegradability.

The viability of PSB was better in powdered than granulated formulation, whereas the availability of phosphorus was higher in granulated than powdered formulation (Fig. 3). The larger surface area and higher availability of moisture per unit carrier help in better survival of PSB in the powdered formulation . On the contrary, restricted aeration might be the cause of poor viability of PSB in the granulated formulation. This is in agreement with earlier reports (Roughley & Vincent, 1967; Dommergues et ai, 1979).

The increase in dry weight of nodule, dry matter (shoot) and grain yield, Nand P uptake of shoot and seed was significantly higher with powdered and granulated soil formulations compared to seed for­mulations (Figs 4 & 5). Soil implant formulations en­sured high titre of PSB and consequent availbility of P from insoluble rock phosphate in the rhizosphere on account of close contact of PBS with rock phosphate. Conversely, only a fraction of applied rock phosphate in soil comes in close contact of PSB inoculated on seed.

Soybean is sown in India during the hottest months, June-July, when the temperatures often exceed 40°C. It is likely that PSB placed in soil through seed in­oculation desiccates and hence fails to respond as soil inoculant. Inocula on seed surface are exposed to the toxic factors present on the seed coat, heat and desic­cation, and die rapidly if seeds are not sown deep in so il immediately after inoculation (Dadarwal & Sen, 1971 , 1973). In the soil implant inoculant, organisms remain protected from high temperature and desicca­tion as they are placed deep into soi l away from the deleterious effect of toxic seed exudates (Brockwell, 1977).

The increase in the nodulation and growth of soy­bean was significant and positively core1ated with PSB inoculation (Fig. 5; Table 2). PSB produces or­ganic acids, which solubilize inorganic phosphates in so il (Asea et ai, 1988; IIlmer et ai, 1995). The benefi­cial effect of growth promoting substances such as auxins, gibberellins and cytokinins released by PSB in so il has been demonstrated ( Brown, 1972; Barea et ai, 1976; Sattar & Gaur, 1987; De Freitas et at, 1997). The increase in nodulation and growth of soybean may, therefore, be attributed to improved P availabil­ity and release of growth promoting substances in soil. A number of organisms, known to solubilize phosphorus from insoluble inorganic phosphates, vary

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VIVEGANANDAN & JAUHRT: ROCK PHOSPHATE BASED SOIL IMPLANT FORMULATION

Pseudomonas striata {P-27) Bacillus po/ymyxa (H-5) Farmyard manure

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Days after inoculation

150 1 80

150 180

183

Fi g. I -- Surviva l of Pseudomonas striata (P-27) and Bacillus po/ymyxa (H-S) at di f feren t concentrations of farmyard ma­nure and vermicompost in rock phosphate.

184

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0 0

INDIAN J BIOTECHNOL, APRIL 2002

Pseudomonas striata (P-27) Bacillus po/ymyxa (H-5)

• A

+

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-20 Y=145.52B-3.371X, R' =0.0576 -20 Y = 117.414-4.',4X, A2 = 0.0329

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Fig. 2 - Avai lable phosphorus in inocu lants of Pseudomonas striata (P-27) and Bacillus polymyxa (H-5) prepared with different con­centrat ions of farmyard manure and vermicompost in rock phosphate_

VIVEGANANDAN & JAUHRI: ROCK PHOSPHATE BASED SOIL IM PLANT FORMULATION

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185

Fi g. 3 __ Surviva l of Pselldo llloll llS slri(lIC/ (P-27) and Baci lllls pO/YlIIyxa (H-S), loss of mo isture and available phosphorus in granular

and powdered formulati ons of rock phosphate .

186 INDIAN J BIOTECHNOL, APRIL 2002

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Soli Inoculant (Granulated)

Fig. 4 - Nodulation, dry matter and grai n yields in response to inoc ulation with different formulations of Pseudomonas striata (P-27) and Bacillus polymyxa (H-5) in soybean.

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40

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60

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5011 Inoculant (Granulatad)

Fig. 5 - Nand P uptake of shoot and seed in response to inocu­lation with different formulations of Pseudomonas striata (P-27) and Bacillus polymyxa (H-5) in soybean.

Table 2 -- Correlation between plant parameters of soybean val'. Pusa-22

S. No.

I 2 3 4 5 6 7

Nodule wt (mg/ plant)

1.000

Significant at 5 %

Shoot wt (g/pot)

2

0.972 1.000

Grain yield (g/pot) N-uptake

(mg/pot) 3 4

0 .920 0.928 0.951 0.978 1.000 0.924

1.000

Shoot Seed P-uptake N-uptake P-uptake (mg/pot) (mg/pot) (mg/pot)

5 6 7

0.954 0.927 0.942 0.978 0.99 1 0.983 0.961 0.952 0.975 0.952 0.978 0.965 1.000 0.977 0.981

1.000 0.986 1.000

VIVEGANANDAN & JAUHRI: ROCK PHOSPHATE BASED SOIL IMPLANT FORMULATION 187

in their capacity of phosphorus solubilization in soil (Somani et ai, 1989). The variations observed in nodulation and growth of soybean with different strains of PSB 's may, therefore, be attributed to their intrinsic capability to release phosphorus from insolu­ble rock phosphate in soil.

It is concluded that the efficacy of a low-grade rock phosphate can be improved by developing soil im­plant formulations of PSB . Addition of organic addi ­tives improves the viability of PSB and availability of P in rock phosphate. Vermicompost is better than FYM as an additive for rock phosphate based inocu­lants. To attain requisite number of PSB in a formula­tion, vermicompost concentration should be restricted to 30% in rock phosphate. The shelf life of the for­mulation is satisfactory since PSB viability can be maintained up to 90 days of storage at ambient tem­perature.

Acknowledgement The authors thank the Head, Division of Microbi­

ology, IARI, New Delhi for providing facilities and to Indian Council of Agricultural Research, New Delhi for providing fellowship to the senior author during the tenure of his doctoral programme.

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