Biofertilizing potential of feather hydrolysate produced by indigenous keratinolytic Amycolatopsis sp. MBRL 40 for rice cultivation under field conditions

Author’s Accepted Manuscript Biofertilizing potential of feather hydrolysate produced by indigenous keratinolytic Amycolatopsis sp. MBRL 40 for rice cultivation under field conditions K. Tamreihao, Laishram Jaya Devi, Rakhi Khunjamayum, Saikat Mukherjee, Roshan Singh Ashem, Debananda S. Ningthoujam

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To appear in: Biocatalysis and Agricultural Biotechnology Received date: 16 July 2016 Revised date: 11 April 2017 Accepted date: 12 April 2017 Cite this article as: K. Tamreihao, Laishram Jaya Devi, Rakhi Khunjamayum, Saikat Mukherjee, Roshan Singh Ashem and Debananda S. Ningthoujam, Biofertilizing potential of feather hydrolysate produced by indigenous keratinolytic Amycolatopsis sp. MBRL 40 for rice cultivation under field c o n d i t i o n s , Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2017.04.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biofertilizing potential of feather hydrolysate produced by indigenous keratinolytic Amycolatopsis sp. MBRL 40 for rice cultivation under field conditions K. Tamreihao, Laishram Jaya Devi, Rakhi Khunjamayum, Saikat Mukherjee, Roshan Singh Ashem, Debananda S. Ningthoujam* State Biotech Hub, Microbial Biotechnology Research Laboratory, Department of Biochemistry, Manipur University, Canchipur-795003, India *

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Abstract Extracellular keratinase-producing strain Amycolatopsis sp. MBRL 40 exhibited antifungal activity against four important rice fungal pathogens and plant growth promoting traits such as indole 3-acetic acid (IAA) production and phosphate (P) solubilization. The strain could produce IAA in the degraded feather medium (3 µg/ml) and increased production was observed when the medium was supplemented with 0.2% tryptophan (7 µg/ml). The strain could solubilize significant amounts of tricalcium P (117 µg/ml). Rice seeds treated with MBRL 40 showed higher germination percentages and vigor indices, and enhanced seedling growth over control under gnotobiotic conditions. Under field conditions, rice plants grown in presence of dried hydrolysate pellet (P), hydrolysate filtrate (S) and feather hydrolysate with bioinoculant (B+F) exhibited significantly enhanced growth and higher number of tillers per plant over plants grown in the absence of urea (C-). However, P treated plants did not have enhanced growth in all the

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parameters in comparison to plants treated with S and B+F. Rice grown in presence of B+F was found to exhibit better growth over plants grown in presence of P and S. Rice grown in presence of urea (C+) and B+F showed similar levels of growth promotion. Keywords: Amycolatopsis sp. MBRL 40; chicken feather hydrolysate; antifungal; plant growth promoting; vigor index; Manipur 1. Introduction World population is projected to grow from 7 to 9 billion in 2050 and, hence, agricultural productivity needs to increase by 60% in 2030-2050 relative to production levels in 2005 -2007( Schroder, 2014). Replacement of hazardous synthetic fertilizers with eco-friendly bio-fertlizers or slow release nitrogenous organic fertilizers could be an alternative option for enhancing agricultural productivity. Keratinolytic bacteria can serve as potential recycling agents for keratinous wastes (Bose et al., 2014). Degraded chicken feather hydrolysate generated by keratinolytic bacteria can act as slow release nitrogen (N) fertilizers by conserving and recycling nutrients besides reducing waste discharge and use of synthetic fertilizers (Jeong et al., 2010). Feathers represent 5 to 7% of the total weight of mature chickens thereby producing substantial amounts of poultry wastes (Govinden and Puchooa, 2012). Feathers contain about 80 to 90% keratin on dry mass basis (Ghosh et al., 2008) and feather as a whole contains about 15% N (Papadopoulos et al., 1986). Thus, feather hydrolysate prepared with keratinolytic bacteria can be a better alternative to synthetic fertilizers for development of slow release N fertilizers. Degraded feather produce essential amino acids that serve as precursors for the plant growth promoting compounds (Rai and Mukherjee, 2015). Indole-3-acetic acid (IAA) is a major plant growth hormone which promotes root growth and thus, ultimately increases the accessibility of

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nutrients and water for the plants. Degraded feather could generate tryptophan, for a precursor of IAA (Bhange et al., 2016). Bacteria especially actinobacteria with plant growth promoting traits such as production of IAA and siderophore and solubilization of phosphate (P) have been reported to enhance the growth of plants (Passari et al., 2015). Moreover, keratinolytic bacteria having both antifungal and plant growth promoting activities could offer a number of economic and environmental advantages over synthetic chemical-based applications (Jeong et al., 2010). The present study deals with studies of antifungal and plant growth promoting activities of the indigenous extracellular keratinase producing strain Amycolatopsis sp. MBRL 40 (Ningthoujam et al., 2016). It also deals with studies growth promotion of rice plants under field conditions by feather hydrolysate as slow release N fertilizers in presence and absence of the strain. Comparison of growth promotion by feather hydrolysates and synthetic N fertilizers is also incorporated in this paper. 2. Materials and methods 2.1.

Antifungal activities Antifungal activities of the strain were assayed using dual culture method (Khamna et al.,

2009) against Pyricularia oryzae (MTCC 1477), Rhizoctonia solani (MTCC 4633), Fusarium oxysporum (MTCC 287) and Curvularia oryzae (MTCC 2605). Agar plugs (6 mm) of 5 d old MBRL 40 strain grown on Starch Casein Nitrate agar (SCNA) were placed at the corners of the Potato Dextrose agar (PDA) plates leaving 1 cm from the margins. The plates were incubated at 30°C for 24 hr. Fungal plugs (6 mm) were then placed at the centers of the plates. Plates containing fungal plugs without the isolates were kept as controls. The inhibition zone was measured after the fungal mycelia in the control plates reached the edges of the plates. Colony

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growth inhibition was calculated using the formula: (C - T)/C × 100, where C is the growth of the test pathogen in the control plate (measured in mm radius), and T is the growth of test pathogen in the test plate (measured in mm). Ammonia production was screened according to Cappuccino and Sherman (1992) with some modifications. The strain was inoculated in 10 ml peptone water containing 0.5% (w/v) chicken feather and kept in a shaker (150 rpm, 30 °C) for 4 d. 0.5 ml of Nessler’s reagent was then added to each tube. Development of brown to yellow color indicated ammonia production. 2.2. Plant growth promoting traits 2.2.1. IAA production The strain was inoculated in FBM-4 consisting of 0.5% (w/v) chicken feather, 0.2% (w/v) yeast extract, 0.91% (w/v) corn flour and 0.5% (w/v) soyabean meal (Ningthoujam et al., 2016) in absence and presence of 0.2% L-tryptophan (Trp). Cultures were kept incubated on a shaker (150 rpm, 30 °C, 5 d), centrifuged (10,000 rpm, 10 min) and 1 ml of the supernatant was mixed with 2 ml of Salkowski reagent. The appearance of pink color indicated IAA production. Optical density (OD) was read at 530 nm and the amount of IAA produced was calculated by comparing with the standard IAA curve. 2.2.2. P solubilization Qualitative assay for phosphate solubilization was done using NBRIP-BPB medium (Mehta and Nautiyal, 2001). A halo zone surrounding the colony after 4 d of incubation at 30 °C indicated P solubilization. Quantitative estimation of P solubilization was done by inoculating the strain in FBM-4 containing 0.5% of tricalcium phosphate (TCP). Cultures were kept incubated on a shaker (150 rpm, 30 °C) for 5 d, centrifuged (10,000 rpm, 10 min) and the

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supernatant was analyzed for P concentration. The amount of P in the culture supernatant was estimated using the method of Fiske and Subbarow (1925) and expressed as equivalent P (μg/ml). KH2PO4 was used as the standard. 2.2.3. Siderophore Qualitative siderophore production was assayed according to You et al. (2005) with few modifications. Agar plug (6 mm) of strain MBRL 40 was inoculated in SCNA (without iron) amended with CAS-substrate and kept incubated at 30 °C for 5 d. Halo zone with orange color surrounding the colony was considered as positive indicator for siderophore production. 2.3.

In vitro seed germination test (Vigor index) The strain was grown in SCN for 5 d, centrifuged (10,000 rpm, 10 min) and the pellet

collected was washed thrice with sterile distilled water (SDW). Rice seeds (Variety: Tampha) were surfaced sterilized with 70% ethanol for 5 min followed by 0.2% sodium hypochlorite for 5 min and rinsed four times with SDW. Sterilized seeds were soaked in the cell suspensions and kept under shaking conditions (150 rpm, 2 h). Sterilized seeds soaked in SDW were taken as control. The seeds were then transferred to sterile plates containing wetted filter papers at the rate of 10 seeds per plate. Plates were incubated at 28-30 °C for 5 d. The number of germinated seeds, root lengths, and shoot lengths, and fresh and dry weights of seedlings were noted and compared with controls. Four replications were done per treatment and the experiment was repeated twice. Vigor index was calculated using the formula (Baki and Anderson, 1973): Percent germination × Seedling length (i.e. shoot length + root length).

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2.4.

Field trials A field trial was conducted in the Manipur University campus. The nature of the soil was

red gravelly. The field was divided into 5 plots each of 18 sq ft (6 x 3) separated by a space of 1 ft. Five different treatments were done as shown in Table 1. The FBM-4 medium containing feather hydrolysate was centrifuged (10,000 rpm, 5 min) and the filtrate collected was kept in a bottle (1 L). The hydrolysate pellet collected was kept dried for 5 d at 70 0C. Soils were treated with 10 g of dried hydrolysate pellet, 1 L of hydrolysate filtrate and 1 L of degraded medium containing feather hydrolysate and bioinoculant in each designated plot. Rice (Variety: Tampha) seeds were placed in each plot at a seed to seed spacing of 6 cm apart (approx.) at a depth of 1 to 2 cm. After 20 d, rice plants were thinned in order to keep plant to plant spacing of 12 cm. Soils were treated again with various forms of feather hydrolysate as stated above. Synthetic fertilizer treatments were also done as shown in Table 1. After 45 d, 15 plants from each plot were uprooted randomly and different growth parameter measurements were performed. 2.5.

Statistical analysis Data were subjected to one-way ANOVA followed by independent t-test at P≤ 0.05 using

the SPSS 17 software (SPSS Inc). 3. Results 3.1.

Antifungal activities MBRL 40 could significantly inhibit the mycelial growth of test pathogens. It showed

highest inhibition against Rhizoctonia solani (85.71%) and lowest against Pyricularia oryzae (46.5%) (Fig. 1). Strain MBRL 40 showed positive results for ammonia production.

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3.2.

Plant growth promoting traits MBRL 40 showed positive results for IAA production and P solubilization but negative

for siderophore production. Production of IAA increased when the medium was supplemented with Trp. The strain could produce 3±0.08 µg/ml of IAA in the absence of Trp and 7±0.07 µg/ml of IAA when the medium was supplemented with 2% Trp. The strain could solubilize significant amount of TCP (117±0.52 µg/ml). 3.3.

Seed germination MBRL 40 treated seeds showed higher germination percentage and vigor index over the

control. Rice seedlings from seeds treated with the strain showed a significant increase (Р ≤ 0.05) in root length and biomass dry weight over the control (Table 2) (Supplementary figure, S1). 3.4.

Field trials Rice plants grown in plot C+ (positive control), S (dried hydrolysate pellet) and B+F

(feather hydrolysate with bioinoculant) showed significant increase (Р ≤ 0.05) in root and shoot length, fresh and dry root weights, fresh and dry shoot weights, and higher number of tillers over plants grown in plot P (dried hydrolysate pellet) and C- (negative control). Plants grown in plot P exhibited significant increase (Р ≤ 0.05) in shoot lengths and fresh root weights over plants grown in C-. Rice plants in plots C+ and B+F showed similar growth promotion. However, a higher number of tillers per plant was observed in plants grown in plot B+F. Feather hydrolysate with MBRL 40 (B+F) treated plants also showed significant increase (Р ≤ 0.05) in fresh shoot and dry root weights over plants treated with hydrolysate filtrate (S). A higher number of tillers

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per plant was observed in plants treated with various forms of feather hydrolysate over the negative control plants (Table 3) (Fig. 2) (see also Supplementary figures S2, S3, S4, and S5). 4. Discussion The keratinolytic strain MBRL 40 exhibited significant antifungal and PGP activities. The strain has potential for development as a biofertilizer/biocontrol agent for rice cultivation. Keratinolytic Stenotrophomonas maltophilia has been reported earlier to exhibit antifungal activities against Fusarium oxysporum, Pythium ultimum and Botrytis cinerea (Jeong et al., 2010). Strain MBRL 40 was found to produce ammonia. Ammonia has been reported to play an important role as a biocontrol agent (Bhange et al., 2016; Trivedi et al., 2008). Howell et al. (1988) observed that ammonia production by the biocontrol strain Enterobacter cloacae controlled damping-off disease caused by Pythium sp. Ammonia, besides its role in biocontrol, may also be a factor in plant growth promotion by accumulating nitrogen in the soil and enhancing N availability to the plant (Passari et al., 2016). MBRL 40 produced IAA in the optimal feather medium without Trp supplementation, indicating simultaneous Trp release from feather degradation. However, the amount of IAA production increased when the medium was supplemented with Trp. This is in agreement with the reports of Jeong et al. (2010) and Bhange et al. (2016) in which keratinolytic Stenotrophomonas maltophilia and Bacillus subtilis respectively produced IAA in the optimal feather medium without Trp supplementation and IAA production were found to increase when the media were supplemented with Trp. Strain MBRL 40 could solubilize significant amount of inorganic P. Similarly, keratinolytic Bacillus subtilis has been reported to solubilize inorganic P (Bhange et al., 2016).

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Treatment of agricultural crops such as carrot, Chinese cabbage, gram and bean with chicken feather hydrolysate has been reported to enhance the growth of the crops under gnotobiotic and pot trial conditions (Kim et al., 2005; Rai and Mukherjee, 2015; Bhange et al., 2016). Rice plants treated with dried feather hydrolysate pellet prepared with MBRL 40 exhibited more growth promotion than plants grown in the absence of synthetic nitrogen fertilizers. This might be due to the release of some nitrogenous compounds from the degraded feather. The hydrolysate filtrate significantly enhanced the growth of rice plants over dried hydrolysate pellet and control plants grown in the absence of nitrogen source. The feather hydrolysate filtrate treated soil has been reported to exert a beneficial effect on seed germination and growth of Bengal gram under pot trial conditions (Rai and Mukherjee, 2015). Plants grown in presence of feather hydrolysate with bacterial suspension exhibited higher growth promotion than those grown in presence of dried pellet and hydrolysate filtrate. This may be due to enhancement of seed germination and PGP activities such as IAA production and P solubilization by strain MBRL 40 which may ultimately contribute to better growth of the rice plants. Similarly, Mung bean seeds treated with bacterial suspension and feather hydrolysate gave significant growth promotion effects over seeds treated individually with bacterial suspension and water under gnotobiotic conditions (Bhange et al., 2016). Plants treated with feather hydrolysate and bacterial suspension showed a similar level of growth promotion compared to plants treated with synthetic nitrogen fertilizers. Rice plants grown in presence of hydrolysate filtrate, and feather hydrolysate with bioinoculant showed higher number of tillers per plant over other treatments. The tillering number is one of the most important indicators of crop yield potential as it is closely related with the number of panicles per unit area in the rice

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plants (Chen et al., 2008). To our knowledge, this is the first report of rice growth promotion by treatment with various forms of chicken feather hydrolysates under field conditions. 5. Conclusions Indigenous keratinolytic Amycolatopsis strain MBRL 40 exhibited antifungal and plant growth promoting activities. The feather hydrolysate produced by MBRL 40 significantly enhanced the growth of rice plants under field conditions. Feather hydrolysate with bacterial suspension showed a similar level of growth promotion as that of synthetic nitrogen fertilizers. This investigation yielded baseline data that may lead to development of low-cost fertilizers from waste chicken feathers with the long term objectives of poultry waste valorization, recycling, remediation and partial replacement of synthetic fertilizers for rice cultivation. Acknowledgements This work was supported by grants from the Department of Biotechnology (DBT), Government of India, under the State Biotech Hub (SBT Hub) scheme (BT/04/NE/2009). References Baki, A.A., Anderson, J.D., 1973. Vigor determination in soybean seed by multiple criteria. Crop Sc. 13(6), 630-633. Bhange, K., Chaturvedi, V., Bhatt, R., 2016. Ameliorating effects of chicken feathers in plant growth promotion activity by a keratinolytic strain Bacillus subtilis PF1. Bioresour. Bioprocess. 3, 13. Bose, A., Pathan, S., Pathak, K., Keharia, H., 2014. Keratinolytic protease production by Bacillus amyloliquefaciens 6B using feather meal as substrate and application of feather

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hydrolysate as organic nitrogen input for agricultural soil. Waste Biomass Valor. 5, 595605. Cappucino, J.C., Sherman, N., In: Microbiology: A Laboratory Manual. New York, 1992. Chen, S., Zeng, F-R., Pao, Z-Z., Zhang, G-P., 2008. Characterization of high-yield performance as affected by genotype and environment in rice. J. Zhejiang Univ. Sci. B. 9(5), 363370. Fiske, C.H., Subbarow, Y., 1925. A colorimetric determination of phosphorus. J. Biol. Chem. 66(2), 375-400. Ghosh, A., Chakrabarti, K., Chattopadhyay, D., 2008. Degradation of a raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW. J. Ind. Microbiol. Biotechnol. 35(8), 825–834. Govinden, G., Puchooa, D., 2012. Isolation and characterization of feather degrading bacteria from Mauritian soil. Afr. J. Biotechnol. 11(71), 13591-13600. Howell, C.R., Beier, R.C., Stipanovic, R.D., 1988. Production of ammonia by Enterobacter cloacae and its possible role in the biological control of Pythium preemergence damping-off by the bacterium. Phytopathology 78, 1075-1078. Jeong, J-H., Lee, O-M., Jeon, Y-D., Kim, J-D., Lee, N-R., Lee, C-Y., Son, H-J., 2010. Production of

keratinolytic enzyme by a

newly isolated feather-degrading

Stenotrophomonas maltophilia that produces plant growth-promoting activity. Process Biochem. 45, 1738-1745.

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Khamna, S., Yokota, A., Lumyong, S., 2009. Actinomycetes isolated from medicinal plant rhizospheric soils: diversity and screening of antifungal compounds, indole-3-acetic acid and siderophore production. World J. Microbiol. Biotechnol. 25, 649-655. Kim., Man, J., Choi, Y.M., Suh, H.J., 2005. Preparation of feather digests as fertilizer with Bacillus pumilis KHS-1. J. Microbiol. Biotechnol. 15 (3), 472-476. Mehta, S., Nautiyal, C.S., 2001. An efficient method for qualitative screening of phosphatesolubilizing bacteria. Cur. Microbiol. 43, 51-56. Ningthoujam, D.S., Devi, L.J., Devi, P.J., Kshetri, P., Tamreihao, K., Mukherjee, S., Devi, S.S., Betterson, N., 2016. Optimization of keratinase production by Amycolatopsis sp. strain MBRL 40 from a limestone habitat. J. Bioprocess Biotech. 6, 282. Papadopoulos, M.C., El-boushy, A.R., Roodbeen, A.E., Ketelars, E.H., 1986. Effects of processing time and moisture content on amino acid composition and nitrogen characteristics of feather meal. Animal Feed Sci. Tech. 14(3-4), 279-290. Passari, A.K., Mishra, V.K., Gupta, V.K., Yadav, M.K., Saikia, R., Singh, B.P., 2015. In vitro and in vivo plant growth promoting activities and DNA fingerprinting of antagonistic endophytic actinomycetes associates with medicinal plants. PLoS ONE 10(9), e0139468. Passari, A.K., Chandra, P., Zonthanpuia, Mishra, V.K., Leo, V.V., Gupta, V.K., Kumar, B., Singh, B.P., 2016. Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plantgrowth-promoting effect. Res. Microbiol. 167, 692-705.

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Rai, S.K., Mukherjee, A.K., 2015. Optimization for production of liquid nitrogen fertilizer from the degradation of chicken feather by iron-oxide (Fe3O4) magnetic nanoparticles coupled β-keratinase. Biocatalysis and Agricultural Biotechnology. 4(4), 632-644. Schröder, J.J., 2014. The position of mineral nitrogen fertilizer in efficient use of nitrogen and land: a review. Natural Resources. 5, 936-948. Trivedi, P., Pandy, A., Palni, L.M., 2008. In vitro evaluation of antagonistic properties of Pseudomonas corrugate. Microbiol. Res. 163(3), 329-336. You, J.L., Cao, L.X., Liu, G.F., Zhou, S.N., Tan, H.M., Lin, Y.C., 2005. Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments. World J. Microbiol. Biotechnol. 21(5), 679-682.

Figure legends Figure 1 Mycelial growth inhibition of the fungal test pathogens by strain MBRL 40, Note: MTCC 4633, Rhizoctonia solani; MTCC 1477, Pyricularia oryzae; MTCC 287, Fusarium oxysporum; MTCC 2605, Curvularia oryzae

Figure 2 Rice plant growth promotion under different field treatments, Note: C-, Rice grown in presence of superphosphate and potash in absence of urea (negative control); C+, Rice grown in presence of superphosphate, potash and urea (positive control); P, Rice grown in presence of dried feather hydrolysate pellet, superphosphate and potash; S, Rice grown in presence of hydrolysate filtrate, superphosphate and potash; B+F, Rice plant treated with feather hydrolysate , bioinoculant, superphosphate and potash

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Table 1. Five different treatments of rice plants under field conditions Designated Plot C-

Treatment

Components added

Negative control

20g potash (60 % K, 46 % Cl), 30 g super phosphate (15 % P, 24 % Ca)

C+

Positive control

50g Urea (46 % N), 20g potash, 30 g super phosphate

P

Dried hydrolysate

20 g cell-free dried feather pellet, 20g potash, 30 g super

pellet

phosphate

Hydrolysate filtrate

2L Cell-free supernatant, 20g potash, 30 g super

S

phosphate B+F

Feather hydrolysate

2L degraded feather medium with bioinoculant, 20g

with bioinoculant

potash, 30 g super phosphate

Table 2. In vitro rice seed germination by strain MBRL 40 Treatment

Germination percent 86.7

Root length* (cm) 2.98±1.64a

Shoot length* (cm) 2.17±1.00a

Control MBRL 40

Vigor Index 446.50

0.04±0.012a 0.22±0.002a

96.7

5.70±2.14b

2.82±0.62a

823.88

0.47±0.007a 0.23±0.000b

*Values with the same letter within a column are not significant at P≤ 0.05

Fresh weight*

Dry weight*

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Table 3. Growth characteristics of rice plants under five different field treatments Treat ment C-

C+

P

S

B+F

Length (in cm)

Root* 6.98±2.53a

Fresh weight (in gram)

Dry weight (in gram)

No. of tillers per plant*

Shoot* 32.21±8.42

Root* 0.65±0.54

Shoot* 2.20±1.99

Root* 0.44±0.35

Shoot* 1.12±0.75

1.53±1.24

a

a

a

a

a

a

10.53±1.90

45.99±4.36

0.98±0.50

4.40±1.63

0.64±0.40

2.24±0.86

2.46±1.30

b

b

c

c

c

b

a

7.76±1.08a

37.28±4.86

0.74±0.45

2.40±1.09

0.48±0.33

1.14±0.54

1.80±1.02

b

b

a

a

a

a

44.63±4.81

0.98±0.48

3.84±1.76

0.59±0.32

2.13±1.15

2.27±1.03

b

c

b

b

b

a

10.87±1.83

48.86±5.16

0.92±0.44

4.28±1.58

0.62±0.30

2.32±0.87

3.06±1.33

b

b

c

c

c

b

a

9.44±1.24b

*Values with the same letter within a column are not significant at P≤ 0.05

16 Highlights  Keratinolytic strain Amycolatopsis sp. MBRL 40 exhibited antifungal activity against four major rice fungal pathogens  The strain showed positive results for plant growth promoting traits such as IAA production and phosphate solubilization  Rice seeds treated with the strain showed higher germination percentages and vigor indices and showed seedling growth over control under gnotobiotic conditions  Under field conditions, rice plants grown in presence of dried feather hydrolysate pellet, hydrolysate filtrate and feather hydrolysate with bioinoculant exhibited significantly enhanced growth and showed higher number of tillers per plant over plants grown in absence of nitrogen fertilizers (urea)  Plants individually grown in presence of feather hydrolysate with bioinoculant and urea showed similar levels of growth promotion.

Fig. 1

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Fig. 2