Indian J Microbiol (Oct–Dec 2014) 54(4):413–418

DOI 10.1007/s12088-014-0468-6

ORIGINAL ARTICLE

Plant Growth Promoting Bacteria from Cow Dung Based

Biodynamic Preparations
T. K. Radha • D. L. N. Rao

Received: 5 February 2014 / Accepted: 26 April 2014 / Published online: 14 May 2014
Ó Association of Microbiologists of India 2014

Abstract Indigenous formulations based on cow dung understanding the beneficial effects of biodynamic prepa-
fermentation are commonly used in organic farming. Three rations and industrial deployment of the strains.
biodynamic preparations viz., Panchagavya (PG), BD500
and ‘Cow pat pit’ (CPP) showed high counts of lactobacilli Keywords Bacillus spp.
Biofertilizer
BD500
Cow pat
(109 ml-1) and yeasts (104 ml-1). Actinomycetes were pit
Fermentation
Lysinibacillus sp.
Panchagavya
present only in CPP (104 ml-1) and absent in the other two.
Seven bacterial isolates from these ferments were identified
by a polyphasic approach: Bacillus safensis (PG1), Bacillus Introduction
cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysiniba-
cillus xylanilyticus (BD3) and Bacillus licheniformis Many agricultural products based on indigenous fermen-
(CPP1). This is the first report of L. xylanilyticus and B. tation technologies are used in organic farming like bio-
licheniformis in biodynamic preparations. Only three car- dynamic preparations and liquid manures. Cow dung is an
bon sources—dextrose, sucrose and trehalose out of 21 integral component of all these preparations and serves as a
tested were utilized by all the bacteria. None could utilize source of inoculum of beneficial microorganisms. ‘Panc-
arabinose, dulcitol, galactose, inositol, inulin, melibiose, hagavya’ (PG; Sanskrit for a blend of ‘five products from
raffinose, rhamnose and sorbitol. All the strains produced cow’) is a traditional product prepared in India by fer-
indole acetic acid (1.8–3.7 lg ml-1 culture filtrate) and menting cow dung, cow urine, milk, curd and clarified
ammonia. None could fix nitrogen; but all except B. safensis butter (ghee) [1]. BD500, a biodynamic preparation that is
and B. licheniformis could solubilize phosphorous from also called as horn manure and ‘Cow pat pit’ (CPP) are
insoluble tri-calcium phosphate. All the strains except L. preparations from cow dung that are used in organic
xylaniliticus exhibited antagonism to the plant pathogen farming [2, 3]. Biodynamic products are included in the list
Rhizoctonia bataticola whereas none could inhibit Sclero- of materials and techniques permitted in organic farming
tium rolfsi. In green house experiment in soil microcosms, by an EC regulation (834/2007). These contain macro- and
bacterial inoculation significantly promoted growth of micro-nutrients, amino acids, growth promoting substances
maize; plant dry weight increased by *21 % due to inoc- like indole acetic acid, gibberellins and beneficial micro-
ulation with B. cereus (PG2). Results provide a basis for organisms. Beneficial effects of biodynamic preparations
have been reported on lentil and wheat [4]. Spraying a 3 %
solution of PG along with soil application of biogas slurry
improved the yields of maize and sunflower [1]. Biody-
Electronic supplementary material The online version of this
article (doi:10.1007/s12088-014-0468-6) contains supplementary namic sprays increased the yields of cereals and vegetables,
material, which is available to authorized users. during the years when yields were low [5].
Presence of naturally occurring beneficial microorgan-
T. K. Radha
D. L. N. Rao (&)
isms, predominantly bacteria, yeast, actinomycetes, and
Indian Institute of Soil Science, Nabi Bagh,
Bhopal 462038, Madhya Pradesh, India certain fungi have been reported in cow dung [6]. Research
e-mail: [email protected] related to isolation and characterization of the beneficial

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attributes of the bacteria present in biodynamic prepara- Rogosa Sharpe (MRS) agar, malt agar (with chloram-
tions are few [7, 8]. Definitive proof is required whether the phenicol added after sterilization at 0.5 %) and Actino-
bacteria in such formulations have any PGPR attributes, mycetes isolation agar for the enumeration of bacteria,
and if so whether they can improve plant growth under lactobacilli, yeast and actinomycetes respectively. Based
defined conditions in soil microcosms, that overcome the on morphological variations viz., shape, elevation, texture
drawbacks of field experiments by eliminating the errors and margin, seven isolates—four from PG, two from
arising from spatial variability of soil physico-chemical BD500 and one from CPP were short listed for evaluation.
and fertility properties that occur in field gradients. There They were characterised for gram reaction, endospore
are no reports on the definitive identification of the bene- formation and selected biochemical attributes viz, catalase,
ficial bacteria in such biodynamic preparations using nitrate reduction, casein hydrolysis, starch hydrolysis, H2S
molecular methods. In the present work the microbial production, urease activity, oxidase, citrate utilization,
composition of three biodynamic preparations viz., PG, Voges–Proskauer test and gelatin liquefaction [10].
BD500 and CPP was analyzed. Seven bacterial isolates
were identified by morphological, biochemical and Strain Identity
molecular features and their plant growth promoting attri-
butes were evaluated in the laboratory and their potential The isolates were tested for their ability to utilize 21 carbon
for improving plant growth was tested in vivo on maize compounds as sole sources of energy; monosaccharides
crop in soil microcosms. (adonitol, arabinose, dextrose, dulcitol, fructose, galactose,
inositol, mannitol, mannose, rhamnose, sorbitol, xylose),
disaccharides (cellobiose, lactose, maltose, melibiose, sal-
Materials and Methods icin, sucrose, trehalose), trisaccharides (raffinose) and
polysaccharides (inulin). The isolates were inoculated in
Biodynamic Preparations test tubes containing basal medium with phenol red indi-
cator (Himedia laboratories, India). Carbohydrate discs
‘Panchagavya’ was prepared by modification of the containing 25 mg of the carbon source impregnated in
method described by Suresh Kumar et al. [9]. A mixture of them were suspended in the broth. After incubation at
5 kg fresh cow dung and 500 g clarified butter (ghee) made 24–48 h the tubes were observed for change in color of the
from cow milk was incubated in a 20 l plastic container for medium from red to yellow which was taken as positive for
4 days at room temperature. Twice a day, the mixture is utilization.
stirred for 20 min with a wooden stick, 10 min in clock- The near full length 16S rRNA gene sequences of the
wise and 10 min in the opposite direction. On the 5th day, bacterial strains were custom sequenced on ABI 373091
3 l of cow urine, 2 l of cow milk, 2 l of curd made from Genetic Analyzer at Xcelris Labs Ltd., Ahmedabad, Guj-
cow milk, 500 g sugarcane jaggery, 3 l sugarcane juice, 12 arat, India. The sequences were deposited in the NCBI
nos. of ripened banana, 3 l of tender coconut water and GenBank data base; accession numbers are given in
100 g brewer’s yeast (Saccharomyces cerevisae) were Table 2. Nearest identities of the strains were obtained by
added. The contents were incubated for 15 days along with comparing sequences of 16S rRNA gene with database in
two stirrings daily as described above for 20 min to facil- GenBank (http://www.ncbi.nlm.nih.gov/) using the
itate aeration. BD500 is a humus mixture prepared by BLASTn program. The strains were also deposited in the
filling the horn of a cow with cow manure and burying it in culture collection of National Bureau of Agriculturally
the ground (40–60 cm below the surface) in the autumn. It Important Microorganisms (NBAIM), Mau. U.P., acces-
is left to decompose during the winter and recovered for sion numbers are given in Table 2.
use the following spring. For preparation of CPP fresh cow
dung obtained from indigenous, pasture grazing and lac- Growth Promoting Characteristics and Plant Bioassay
tating cows is fermented along with crushed eggshell
(source of calcium) and basalt dust (source of silica) mixed The isolates were tested for plant growth promoting attri-
and placed in a 12 inch deep pit along with biodynamic butes like diazotrophy: growth on Jensen’s N free medium;
preparations (BD502–BD507) for catalysing the compost- P solubilisation on Pikovskaya agar followed by spectro-
ing process. In the present study, the commercial formu- photometric quantification of P solubilized in broth after
lations (dried powder) of BD500 and CPP were diluted 10 days growth at 28 ± 2 °C in shake cultures at 125 rpm;
1:10 with sterile water and used for analysis of microbio- ammonia production; indole acetic acid production [11],
logical populations and isolation of bacteria. siderophore production was ascertained by growth in
The biodynamic preparations were serially diluted ten- chrome azurol S (CAS) medium [12] after 48–72 h. growth
fold and plated on Luria–Bertani (LB) agar, de Man at 28 °C The isolates screened for their ability to utilize

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ACC (1-aminocyclopropane-1-carboxylic acid) as a sole N numbers e.g., Azospirillum (1010 ml-1), Azotobacter
source by using MDF (modified nitrogen free-Dworkin and (109 ml-1) and Pseudomonas (106 ml-1) [14]. Lactoba-
Foster) medium [13]. Screening of bacterial culture against cillus was also detected in PG but not counted [14]. In our
plant fungal pathogens Rhizoctonia bataticola and Sclero- study lactobacilli were present in very high numbers in PG
tium rolfsi was done using dual culture technique. The (2.0 9 1010 ml-1) due to addition of milk and milk pro-
bacterial isolates were screened in green house for their ducts during the fermentation. Rupela et al. [2] evaluated
ability to promote plant growth in soil microcosms. 330 ml six other biodynamic preparations and found the popula-
paper cups were filled with 300 g soil (Vertisol). Seeds of tion of bacteria to range from 3.24 log10 ml-1 (in BD502)
maize (var. hybrid Marvel) were surface sterilized by to 6.90 log10 ml-1 (in BD500). Yeasts counts were ten-fold
dipping in 95 % ethanol for 5 min and 0.1 % HgCl2 for higher in PG as compared to the more humified prepara-
3 min and finally washed 5 times in sterile distilled water. tions like BD500 and CPP because of the low pH as well as
The bacterial strains were grown on LB broth for 6 days the fact that yeast was added as inoculum. In our study,
with shaking. Farm yard manure was used as a carrier actinomycetes were absent in PG and BD500 (Table 1); in
material to prepare the inoculants. FYM was air dried for the former it may due to the very low pH (3.7) and also
3–4 days and passed through 0.2 mm sieve and sterilized competition for substrate from the fast growing bacilli.
three times by steam sterilization (121 °C for 20 min) on Substrate competition might also be responsible for its
successive days and then two times dry heat sterilization absence in BD500. But in CPP actinomycetes were
(160 °C for 3 h each time). Forty millilitre of the culture detected possibly due to their stimulation by the added
broth was added to 100 g FYM in a plastic pouch, mixed calcium source. Stalin et al. [3] enumerated the micro
by hands and sealed. One gram of each inoculant was organisms from several organic and biodynamic manures,
added to 10 ml of 1 % carboxy-methyl cellulose (CMC). among them CPP manure contained the highest bacterial
Then 24 seeds of maize were transferred to the CMC- load (4.8 9 106 cfu g-1); Bacillus subtilis was predomi-
culture suspension and kept overnight. The seeds were nant in CPP manure.
removed aseptically and air dried in a laminar air flow The 16S rRNA gene sequence of the bacteria revealed
work station. The inoculant coated seeds were sown at four that all the strains isolated from PG belonged to Bacillus
seeds per cup in five replications. After germination, the safensis or Bacillus cereus. Strains from BD500 belonged
plants were thinned to maintain three plants in each cup. to B. subtilis and Lysinibacillus xylanilyticus. The strain
After 15 days urea was applied as solution to the cups at from CPP belonged to Bacillus licheniformis. In milk and
40 lg N g-1 soil. The cups were watered regularly with tap milk products, B. licheniformis and B. cereus are the most
water (boiled for 30 min and cooled) to maintain optimum frequently isolated bacilli [15]. Recently Giannattasi et al.
moisture. The shoot and root length of the plants and their [7] characterized the bacterial and fungal communities of
dry mass was recorded at 5 weeks growth stage (37 days BD500 using ARISA (Automated rRNA Intergenic Spacer
after sowing). Analysis) fingerprints. BD500 was found to harbor a bac-
terial community of 2.38 9 108 cfu g-1 dry weight dom-
inated by Gram positives with minor instances of
Results and Discussion Actinobacteria and Gamma proteobacteria. The culturable
fraction was dominated by Bacillus species.
The micro-flora of the biodynamic preparations consisted The composition of these ferments reflects the compo-
pre-dominantly of lactobacilli (109 ml-1) and yeasts sition of cowdung used in fermentation. Swain and Ray [6]
(104 ml-1) (Table 1). The microflora was thus over- identified Bacillus, Corynebacterium, Lactobacillus, Leu-
whelmingly bacterial whereas yeasts formed only 0.001 %. conostoc, Bifidobacterium, Enterococcus and Streptococ-
In PG, plant growth promoting bacteria that are widely cus in cowdung. The predominant fungal genera were
used as biofertilizers have been reported to occur in high Aspergillus, Rhizopus, Trichoderma with the remaining

Table 1 Microbial Populations of biodynamic preparations in specific media (cfu ml-1)

Sample pH Lactobacillus spp. (9109) Bacteria (9106) Yeast (9104) Actinomycetes (9103)
(MRS agar) (LB agar) (Malt agar) (Actinomycetes isolation agar)

PG 3.7 20.0 ± 2.4 3.8 ± 0.7 15.0 ± 1.2 Nil

BD500a 6.9 3.0 ± 0.4 0.4 ± 0.04 5.0 ± 1.0 Nil
CPPa 6.6 2.0 ± 4.0 8.2 ± 1.2 1.0 ± 0.35 10.0 ± 1.2
a
Samples were one-tenth dilution of the powder formulations; ±SEM

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Table 2 Genomic identity of the bacterial strains isolated from biodynamic preparations and their morphological and biochemical
characteristics
Code Isolates Colony characteristics 16S rRNA NCBI NBAIM NR CH O CU VP
homology (%) acc. no. acc. no.

PG1 Bacillus safensis White, spreading, feathery 100.0 KF 804070 B-01476 2 1 1 1 1

PG2 B. cereus White, raised, smooth margin 99.1 KF 804071 B-01477 1 1 2 2 1
PG4 B. cereus Off white, round, smooth margin 99.1 KF 804072 B-01478 1 1 2 2 1
PG5 B. cereus Off white, flat, round, smooth margin 99.5 KF 804073 B-01479 1 1 2 2 1
BD2 B. subtilis Off white, spreading, irregular margin 99.2 KF 804074 B-01480 1 1 2 1 1
BD3 Lysinibacillus Off white, round, smooth margin, 95.3 KF 804075 B-01481 2 2 1 2 2
xylanilyticus shiny, raised, translucent
CPP1 B.licheniformis White, spreading, irregular margin 98.6 KF 804076 B-01482 1 2 1 1 1
All the strains were gram positive, endospore formers, motile, catalase positive, positive for starch hydrolysis and gelatin liquefaction. All were
negative for H2S production and urease
NR nitrate reduction, CH casein Hydrolysis, O oxidase, CU citrate utilization, VP voges–Proskauer test

belonging to unidentified yeasts and other species. solubilize P from insoluble tri-calcium phosphate produc-
Recently bacteria were identified in cow dung through ing clearing zones of 10–14 mm diameter in petri plates.
metagenomic approach [16] and found to belong mainly to The amount of P solubilized ranged from 24.3 to
the phyla Bacteroidetes (38.3 %), Firmicutes (29.8 %), 45.5 lg ml-1 of culture filtrate which amounted to solu-
Proteobacteria (21.3 %) and Verrucomicrobia (2 %). L. bilization of 2.4–4.4 % of added insoluble tricalcium
xylanilyticus, a novel species has been reported from forest phosphate P (Table 3). None of the strains produced sid-
humus [17] but ours is the first report on its isolation from erophores or exhibited ACC deaminase activity. All the
biodynamic preparations. Also this is the first report of B. species except L. xylaniliticus exhibited antagonism to the
licheniformis in biodynamic preparations. plant pathogenic fungus Rhizoctonia bataticola. None of
All the seven bacterial strains isolated in this study were the strains inhibited the disease causing fungus Sclerotium
gram positive, endospore forming rods that exhibited white rolfsi. Our results agree in few respects with Sreenivasa
to off white colony colour in agar plates. All were catalase et al. [22] who found that bacteria from ‘Beejamrutha’
positive and also positive for starch hydrolysis and gelatin (fermented cow urine) were capable of P solubilization and
liquefaction. All were negative for H2S production and production of growth promoting substances like IAA and
urease activity. Results of other biochemical tests are given GA. They also found the bacteria to be capable of N2-
in Table 2. Only three carbon sources out of 21 tested were fixation and suppressing Sclerotium sp.
utilized by all the bacterial strains: dextrose, sucrose and Among the seven strains tested in the green house, only
trehalose. None of the strains could utilize arabinose, dul- two strains of Bacillus cereus (PG2 and PG4) could
citol, galactose, inositol, inulin, melibiose, raffinose, increase the shoot length of maize significantly (Fig. 1).
rhamnose, and sorbitol. Cellobiose was utilized only by B. Improvement in root length was significant with the all the
cereus. Maltose was utilized by B. cereus and L. xylani- isolates except B. subtilis (BD2). Total dry weight of the
lyticus. Mannose was utilized by both B. cereus and plants was significantly increased by all the isolates. There
B.safensis but mannitol was utilized only by B. licheni- was a consistently significant increase in shoot and root
formis. The results on the utilization of carbon sources length, and shoot and root dry weight by 23.3, 71.6 and
were in conformity with the earlier reports on the catabolic 20.5, 20.7 % respectively over uninoculated control due to
ability of B. subtilis and B.cereus [18]; B.licheniformis [19] inoculation with B. cereus (PG2). Increase in plant growth
and B.safensis [20]. The sole exception was the inability of may be due to the production of auxin, P solubilization and
B.cereus in the present study to utilize citrate which did not biocontrol ability. Our result support the work of Nagaraj
agree with Collins and Lyne [18] but agreed with another Naik and Sreenivasa [8] who found that the wheat seeds
recent report [21]. treated with the isolates (unidentified) obtained from PG
All the bacterial strains were able to produce indole increased the germination, seedling length and seedling
acetic acid (IAA) under in vitro conditions ranging from vigour index.
1.8–3.7 lg ml-1 (Table 3). All the strains produced Plant growth promoting rhizobacteria are known to
ammonia but none of them fixed atmospheric nitrogen. All influence the growth, development, and yield of crops
the species except B. safensis and B. licheniformis could either directly or indirectly through various mechanisms.

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Table 3 Attributes of the bacterial strains for promoting plant growth

Strain Isolates Ammonia IAA production P solubilization Antagonism to Rhizoctonia
code production (lg/ml) bataticola
% P solubilized pH of broth
in 10 days after 10 days

PG1 Bacillus safensis ??? 3.75 3.26 4.83 1

PG2 B. cereus ?? 3.25 2.98 4.52 1
PG4 B. cereus ??? 3.00 4.44 4.68 1
PG5 B. cereus ??? 3.25 4.12 4.52 1
BD2 B. subtilis ??? 3.00 3.03 4.89 1
BD3 Lysinibacillus xylanilyticus ?? 2.75 3.34 4.81 2
CPP1 B. licheniformis ?? 1.75 2.37 4.96 1
All strains negative for nitrogen fixation, siderophore production, ACC deaminase activity

Fig. 1 Effect of the inoculation of bacterial strains isolated from CPP1 B.licheniformis. LSD values p = 0.05 are 2.8 and 8.0 cm
biodynamic preparations on the growth of maize in soil microcosms plant-1 for shoot (S) and root (R) length; 30.1 and 21.7 mg DW
at 6 weeks. PG1 Bacillus safensis; PG2 B. cereus; PG4 B. cereus; plant-1 for shoot (S) and (R) root biomass
PG5 B. cereus; BD2 B. subtilis; BD3 Lysinibacillus xylanilyticus;

Direct effects include production of plant hormones such as based biodynamic preparations show that they are domi-
auxins, gibberellins and cytokinins, supplying biologically nated by Bacillis spp. This is a new report of the occur-
fixed nitrogen or solubilizing insoluble phosphates. Indirect rence of L. xylanilyticus and B. licheniformis in
mechanisms include suppression of bacterial, fungal and biodynamic preparations. The isolated bacterial strains
nematode pathogens by production of siderophores, HCN, exhibited plant growth promoting attributes like IAA pro-
ammonia, antibiotics, volatile metabolites etc., by induced duction, P solubilization, antagonism to R. bataticola and
systemic resistance and by competing with the pathogen improved the growth of maize plants. The results provide a
for nutrients or for colonization of space [23]. Bacillus basis for understanding the beneficial effects of biody-
species are a major component of the heterotrophic soil namic preparations and for deploying the strains in indus-
microflora and are known to influence plant growth through trial production of biofertilizer and bio-control agents.
production of auxins [24] and gibberellins [25]. B. subtilis
isolated from cowdung exhibited biocontrol activity Acknowledgments We thank Dr. P. Ramesh, Sr. Scientist, Network
Project on Organic Farming, IISS, Bhopal for supplying the biody-
against plant pathogenic fungi Fusarium oxysporum and namic preparations.
Botryodiplodia theobromae and the strain also promoted
root elongation in seedlings of Cicer arietinum up to
70–74 % as compared to untreated seeds [6]. Further
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