Plant Growth Promoting Bacteria from Cow Dung Based Biodynamic Preparations

Abstract

Indigenous formulations based on cow dung fermentation are commonly used in organic farming. Three biodynamic preparations viz., Panchagavya (PG), BD500 and ‘Cow pat pit’ (CPP) showed high counts of lactobacilli (10⁹ ml⁻¹) and yeasts (10⁴ ml⁻¹). Actinomycetes were 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 cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysinibacillus xylanilyticus (BD3) and Bacillus licheniformis (CPP1). This is the first report of L. xylanilyticus and B. licheniformis in biodynamic preparations. Only three carbon sources—dextrose, sucrose and trehalose out of 21 tested were utilized by all the bacteria. None could utilize arabinose, dulcitol, galactose, inositol, inulin, melibiose, raffinose, rhamnose and sorbitol. All the strains produced indole acetic acid (1.8–3.7 μg ml⁻¹ culture filtrate) and ammonia. None could fix nitrogen; but all except B. safensis and B. licheniformis could solubilize phosphorous from insoluble tri-calcium phosphate. All the strains except L. xylaniliticus exhibited antagonism to the plant pathogen Rhizoctonia bataticola whereas none could inhibit Sclerotium rolfsi. In green house experiment in soil microcosms, bacterial inoculation significantly promoted growth of maize; plant dry weight increased by ~21 % due to inoculation with B. cereus (PG2). Results provide a basis for understanding the beneficial effects of biodynamic preparations and industrial deployment of the strains.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0468-6) contains supplementary material, which is available to authorized users.

Introduction

Many agricultural products based on indigenous fermentation technologies are used in organic farming like biodynamic preparations and liquid manures. Cow dung is an integral component of all these preparations and serves as a source of inoculum of beneficial microorganisms. ‘Panchagavya’ (PG; Sanskrit for a blend of ‘five products from cow’) is a traditional product prepared in India by fermenting cow dung, cow urine, milk, curd and clarified butter (ghee) [1]. BD500, a biodynamic preparation that is also called as horn manure and ‘Cow pat pit’ (CPP) are preparations from cow dung that are used in organic farming [2, 3]. Biodynamic products are included in the list of materials and techniques permitted in organic farming by an EC regulation (834/2007). These contain macro- and micro-nutrients, amino acids, growth promoting substances like indole acetic acid, gibberellins and beneficial microorganisms. 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]. Biodynamic sprays increased the yields of cereals and vegetables, during the years when yields were low [5].

Presence of naturally occurring beneficial microorganisms, predominantly bacteria, yeast, actinomycetes, and certain fungi have been reported in cow dung [6]. Research related to isolation and characterization of the beneficial attributes of the bacteria present in biodynamic preparations are few [7, 8]. Definitive proof is required whether the bacteria in such formulations have any PGPR attributes, and if so whether they can improve plant growth under defined conditions in soil microcosms, that overcome the drawbacks of field experiments by eliminating the errors arising from spatial variability of soil physico-chemical and fertility properties that occur in field gradients. There are no reports on the definitive identification of the beneficial bacteria in such biodynamic preparations using molecular methods. In the present work the microbial composition of three biodynamic preparations viz., PG, BD500 and CPP was analyzed. Seven bacterial isolates were identified by morphological, biochemical and molecular features and their plant growth promoting attributes were evaluated in the laboratory and their potential for improving plant growth was tested in vivo on maize crop in soil microcosms.

Materials and Methods

Biodynamic Preparations

‘Panchagavya’ was prepared by modification of the method described by Suresh Kumar et al. [9]. A mixture of 5 kg fresh cow dung and 500 g clarified butter (ghee) made from cow milk was incubated in a 20 l plastic container for 4 days at room temperature. Twice a day, the mixture is stirred for 20 min with a wooden stick, 10 min in clock-wise and 10 min in the opposite direction. On the 5th day, 3 l of cow urine, 2 l of cow milk, 2 l of curd made from cow milk, 500 g sugarcane jaggery, 3 l sugarcane juice, 12 nos. of ripened banana, 3 l of tender coconut water and 100 g brewer’s yeast (Saccharomyces cerevisae) were added. The contents were incubated for 15 days along with two stirrings daily as described above for 20 min to facilitate aeration. BD500 is a humus mixture prepared by filling the horn of a cow with cow manure and burying it in the ground (40–60 cm below the surface) in the autumn. It is left to decompose during the winter and recovered for use the following spring. For preparation of CPP fresh cow dung obtained from indigenous, pasture grazing and lactating cows is fermented along with crushed eggshell (source of calcium) and basalt dust (source of silica) mixed and placed in a 12 inch deep pit along with biodynamic preparations (BD502–BD507) for catalysing the composting process. In the present study, the commercial formulations (dried powder) of BD500 and CPP were diluted 1:10 with sterile water and used for analysis of microbiological populations and isolation of bacteria.

The biodynamic preparations were serially diluted tenfold and plated on Luria–Bertani (LB) agar, de Man Rogosa Sharpe (MRS) agar, malt agar (with chloramphenicol added after sterilization at 0.5 %) and Actinomycetes isolation agar for the enumeration of bacteria, lactobacilli, yeast and actinomycetes respectively. Based on morphological variations viz., shape, elevation, texture and margin, seven isolates—four from PG, two from BD500 and one from CPP were short listed for evaluation. They were characterised for gram reaction, endospore formation and selected biochemical attributes viz, catalase, nitrate reduction, casein hydrolysis, starch hydrolysis, H₂S production, urease activity, oxidase, citrate utilization, Voges–Proskauer test and gelatin liquefaction [10].

Strain Identity

The isolates were tested for their ability to utilize 21 carbon compounds as sole sources of energy; monosaccharides (adonitol, arabinose, dextrose, dulcitol, fructose, galactose, inositol, mannitol, mannose, rhamnose, sorbitol, xylose), disaccharides (cellobiose, lactose, maltose, melibiose, salicin, sucrose, trehalose), trisaccharides (raffinose) and polysaccharides (inulin). The isolates were inoculated in test tubes containing basal medium with phenol red indicator (Himedia laboratories, India). Carbohydrate discs containing 25 mg of the carbon source impregnated in them were suspended in the broth. After incubation at 24–48 h the tubes were observed for change in color of the medium from red to yellow which was taken as positive for utilization.

The near full length 16S rRNA gene sequences of the bacterial strains were custom sequenced on ABI 3730×1 Genetic Analyzer at Xcelris Labs Ltd., Ahmedabad, Gujarat, India. The sequences were deposited in the NCBI GenBank data base; accession numbers are given in Table 2. Nearest identities of the strains were obtained by comparing sequences of 16S rRNA gene with database in GenBank (http://www.ncbi.nlm.nih.gov/) using the BLASTn program. The strains were also deposited in the culture collection of National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau. U.P., accession numbers are given in Table 2.

Table 2.

Genomic identity of the bacterial strains isolated from biodynamic preparations and their morphological and biochemical characteristics

Code	Isolates	Colony characteristics	16S rRNA homology (%)	NCBI acc. no.	NBAIM acc. no.	NR	CH	O	CU	VP
PG1	Bacillus safensis	White, spreading, feathery	100.0	KF 804070	B-01476	−	+	+	+	+
PG2	B. cereus	White, raised, smooth margin	99.1	KF 804071	B-01477	+	+	−	−	+
PG4	B. cereus	Off white, round, smooth margin	99.1	KF 804072	B-01478	+	+	−	−	+
PG5	B. cereus	Off white, flat, round, smooth margin	99.5	KF 804073	B-01479	+	+	−	−	+
BD2	B. subtilis	Off white, spreading, irregular margin	99.2	KF 804074	B-01480	+	+	−	+	+
BD3	Lysinibacillus xylanilyticus	Off white, round, smooth margin, shiny, raised, translucent	95.3	KF 804075	B-01481	−	−	+	−	−
CPP1	B.licheniformis	White, spreading, irregular margin	98.6	KF 804076	B-01482	+	−	+	+	+

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

Growth Promoting Characteristics and Plant Bioassay

The isolates were tested for plant growth promoting attributes like diazotrophy: growth on Jensen’s N free medium; P solubilisation on Pikovskaya agar followed by spectrophotometric quantification of P solubilized in broth after 10 days growth at 28 ± 2 °C in shake cultures at 125 rpm; ammonia production; indole acetic acid production [11], siderophore production was ascertained by growth in chrome azurol S (CAS) medium [12] after 48–72 h. growth at 28 °C The isolates screened for their ability to utilize ACC (1-aminocyclopropane-1-carboxylic acid) as a sole N source by using MDF (modified nitrogen free-Dworkin and Foster) medium [13]. Screening of bacterial culture against plant fungal pathogens Rhizoctonia bataticola and Sclerotium rolfsi was done using dual culture technique. The bacterial isolates were screened in green house for their ability to promote plant growth in soil microcosms. 330 ml paper cups were filled with 300 g soil (Vertisol). Seeds of maize (var. hybrid Marvel) were surface sterilized by dipping in 95 % ethanol for 5 min and 0.1 % HgCl₂ for 3 min and finally washed 5 times in sterile distilled water. The bacterial strains were grown on LB broth for 6 days with shaking. Farm yard manure was used as a carrier material to prepare the inoculants. FYM was air dried for 3–4 days and passed through 0.2 mm sieve and sterilized three times by steam sterilization (121 °C for 20 min) on successive days and then two times dry heat sterilization (160 °C for 3 h each time). Forty millilitre of the culture broth was added to 100 g FYM in a plastic pouch, mixed by hands and sealed. One gram of each inoculant was added to 10 ml of 1 % carboxy-methyl cellulose (CMC). Then 24 seeds of maize were transferred to the CMC-culture suspension and kept overnight. The seeds were removed aseptically and air dried in a laminar air flow work station. The inoculant coated seeds were sown at four seeds per cup in five replications. After germination, the plants were thinned to maintain three plants in each cup. After 15 days urea was applied as solution to the cups at 40 µg N g⁻¹ soil. The cups were watered regularly with tap water (boiled for 30 min and cooled) to maintain optimum moisture. The shoot and root length of the plants and their dry mass was recorded at 5 weeks growth stage (37 days after sowing).

Results and Discussion

The micro-flora of the biodynamic preparations consisted pre-dominantly of lactobacilli (10⁹ ml⁻¹) and yeasts (10⁴ ml⁻¹) (Table 1). The microflora was thus overwhelmingly bacterial whereas yeasts formed only 0.001 %. In PG, plant growth promoting bacteria that are widely used as biofertilizers have been reported to occur in high numbers e.g., Azospirillum (10¹⁰ ml⁻¹), Azotobacter (10⁹ ml⁻¹) and Pseudomonas (10⁶ ml⁻¹) [14]. Lactobacillus was also detected in PG but not counted [14]. In our study lactobacilli were present in very high numbers in PG (2.0 × 10¹⁰ ml⁻¹) due to addition of milk and milk products during the fermentation. Rupela et al. [2] evaluated six other biodynamic preparations and found the population of bacteria to range from 3.24 log₁₀ ml⁻¹ (in BD502) to 6.90 log₁₀ ml⁻¹ (in BD500). Yeasts counts were ten-fold higher in PG as compared to the more humified preparations like BD500 and CPP because of the low pH as well as the fact that yeast was added as inoculum. In our study, actinomycetes were absent in PG and BD500 (Table 1); in the former it may due to the very low pH (3.7) and also competition for substrate from the fast growing bacilli. Substrate competition might also be responsible for its absence in BD500. But in CPP actinomycetes were detected possibly due to their stimulation by the added calcium source. Stalin et al. [3] enumerated the micro organisms from several organic and biodynamic manures, among them CPP manure contained the highest bacterial load (4.8 × 10⁶ cfu g⁻¹); Bacillus subtilis was predominant in CPP manure.

Table 1.

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

Sample	pH	Lactobacillus spp. (×109) (MRS agar)	Bacteria (×106) (LB agar)	Yeast (×104) (Malt agar)	Actinomycetes (×103) (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

^aSamples were one-tenth dilution of the powder formulations; ±SEM

The 16S rRNA gene sequence of the bacteria revealed that all the strains isolated from PG belonged to Bacillus safensis or Bacillus cereus. Strains from BD500 belonged to B. subtilis and Lysinibacillus xylanilyticus. The strain from CPP belonged to Bacillus licheniformis. In milk and milk products, B. licheniformis and B. cereus are the most frequently isolated bacilli [15]. Recently Giannattasi et al. [7] characterized the bacterial and fungal communities of BD500 using ARISA (Automated rRNA Intergenic Spacer Analysis) fingerprints. BD500 was found to harbor a bacterial community of 2.38 × 10⁸ cfu g⁻¹ dry weight dominated by Gram positives with minor instances of Actinobacteria and Gamma proteobacteria. The culturable fraction was dominated by Bacillus species.

The composition of these ferments reflects the composition of cowdung used in fermentation. Swain and Ray [6] identified Bacillus, Corynebacterium, Lactobacillus, Leuconostoc, Bifidobacterium, Enterococcus and Streptococcus in cowdung. The predominant fungal genera were Aspergillus, Rhizopus, Trichoderma with the remaining belonging to unidentified yeasts and other species. Recently bacteria were identified in cow dung through metagenomic approach [16] and found to belong mainly to the phyla Bacteroidetes (38.3 %), Firmicutes (29.8 %), Proteobacteria (21.3 %) and Verrucomicrobia (2 %). L. xylanilyticus, a novel species has been reported from forest humus [17] but ours is the first report on its isolation from biodynamic preparations. Also this is the first report of B. licheniformis in biodynamic preparations.

All the seven bacterial strains isolated in this study were gram positive, endospore forming rods that exhibited white to off white colony colour in agar plates. All were catalase positive and also positive for starch hydrolysis and gelatin liquefaction. All were negative for H₂S production and urease activity. Results of other biochemical tests are given in Table 2. Only three carbon sources out of 21 tested were utilized by all the bacterial strains: dextrose, sucrose and trehalose. None of the strains could utilize arabinose, dulcitol, galactose, inositol, inulin, melibiose, raffinose, rhamnose, and sorbitol. Cellobiose was utilized only by B. cereus. Maltose was utilized by B. cereus and L. xylanilyticus. Mannose was utilized by both B. cereus and B.safensis but mannitol was utilized only by B. licheniformis. The results on the utilization of carbon sources were in conformity with the earlier reports on the catabolic ability of B. subtilis and B.cereus [18]; B.licheniformis [19] and B.safensis [20]. The sole exception was the inability of B.cereus in the present study to utilize citrate which did not agree with Collins and Lyne [18] but agreed with another recent report [21].

All the bacterial strains were able to produce indole acetic acid (IAA) under in vitro conditions ranging from 1.8–3.7 µg ml⁻¹ (Table 3). All the strains produced ammonia but none of them fixed atmospheric nitrogen. All the species except B. safensis and B. licheniformis could solubilize P from insoluble tri-calcium phosphate producing clearing zones of 10–14 mm diameter in petri plates. The amount of P solubilized ranged from 24.3 to 45.5 µg ml⁻¹ of culture filtrate which amounted to solubilization of 2.4–4.4 % of added insoluble tricalcium phosphate P (Table 3). None of the strains produced siderophores or exhibited ACC deaminase activity. All the species except L. xylaniliticus exhibited antagonism to the plant pathogenic fungus Rhizoctonia bataticola. None of the strains inhibited the disease causing fungus Sclerotium rolfsi. Our results agree in few respects with Sreenivasa et al. [22] who found that bacteria from ‘Beejamrutha’ (fermented cow urine) were capable of P solubilization and production of growth promoting substances like IAA and GA. They also found the bacteria to be capable of N₂-fixation and suppressing Sclerotium sp.

Table 3.

Attributes of the bacterial strains for promoting plant growth

Strain code	Isolates	Ammonia production	IAA production (µg/ml)	P solubilization		Antagonism to Rhizoctonia bataticola
				% P solubilized in 10 days	pH of broth after 10 days
PG1	Bacillus safensis	+++	3.75	3.26	4.83	+
PG2	B. cereus	++	3.25	2.98	4.52	+
PG4	B. cereus	+++	3.00	4.44	4.68	+
PG5	B. cereus	+++	3.25	4.12	4.52	+
BD2	B. subtilis	+++	3.00	3.03	4.89	+
BD3	Lysinibacillus xylanilyticus	++	2.75	3.34	4.81	−
CPP1	B. licheniformis	++	1.75	2.37	4.96	+

All strains negative for nitrogen fixation, siderophore production, ACC deaminase activity

Among the seven strains tested in the green house, only two strains of Bacillus cereus (PG₂ and PG₄) could increase the shoot length of maize significantly (Fig. 1). Improvement in root length was significant with the all the isolates except B. subtilis (BD₂). Total dry weight of the plants was significantly increased by all the isolates. There was a consistently significant increase in shoot and root length, and shoot and root dry weight by 23.3, 71.6 and 20.5, 20.7 % respectively over uninoculated control due to inoculation with B. cereus (PG2). Increase in plant growth may be due to the production of auxin, P solubilization and biocontrol ability. Our result support the work of Nagaraj Naik and Sreenivasa [8] who found that the wheat seeds treated with the isolates (unidentified) obtained from PG increased the germination, seedling length and seedling vigour index.

Fig. 1.

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

Plant growth promoting rhizobacteria are known to influence the growth, development, and yield of crops either directly or indirectly through various mechanisms. Direct effects include production of plant hormones such as auxins, gibberellins and cytokinins, supplying biologically fixed nitrogen or solubilizing insoluble phosphates. Indirect mechanisms include suppression of bacterial, fungal and nematode pathogens by production of siderophores, HCN, ammonia, antibiotics, volatile metabolites etc., by induced systemic resistance and by competing with the pathogen for nutrients or for colonization of space [23]. Bacillus species are a major component of the heterotrophic soil microflora and are known to influence plant growth through production of auxins [24] and gibberellins [25]. B. subtilis isolated from cowdung exhibited biocontrol activity against plant pathogenic fungi Fusarium oxysporum and 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 screening of desirable attributes of plant growth promoting organisms from biodynamic preparations would include stability and good survival in soil.

In conclusion, our results on the identification and characterization of culturable bacteria from cow dung based biodynamic preparations show that they are dominated by Bacillis spp. This is a new report of the occurrence of L. xylanilyticus and B. licheniformis in biodynamic preparations. The isolated bacterial strains exhibited plant growth promoting attributes like IAA production, P solubilization, antagonism to R. bataticola and improved the growth of maize plants. The results provide a basis for understanding the beneficial effects of biodynamic preparations and for deploying the strains in industrial production of biofertilizer and bio-control agents.