effect of different formulations of liquid manures on the ...bbrc.in/bbrc/papers/s/volume 7 - no 1 -...

Microbiological CommunicationBiosci. Biotech. Res. Comm. 7(1): 18-26 (2014)

Effect of different formulations of liquid manures on

the biodiversity of benefi cial microbes

*Sonali Phate1, Tarak Kate2 and G. N. Wagh3

1Principal Investigator, Dharamitra, Wardha2Chairman, Dharamitra, Wardha, India3Associate Professor and Head Department of Microbiology, J.B. College of Science, Wardha – 442001, India

ABSTRACT

Seven different promising formulations of liquid manures were identifi ed and assayed for their nutrient level, micro-bial content and amount of extracellular enzymes secreted by them. Although the chemical nutrient status of the liquid manures was noted to be low, the population of the benefi cial microbes was high. The study also showed that these liquid manures exhibit high levels of enzymatic activity which might be responsible for mineralization of some plant nutrients which in turn might be infl uencing the growth of plants. The two years study on effect of application of liquid manures to the soil on the population of benefi cial microorganisms conducted showed that even if status of chemical nutrients was low in the organic growth promoters, populations of benefi cial microorganisms (PGPR) present in them were high which helped in soil amelioration. Sanjeevak 5 and Panchagavya are the best formulations of liquid manure showing highest microbial count and presence of high amount of extracellular enzymes. Among all the liquid manures studied for their effect on seed germination and root growth for different crops, T5 treatment was noted to have pronounced effect on root growth in case of crops like cotton, pigeon pea and wheat while bet-ter growth observed in the liquid manure treatments T6 and T7 for corps like cotton and Bengal gram, respectively. Panchagavya treatment was seen to be more favorable for the proper growth of Actinomycetes and PSM in the soil, while Amritpani treatment was seen to be favored by microbes like Azotobacter and Pseudomonas closely followed by Sanjeevak 5 treatment.

KEY WORDS: LIQUID MANURES, PLANT GROWTH PROMOTING BACTERIA, SANJEEVAK, PANCHAGAVYA, AMRITPANI.

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ARTICLE INFORMATION:

*Corresponding AuthorReceived 23rd March, 2014Accepted after revision 18th June, 2014 BBRC Print ISSN: 0974-6455Online ISSN: 2321-4007 © A Society of Science and Nature Publication, 2014. All rights reserved.Online Contents Available at: http//www.bbrc.in

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Sonali Phate, Tarak Kate and Wagh

INTRODUCTION

Nutrient management is one of the major factors gov-erning crop productivity. The excess use of chemical fertilizers in agriculture and horticulture has resulted in serious environmental problems (Rabindra, 2003). How-ever, use of organic manures to enhance soil fertility and herbal extracts to protect crop can be made more effi -cient by the scientifi c validation of the practices (Rupela, 2006). The research conducted on use of organic manure plus minerals showed positive effect on nitrogen miner-alization and microbial biomass in paddy soils (Zhang, 2009) compared to that of application of only chemi-cal fertilizers. In the organically fertilized soils the fugal population increases quantitatively as well as qualita-tively (Cwalina, 2007). It was proved that the application of poultry litter increases the soil microbial population as well as soil enzyme activities (Acosta, 2006). There are many reports suggesting that the manure application showed the strongest effect on the biological parameters (Ondrasek, 2008; Rozkova, 2008) and bioavailability of nutrients (Hanc, 2008). Soil organic matter, soil pH and EC are the major factors infl uencing activities of soil enzymes (Shi, 2008).

The application of organic fertilizers signifi cantly increases the phosphates, dehydrogenase, cellulose and urease activity (Balik, 2007; Balakrishnan, 2007). The study on use of human urine as a fertilizers showed that human urine compared well with urea as a source of N for crops but optimum rates depend on the sensitivity of the crops to soil salinity (Mukeni, 2008). The population of bacteria increases with the addition of liquid dairy and swine manure (Miller, 2009; Sattar, 2014).

Groups of microorganisms studied here are commonly considered as plant growth promoting Rhizo bacteria (PRPR). Several investigations are conducted on under-standing of the diversity, dynamics and importance of soil – PGPR communities and their benefi cial and coop-erative roles in agricultural productivity. PGPR is known to improve plant growth in many ways when compared to synthetic fertilizers, insecticides and pesticides.

They enhance crop growth and can help in sustaina-bility of safe environment and crop productivity (Sattar, 2014). The PGPR appears to promote the plant growth via suppression of plant disease (bio-controls), enhanced nutrient achievement or phytohormone production (bio-fertilizers). The PGPR protect plants from several biotic and abiotic stresses. (Singh, 2013). Pseudomons fl uores-cens produces siderophores and antifungal metabolites. Production of antifungal metabolites by fl uorescent pseudomonas has also been found to suppress soil-borne fungal pathogens (Pal, 2001; Dey, 2004).There are some cases where PGPR promoted plant growth in non-sterile soil by controlling fungal diseases (Cattelan, 1999).

Three alternative methods involving use of liquid manures produced from locally available materials are being successfully tried by innovative farmers under fi eld conditions viz., Sanjeevak, Amarutpani and Pan-chagavva. These liquid manures are mostly fermented products. Jaggary and honey in the admixture acts as a carbon source, cattle dung offers microbial cultures and other ingredients act as nitrogen (protein) source.

The objectives of the study were to analyze the nutri-ent status, benefi cial microbial population and enzy-matic activity of the liquid manures and to evaluate effect of application of liquid manures on native benefi -cial micro-organism of the soil. Free-living soil bacteria benefi cial to plant growth, usually referred to as plant growth promoting rhizobacteria (PGPR), are capable of promoting plant growth by colonizing the plant root (Hayat, 2010). As the ingredients for the preparation of liquid manure can be easily available locally the study will render scientifi c support to this low cost technology. More recent research fi ndings indicate that the treatment of agricultural soils with PGPR inoculation signifi cantly increases agronomic yields as compared to uninoculated soils (Sattar, 2014). Given the negative environmental impact of artifi cial fertilizers and their increasing costs, the use of benefi cial soil microorganisms such as PGPR for sustainable and safe agriculture has increased glo-bally during the last couple of decades. PGPR as biof-ertilizers are well recognized as effi cient soil microbes for sustainable agriculture and hold great promise in the improvement of agricultural yields (Singh, 2013). This study will in turn bring a hope for farmers reducing the input cost and improve the soil health and productivity.

MATERIAL AND METHODS

SAMPLING DESIGN

Seven different formulations of liquid manure were identifi ed from innovative farmers. These liquid manures were prepared and after completion of fermentation were analyzed for their nutrient content, benefi cial microbe population and extracellular enzymes.

CHEMICAL ANALYSIS

The samples after preparation were kept in freezer (+4oC) to stop further reaction and analyzed for NPK and C. Percentage of organic carbon was determined by Walk-ley and Black rapid titration method as described by Jackson (1973). 10 ml of liquid manure was added with 10 ml of 1 N potassium dichromate solution and 20 ml conc. H2SO4. The reaction mixture was allowed to stand for 30 min. The reaction was stopped with addition of

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TABLE 1: Different formulations of organic growth promoters.

Ingredients Sanjeevak 1

Sanjeevak 2

Sanjeevak 3

Sanjeevak 4

Sanjeevak 5

Panchagavya Amritpani

(a) (b)

Cow dung 10 kg 10 kg 60 kg 10 kg 15 kg* 10 kg 20 kg 10 kg

Cow urine 101 51 51 51 151* 101 201 -

Jaggary 250 gm 250 gm 250 gm 1 kg 500 gm 1 kg - -

Flour of pulses - - - 1 kg - 2 kg - -

Ant hill soil - - - - - 1 kg - -

Cow ghee - - - - - - 250 gm 250 gm

Cow milk - - - - - - 250 ml -

Cow curd - - - - - - 250 gm -

Honey - - - - - - - 500 gm

Water 101 101 101 101 151 101 101 2001

200 ml distilled water and titrated against 1N ferrous ammonium sulphate solution. Total nitrogen was esti-mated by macro Kjeldhal acid digestion method using Bal’s modifi cation as described by Piper (1942). The sample was digested with salicylic acid, copper sulphate, potassium sulphate and conc. H2SO4. The digested mix-ture was distilled with 40% NaOH in boric acid solu-tion added with methyl red indicator. The distillate was then titrated with 0.1 N NaOH solutions. Percentage of available nitrogen was estimated by alkaline permanga-nate method as described by Jackson (1973). Total phos-phorus was determined by di-acid extraction of sample. The extract was used for colorimetric determination by amminomolybdate netavanalate yellow colour method using spectrophotometer. Available phosphorus was estimated by Olsen’s method using 0.5 N sodium bicar-bonate extractant of pH 8.5 as described by Jackson (1973). Di-acid extract was used to estimate total potas-sium using fl ame photometer as described by Jackson (1973). The neutral ammonium acetate extract was used for determination of available potassium by fl ame pho-tometer as described by Jackson (1973).

MICROBIAL COMMUNITY COMPOSITION

The samples kept in freezer were analyzed for benefi cial microbial population. Total bacterial count was deter-mined in nutrient agar medium, total fungal count was determined in potato dextrose agar, Actinomycetes count in Starch casein agar, Azotobacter count was determined in Jenson’s medium, Pseudomonas fl uorescence count in modifi ed King B medium and Phosphate solubilizing bacteria count in Pikovsky’s medium (Alef, 1995).

ENZYME ANALYSIS

The samples kept in freezer were analyzed for estimation of amount of extracelluar enzymes. The enzyme dehy-drogenase was assayed by method described by Len-hard (1956) (Anonymous, 2003). The enzyme acid and alkaline phosphatase were assayed by method described by Tabatabai and Bremnei (1969). Phosphatase activ-ity in the soil was determined as follows (Tabatabai and Bremner 1969): the sample was incubated for 2 hours at 25°C in the solution of p-nitrophenylphosphate (Fluka) (1:1 w/v) with added buffer [0.1M TRIS = hydroxymeth-ylamino- methan solution (Merck)]. In order to deter-mine the acidic and alkaline phosphatase, the pH of the buffer was adjusted using concentrated HCl and NaOH, respectively. The formed p-nitro- phenol was determined spectrophotometrically at = 400 nm.

For assay of enzyme polyphenol oxidase the enzyme extract was prepared by homogenizing 0.5 ml of liquid manure in 2.0ml of the extraction medium containing tris HCl, sorbitol and NaCl. The homogenate was centri-fuged at 2000g for 10 minutes and the supernatant was used for the assay.

Phosphate buffer (2.5ml) and 0.3ml of catechol solu-tion were added in the cuvette and the spectrophotom-eter was set at 495nm. The enzyme extract (0.2ml) was added and the change in absorbance was recorded for every 30 seconds up to 5 minutes in a spectrophotom-eter (Esterbauer., 1977).

For assay of enzyme peroxidase a 20% homogenate was prepared in 0.1M phosphate buffer (pH 6.5) with liquid manures. To 3.0ml of pyrogallol solution, 0.1ml of the enzyme extract was added and the spectropho-tometer was adjusted to read zero at 430 nm. To the

EFFECT OF DIFFERENT FORMULATIONS OF LIQUID MANURES ON THE BIODIVERSITY OF BENEFICIAL MICROBES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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TABLE 3: Microbial analysis of liquid manures.

S. No. Treatment Total bacterial CFU / ml

Total fungal CFU / ml

Actinomycetes CFU / ml

Azotobacter CFU / ml

Psudomonas fl uorescence

CFU / ml

PSMCFU /ml

01. Sanjeevak 1 3.0 x 107 1.03 x 106 2.00 x 106 1.21 x 106 9.8 x 102 1.13 x 104

02. Sanjeevak 2 (a) 12.6 x 107 1.46 x 106 2.85 x 106 1.32 x 106 11.1 x 102 1.71 x 104

03. Sanjeevak 2(b) 12.1 x 107 1.11 x 106 2.18 x 106 1.16 x 106 21.1 x 102 1.11 x 104




06. Panchagavya 15.1 x 107 25.0 x 106 2.13 x 106 1.31 x 106 9.6 x 102 20.8 x 104

07. Amritpani 10.3 x 107 0.112 x 106 < 30 colonies < 30 colonies < 30 colonies < 30 colonies

test cuvette, 0.5ml of H2O2 was added and mixed. The change in absorbance was recorded every 30 seconds upto 3 minutes in a spectrophotometer (Reddy, 1995). was extraction with phosphate buffer at 3000 rpm cen-trifugation speed for 5 minutes and developing colour with the help of pyrogallol and hydrogen peroxide.

SEED GERMINATION TRAILS

The effect of liquid manures on seed germination was studied in different crops like cotton, pigeon, soya bean, wheat and chick pea. The seed germination was carried out in micro-humid condition using fi lter papers placed in petri-dishes and moistened, then laden with counted 100 number of soaked seeds and incubated at 32oC 0.5oC for overnight (Abdul-Baki, 1973).

RESULTS AND DISCUSSION

Sanjeevak 5 sample, although was in acidic range, had moderate levels of carbon, nitrogen and phosphorus

TABLE 2: Chemical analysis of some plant nutrients.

S. No. Treatment pH Total carbon(%)

Total nitrogen(%)

Available nitrogen (%)

Total phosphorus (%)

Total potassium (%)

01. Sanjeevak 1 7.91 0.0030 0.0167 0.0084 0.0378 0.0047

02. Sanjeevak 2 (a) 7.83 0.0028 0.0088 0.0068 0.0348 0.0042

03. Sanjeevak 2(b) 7.75 0.0190 0.0070 0.0035 0.0394 0.0050

03. Sanjeevak 3 6.95 0.0024 0.0070 0.0056 0.0434 0.0006

04. Sanjeevak 4 8.21 0.0027 0.0098 0.0069 0.0360 0.0026

05. Sanjeevak 5 6.75 0.0041 0.0111 0.0098 0.0338 0.0081

06. Panchagavya 7.78 0.0046 0.0085 0.0069 0.0426 0.0046

07. Amritpani 5.38 0.0032 0.0078 0.0058 0.0398 0.0038

compared to that of other OGPs; it showed highest con-tent of potassium and available nitrogen (Table - 2).

It was proved that spring barley yield was positively correlated with the activity of the enzymes tested i.e. dehydrogenase, ureases, alkaline phosphatase and acid phosphatase (Jastrazzebska, 2007). Sanjeevak 5 and Panchagavya samples noted to contain highest amount of enzyme dehydrogenase, acid phosphatase and per-oxidase. Sanjeevak 5 also contain highest amount of enzyme polyphenol oxidase (Table - 4).

Alkaline phosphates hydrolyses P from organic com-pounds. It is produced by soil fungi and bacteria (Dick, 2005). Among all the OGPs under study Panchagavya sample was having highest total microbial, fungal and PSM growth closely followed by Sanjeevak 5. The high-est Azotobacter population was found in Sanjeevak 5 sample followed closely by Sanjeevak 4 sample. Highest Psed, fl uoresces count was found in T2(b) formulation (Table 3).

Many studies have showed that dairy slurry and application of organic manure helps to improve soil health and crop production (Stanford, 2009; Grigera


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bial biomass and nutrient status might have resulted in improved seed germination, seedling length and seed vigour in soybean indicating beejamrutha as an effi cient plant growth stimulant (Sreenivasa, 2009).

Our fi ndings on seed germination trials are in agree-ment with these results. In case of soybean highest increase in germination percentage and maximum root growth was noted in T6 treatment followed by that of T3 treatment. T4 and T1 treatments had an adverse effect on seed germination. In case of cotton the highest increase in germination and highest root growth were found with treatment of T5 sample followed by that of T4. In case of pigeon pea highest germination percentage increase was

2006) The results of soil application of liquid manures showed that Panchagavya treatment was more favorable for the proper growth of Actinomycetes and PSM, while Amritpani treatment was seen to be favored by microbes like Azotobacter and Pseudomonas closely followed by Sanjeevak 5 treatments, (Table – 5).

The study on beejamrutha (prepared using the ingre-dients viz cow dung, cow urine, water and lime) con-tains not only general microfl ora, but also certain ben-efi cial biochemical groups such as free living N2-fi xers, P- solubilizers and bacteria producing plant growth pro-moting substances as well as bacteria having biological deterrent activities. Presence of such benefi cial micro-

TABLE 4: Microbial analysis of liquid manures.

S. No. Formulations Dehydrogenase(ppm)

Acid phosphate(ppm)

Alkaline phosphates (ppm)

Polyphenol oxidase (ppm)

Peroxidase(ppm)

01. Sanjeevak 1 24.28 751.22 755.91 56.51 59.23

02. Sanjeevak 2 (a) 24.41 752.21 763.60 32.93 55.44

03. Sanjeevak 2(b) 24.48 751.70 752.65 63.67 45.76

03. Sanjeevak 3 23.28 752.50 763.31 51.15 52.86

04. Sanjeevak 4 24.70 753.41 756.43 118.47 52.00

05. Sanjeevak 5 33.34 754.93 756.78 144.81 60.49

06. Panchagavya 33.70 753.51 756.66 84.49 60.95

07. Amritpani 15.63 753.59 748.75 65.615 24.56

Table 5: Example of soyabean plot soil samples to illustrate the improvement in soil charatarstics.

Parameter Control Chemical Sanjeevak 5 Panchagavya Amritpani

Actinomycetes count CFU / ml 1.13 x 103 2.10 x 103 1.15 x 104 1.92 x 105 1.04 x 104

Azotobacter count CFU / ml 4.5 x 104 4.0 x 103 1.89 x 104 2.12 x 105 2.21 x 105

Pseudomonas Flurosecence count CFU / ml

4.5 x 101 4.0 x 101 2.11 x 102 1.38 x 102 2.17 x 103

PSM count CFU / ml 3.3 x 101 3.1 x 101 2.41 x 102 2.14 x 103 2.05 x 102

TABLE 6: Seed germination trials with organic growth promoters for Soybean Variety: 335.

S. No. Treatment Duration of germination Germination % Avg. length of roots % Increase over control

01. Control 3 days 46 2.8 cm -

02. T1 3 days 58 1.36 cm -

03. T2 3 days 65 3.24 cm 15.71

04. T3 3 days 73 3.92 cm 28.57

05. T4 3 days 34 2.32 cm -

06. T5 3 days 75 3.44 cm 22.86

07. T6 3 days 78 4.02 cm 43.57

08. T7 3 days 69 3.44 cm 22.86

EFFECT OF DIFFERENT FORMULATIONS OF LIQUID MANURES ON THE BIODIVERSITY OF BENEFICIAL MICROBES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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found in T1 treatment followed by T5 and T2 treatments. But maximum root growth was noted in T5 treatment followed by T2 treatment. Hence, T5 and T2 treatments were considered to have positive impact on seed germi-nation and root growth. T6 and T3 treatments showed an adverse effect on seed germination. Maximum root growth was observed in T7 (Amrutpani) treatment for Bengal gram followed by T2 treatment. T6 treatment

had an adverse effect on seed germination. In case of wheat crop, seed germination was not a problem. The maximum root growth was found in T5 treatment fol-lowed by T6 and T4 treatment. Even if the root growth was noted to be equal in both T6 and T4 treatment, T6 treatment showed more number of roots (Tables 6 to 9).

Photographs of seed germination trials with OGPs for soyabean:

TABLE 7: Seed germination trials with organic growth promoters for cotton Variety: Straight line – Shubhra.



02. T1 3 days 85 4.64 cm 31.47

03. T2 3 days 52 3.46 cm 8.81

04. T3 3 days 86 4.96 cm 55.97

05. T4 3 days 87 5.95 cm 87.11

06. T5 3 days 92 6.47 cm 103.46

07. T6 3 days 71 4.48 cm 40.88

08. T7 3 days 73 5.48 cm 72.33

Photographs of seed germination trials with OGPs for cotton: BBRC

24 EFFECT OF DIFFERENT FORMULATIONS OF LIQUID MANURES ON THE BIODIVERSITY OF BENEFICIAL MICROBES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS


TABLE 8: Seed germination trials with organic growth promoters for Pigeon pea Variety: Straight line - Maroti.



02. T1 3 days 95 2.06 cm 22.62

03. T2 3 days 92 2.58 cm 53.57

04. T3 3 days 07 0.25 cm -

05. T4 3 days 88 2.44 cm 45.24

06. T5 3 days 94 2.74 cm 63.10

07. T6 3 days 81 0.8 cm -

08. T7 3 days 78 1.92 cm 14.29

Photographs of seed germination trials with OGPs for pigeon pea:

TABLE 9: Seed germination trials with organic growth promoters for Bengal gram Variety: Vijay.



02. T1 3 days 99 2.87 cm 21.61

03. T2 3 days 99 5.86 cm 148.31

04. T3 3 days 99 4.78 cm 80.93

05. T4 3 days 99 3.16 cm 12.29

06. T5 3 days 34 2.72 cm 15.25

07. T6 3 days 13 0.37 cm -

08. T7 3 days 99 7.8 cm 230.51

Photographs of seed germination trials with OGPs for Bengal gram: BBRC



Photographs of seed germination trials with OGPs for Wheat:

TABLE 10: Seed germination trials with organic growth promoters for wheat Variety: Lok-1.

S. No. Treatment Duration of germination Germination % Avg. length of roots

% Increase over control

01. Control 3 days 100 5 9.06 cm -

02. T1 3 days 100 5 10.16 cm 33.68

03. T2 3 days 100 5 9.32 cm 22.63

04. T3 3 days 100 5 9.74 cm 28.16

05. T4 3 days 100 5 10.46 cm 37.63

06. T5 3 days 100 6 12.62 cm 66.05

07. T6 3 days 100 6 10.46 cm 37.63

08. T7 3 days 100 5 7.6 cm -

CONCLUSION

Our study clearly indicated that Sanjeevak 5 and Pan-chagavya are the best formulations of liquid manure showing highest microbial count and presence of high amount of extracellular enzymes. Among all the liquid manures studied for their effect on root growth dur-ing seed germination for different crops, a treatment of Sanjeevak 5 was noted to have very pronounced effect on root growth in case of crops like cotton, pigeon pea and wheat followed by the treatments of Panchaga-vya and Amritpani for corps like soy bean and Bengal gram, respectively. Panchagavya treatment was seen to be more favorable for the proper growth of Actinomyc-etes and PSM in the soil, while Amritpani treatment was seen to be favored by microbes like Azotobacter and Pseudomonas closely followed by Sanjeevak 5 treat-ment Among all the OGPs studied for their effect on seed germination and root growth for different crops, T5 treatment was noted to have pronounced effect on root growth in case of crops like cotton, pigeon pea and wheat while better growth observed in the ORG treat-ments T6 and T7 for corps like cotton and Bengal gram, respectively.

Now the soil application trials with these liquid manures has been undertaken to understand the effect on productivity of the cropa. Similarly to understand the shelf life of these liquid manures study has been undertaken. Scanning for growth of any plant path-ogenic microorganism needs to done for these liquid manures.

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