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Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers

Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora... Annals of Microbiology, 57 (2) 177-183 (2007) Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers 1 1 1 2 3 1 1 Ghulam JILANI *, Abida AKRAM , Raja M. ALI , Fauzia Y. HAFEEZ , Imran H. SHAMSI , Arshad N. CHAUDHRY , Abid G. CHAUDHRY 1 2 University of Arid Agriculture, Rawalpindi 46300, Pakistan; National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan; College of Agriculture & Biotechnology, Zhejiang University, Hangzhou, China Received 5 December 2006 / Accepted 16 April 2007 Abstract - Field experiment was conducted on fodder maize to explore the potential of integrated use of chemical, organic and biofer- tilizers for improving maize growth, beneficial microflora in the rhizosphere and the economic returns. The treatments were designed to make comparison of NPK fertilizer with different combinations of half dose of NP with organic and biofertilizers viz. biological potas- sium fertilizer (BPF), Biopower, effective microorganisms (EM) and green force compost (GFC). Data reflected maximum crop growth in terms of plant height, leaf area and fresh biomass with the treatment of full NPK; and it was followed by BPF+full NP. The highest uptake of NPK nutrients by crop was recorded as: N under half NP+Biopower; P in BPF+full NP; and K from full NPK. The rhizosphere microflora enumeration revealed that Biopower+EM applied along with half dose of GFC soil conditioner (SC) or NP fertilizer gave the highest count of N-fixing bacteria (Azotobacter, Azospirillum, Azoarcus and Zoogloea). Regarding the P-solubilizing bacteria, Bacillus was having maximum population with Biopower+BPF+half NP, and Pseudomonas under Biopower+EM+half NP treatment. It was con- cluded that integration of half dose of NP fertilizer with Biopower+BPF / EM can give similar crop yield as with full rate of NP fertiliz- er; and through reduced use of fertilizers the production cost is minimized and the net return maximized. However, the integration of half dose of NP fertilizer with biofertilizers and compost did not give maize fodder growth and yield comparable to that from full dose of NPK fertilizers. Key words: N-fixation, diazotrophs, P-solubilizing bacteria, nutrients uptake, economics, biofertilizers. INTRODUCTION Organic materials hold great promise due to their local availability as a source of multiple nutrients and ability to Maize (Zea mays L.) is the most important cereal fodder improve soil characteristics (Khaliq et al., 2006). The and grain crop under both irrigated and rainfed agricultur- improvement of fertility and quality of soil, especially under al systems in the semi-arid and arid tropics (Hussan et al., low input agricultural systems, requires the input of organ- 2003). Green fodder has an immense importance for ani- ic materials (Soumare et al., 2003; Naureen et al., 2005). mal production and development. At present for 21 million Biofertilizers contain different types of microorganisms, heads of livestock in Pakistan, only 51% feed requirements which have an ability to convert nutritionally important ele- of these animals can be met from green fodder. There is ments from unavailable to available form through biologi- still a shortage of 26 million tonne of total digestible nutri- cal processes (Hedge et al., 1999; Vessey, 2003). They ents, and in terms of digestible protein shortage is 1.58 have emerged as an important component of the integrat- million tonne. So, there is a need of 150 million tonne ed nutrient supply system and hold a great promise to green fodder to feed the animals and meet their nutrients improve crop yields. Biofertilizers containing N-fixer requirement (Haqqani et al., 2003). (Azotobacter chroococcum), P-solubilizer (Bacillus mega- Traditional use of chemicals fertilizers in agricultural terium) and K-solubilizer (Bacillus mucilaginous) and production cannot be over-emphasized, but with fertilizer arbuscular mycorrhizal fungi (Glomus mosseae and Glomus costs going up, these need to be supplemented or substi- intraradices) have been developed (Hafeez et al., 2002; tuted with available organic or biofertilizers. Thus integrat- Wu et al., 2005). The P-solubilizing microorganisms (PSM) ed plant nutrients system was introduced which includes render insoluble phosphate into soluble form through the application of organic and biofertilizers to supplement process of acidification, chelation and exchange reactions chemical fertilizers to maintain and increase the soil fertili- (Gull et al., 2004; Son et al., 2006). Therefore, many ty for sustaining increased crop production. It helps for researchers have tried to increase the plant-available phos- making the highly productive cereals mono cropping sys- phate-fraction by means of PSM such as Bacillus (Raj et al., tems more sustainable (Rajendra et al., 1998). 1981), Enterobacter (Laheurte and Berthelin, 1988), Pseudomonas (Suh et al., 1995a, 1995b), Agrobacterium, Aspergillus (Varsha and Patel, 2000; Hameed et al., 2004) * Corresponding author. Phone: 0092-51-9062241; and Pantoea agglomerans (Son et al., 2006). Fax: 0092-51-9290160; E-mail: jilani62@yahoo.com 178 G. Jilani et al. The product of EM (effective microorganisms) contains T BPF as seed coating + full dose of NP fertilizer selected species of microbes including predominant popu- T Biopower as seed coating + half dose of NP fertilizer lations of lactic acid bacteria and yeasts, and smaller num- T Biopower + BPF + half dose of NP fertilizer bers of photosynthetic bacteria, actinomycetes and other T EM (1% solution) for seed soaking, and broadcasted -1 types of organisms (Higa and Wididana, 1991). All of these as soil mixed to 10 L EM with 100 kg soil ha + half are mutually compatible with one another and can coexist dose of NP fertilizer in liquid culture (Higa and Parr, 1994). A latest study indi- T Biopower + EM as in T + half dose of NP fertilizer 8 7 -1 cated that application of EM to cotton increased the effi- T Full dose of GFC (NPK), 250 kg ha -1 ciency of both organic and mineral nutrient sources (Khaliq T Full dose of GFC (SC), 500 kg ha -1 et al., 2006). Economic analysis suggested that the use of T Half dose of GFC (SC), 250 kg ha + Biopower as in 1/2 mineral NPK with EM+OM (organic matter) saved the T -1 mineral N fertilizer by almost 50% compared to a system T Half dose of GFC (SC), 250 kg ha + Biopower + EM with only mineral NPK application. as in T Currently, the need is to get maximum output with min- Each treatment plot size was 7 m x 5 m, and maize vari- imum cost, which is possible only if chemical fertilizers are ety Agaiti-85 (local name meaning the variety to be grown supplemented with organic and biofertilizers. The present in early season) was grown as test crop. The seed was -1 study was, therefore, envisaged with the objective to eval- sown at the rate of 50 kg ha with the help of hand drill on st uate the comparative effect of chemical, organic and 1 August, 2004. The treatment effect was recorded on the th biofertilizers on the growth, yield and nutrients uptake of growth parameters and fodder yield of crop at 60 day maize fodder. The interactions among the microorganisms after sowing. of different microbial products and fertilizers were also Physical and chemical properties of composite soil sam- investigated. ples taken before sowing from the experimental field were determined. The soil was sandy loam in texture, had pH -1 7.3, electrical conductivity 0.30 dS m , organic matter -1 -1 MATERIALS AND METHODS 0.43%, NO -N 1.2 mg kg , available-P 5.2 mg kg , and -1 extractable-K 78.5 mg kg . The plant samples taken at Comparative study of organic and biofertilizers with chem- harvest were analysed for NPK contents and their uptake ical fertilizer was carried out for maize fodder production was calculated by employing the following formula: -1 under rainfed conditions at the research farm of University Nutrients uptake (kg ha ) = -1 of Arid Agriculture, Rawalpindi, Pakistan during the sum- (Nutrient content in plants as %) (Dry yield as kg ha ) mer season of year 2003. The biofertilizers used for seed 100 dressing were: Biological Potassium Fertilizer (BPF, 10 kg –1 -1 ha ), Biopower (2.5 kg ha ) and Effective Microorganisms Isolation of microorganisms. Rhizosphere microorgan- -1 (EM, 2.5 L ha ). While the organic fertilizers were Green isms were isolated from each sample by serial dilution and Force Compost (GFC) enriched with NPK viz. GFC (NPK, spread plate method. One gram soil was dispersed in 10 mL -1 250 kg ha ) and Green Force Compost as soil conditioner of sterile distilled water and thoroughly shaken. One mL of -1 GFC (SC, 500 kg ha ). The nature and composition of above dilution was again transferred to 9 mL of sterile dis- -2 -3 -4 -5 these experimental materials is given under: tilled water to form 10 dilution. Similarly, 10 , 10 , 10 BPF is a biofertilizer inoculum containing K-solubilizing bac- dilutions were made for each soil sample. An aliquot of 0.1 teria Bacillus mucilaginous. mL from each dilution was taken with micropipette (0.01- Biopower is a biofertilizer containing four N-fixing bacteria 0.1 mL range) and plated on agar medium specific for each viz. Azotobacter, Azospirillum, Azoarcus and Zoogloea. microorganism to be isolated. After incubation total num- EM is a mixed inoculum containing mainly Lactobacillus, ber of colonies formed was counted with the help of digital Rhodopseudomonas, Actinomycetes and yeast. colony counter. Total count was determined as colony GFC (NPK) is composted municipal waste enriched with forming units (CFU): -1 NPK fertilizer to have 15-15-15 percent NPK contents. CFU g of moist soil = Mean plate count / (Volume of Green Force Compost (GFC) is a commercial product. sample plated) (Dilution factor) -1 -1 GFC (SC) is composted municipal waste having NPK con- CFU g of dry soil = CFU g of moist soil (100 + percent tents as 3.0-1.2-1.8 percent. This GFC is a soil conditioner moisture in sample / 100) (SC) product. The following N -fixing and P-solubilizing bacteria main- The experiment was laid out in a randomized complete ly found in the rhizosphere were enumerated by using spe- block design with four replications. Urea, single super phos- cific growth media for each species as suggested by phate and sulphate of potash were used as source of NPK, authors quoted against each microorganism. respectively. All the fertilizers were applied in the field Azotobacter: Burk’s medium (Cornish and Page, 2000) -1 o before maize sowing. Soil mixed with EM (10 L ha ) was incubated at 26 C for three days. broadcasted in T , T , and T . The experiment consisted of Azospirillum: Okon medium (Okon et al., 1976) incubated 7 8 12 the following treatments: at 35 C for 2 days. -1 T Full dose of NP fertilizers, 120-90 kg ha , Azoarcus: Jenson nitrogen free biotin medium (Döbereiner, respectively 1995) incubated at 30 C for three days until colour of -1 T Full dose of NPK fertilizers, 120-90-60 kg ha , media changed to blue then these were counted. respectively Zoogloea: Combined carbon medium formulated by Rennie -1 o T Half dose of NP fertilizers, 60-45 kg ha , (1981) incubated at 30 C for 2 days. respectively Bacillus and Pseudomonas: Both these species of P-solubi- lizing bacteria were isolated on Pikovskaya’s agar medium Ann. Microbiol., 57 (2), 177-183 (2007) 179 (Pikovskaya, 1948) containing insoluble tricalcium phos- niques including analysis of variance (ANOVA), and the phate. The plates were incubated at 26 C for 7 days. After treatments means were compared by application of the growth, the colonies of Bacillus and Pseudomonas were Duncan’s multiple range test (Sokal and Rohlf, 1997). isolated and re-cultured on nutrient agar media. The Pseudomonas was incubated at 20 C for 24 hours, while Bacillus at 37 C for 48 hours. RESULTS Purification of microorganisms. Colonies developed on Biometric parameters each specified medium were picked with the help of ster- Vegetative plant growth parameters like plant height, leaf ilised inoculating wire loop and streaked on separate plate. area and biomass yield were measured before harvesting All plates were then incubated at same temperature and for the maize fodder as given in Table 1. Data show that NPK time period as before indicated for each microorganism. fertilizers gave the highest values for these parameters, which had statistically significant difference with the plants Morphological characterisation of microorganisms. from other treatments. It was followed by BPF+full NP treat- Microorganisms of purified colonies were studied for 5 dif- ment which had statistically non significant difference with ferent morphological characters stereomicroscopic feature, full dose of NP fertilizer. The biofertilizers when combined colour, margin of colony, surface form, surface texture, ele- individually with half dose of NP fertilizer could not give vation (Cruickshank et al., 1975). Microorganisms were plant growth similar to full NP or NPK fertilizer. All these identified by Gram staining (Murray et al., 1994). biofertilizers were similar in their effect on plant growth. However, when two biofertilizers were applied together with Biochemical tests for microorganisms. Biochemical half NP fertilizer, they could produce plants with height sta- tests for microorganisms like catalase production, oxidase tistically similar to full dose of NP fertilizer. Thus treatments production, nitrate reduction, triple sugar iron test, and of Biopower+BPF+half NP and Biopower+EM+half NP were sugar fermentation were also performed. All procedures for not significantly different from full NP. Biopower or EM in biochemical tests were taken from Handbook of combination with half NP fertilizer was statistically superior Microbiology (Bisen and Verma, 1994). to only half dose of NP fertilizer. All GFC treatments had lower values than other treatments. Half GFC Economic analysis. Procedures for economic analysis (SC)+Biopower+EM used in combination had not signifi- were adopted from CIMMYT (1988). Expenditures incurred cantly higher value than other GFC treatments. Full GFC -1 on various fertilizing materials and incomes from the (SC) (500 kg ha ) gave the lowest fresh fodder yield among respective treatments were calculated on the basis of mar- all the bio, organic and chemical fertilizer treatments. The ket rates at the time of experiment. Further, the net return plant growth with half GFC (SC)+Biopower+EM was, how- was obtained by finding the difference between total ever, statistically equal to that with half dose of NP fertiliz- income and total expenditure of each treatment. Finally, er. the value to cost ratio (VCR) was calculated for each treat- ment through this formula: Nutrients uptake Expenditure on plant nutrients Nitrogen uptake by crop is shown in Table 2; it had statis- Value cost ratio = ————————————————— tically different values for various treatments. The highest Total income N uptake was observed in full NPK fertilizer treatment -1 (45.0 kg ha ) which was followed by BPF+full NP (40.7 kg -1 Statistical analysis. The data collected for various char- ha ) with a significant difference. Full NP fertilizer occu- acteristics were analysed by application of statistical tech- pied the third position; however it had statistically non sig- TABLE 1 - Comparison of organic, bio and chemical fertilizers for growth and yield of maize fodder Treatments description Plant height Leaf area Fodder yield 2 -1 (cm) (cm ) (kg ha ) -1 c b b T Full NP (120-90 kg ha ) 163 * 456 * 8732 * -1 a a a T Full NPK (120-90-60 kg ha ) 175 472 9540 -1 ef e d T Half NP (60-45 kg ha ) 150 420 7532 b b ab T BPF+full NP 167 461 9170 de d c T Biopower+half NP 155 441 8156 cd bc bc T Biopower+BPF+half NP 158 451 8528 de e c T EM+half NP 155 428 8264 cd cd bc T Biopower+EM+half NP 159 445 8572 -1 g f d T Full GFC (NPK) (250 kg ha ) 143 395 7620 -1 h h e T Full GFC (SC) (500 kg ha ) 132 370 6950 g g d T Half GFC (SC)+Biopower 140 384 7508 fg fg cd T Half GFC (SC)+Biopower+EM 145 389 8026 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05. 180 G. Jilani et al. TABLE 2 - Comparison of organic, bio and chemical fertilizers for nutrients uptake by maize fodder -1 Treatments description Nutrients uptake (kg ha ) Nitrogen Phosphorus Potassium -1 c b d T Full NP (120-90 kg ha ) 36.6*7.29 * 30.9 * -1 a a a T Full NPK (120-90-60 kg ha ) 45.0 7.99 45.2 -1 e e de T Half NP (60-45 kg ha ) 26.7 4.42 28.6 b a b T BPF+full NP 40.7 8.28 40.5 d d cd T Biopower+half NP 30.6 5.28 31.5 cd c cd T Biopower+BPF+half NP 33.7 6.32 34.9 d cd cd T EM+half NP 31.8 5.73 32.9 cd c c T Biopower+EM+half NP 34.2 6.15 35.4 -1 e d d T Full GFC (NPK, 250 kg ha ) 25.9 5.21 30.4 -1 f f e T Full GFC (SC, 500 kg ha ) 22.0 3.61 24.5 ef ef de T Half GFC (SC)+Biopower 24.8 4.11 27.4 de de cd T Half GFC (SC)+Biopower+EM 29.2 4.81 31.3 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05 nificant difference with Biopower+EM+half NP and Biopower+EM+half NP. These were also having the lowest Biopower+BPF+half NP. Generally, non-significant differ- population in chemical fertilizer treatments. The number of ences were observed among the GFC treatments except other two diazotrophs Azoarcus and Zoogloea was also half GFC (SC)+Biopower+EM under which the N uptake higher in the treatments receiving Biopower alone or in -1 was 29.2 kg ha being superior to other GFC treatments. combination especially with EM. The biofertilizers gave The highest values of phosphorus uptake (8.28 and higher number of rhizosphere microflora in the treatments -1 7.99 kg ha ) were recorded in BPF+full NP and full NPK where organic fertilizers were also applied. Regarding the treatment, respectively with non significant difference phosphate solubilizing bacteria Bacillus and Pseudomonas, (Table 2). These were followed by full NP treatment show- their count was statistically higher with the treatments -1 ing 7.29 kg ha P uptake. The treatments (T and T ) in Biopower+BPF+half NP, and Biopower+EM+half NP, 6 8 which bio-fertilizers were used in combination showed non- respectively. The application of BPF either alone or in com- significant difference between themselves. They had signif- bination with other biofertilizers resulted into higher num- icantly higher values than all the GFC treatments. Among ber of Bacillus in the rhizosphere of maize. Contrasting to all GFC treatments, half GFC (SC)+Biopower+EM treat- diazotrophs, the populations of P solubilizers were higher in ment had higher value of phosphorus uptake than others. chemical fertilizer treatments as compared to organic ones. The maximum potassium uptake in maize fodder crop -1 was 45.2 kg ha , obtained from full NPK (Table 2). The Economic analysis BPF+full NP treatment showed significantly lower value The economic analysis on the performance of organic and -1 (40.5 kg ha ) than full NPK but higher than full NP. Full biofertilizers in comparison with chemical fertilizers for maize GFC (NPK) treatment resulted in similar K uptake as with fodder production was performed as in Table 4. The per full NP, however they were inferior to Biopower and EM hectare expenditure on various treatments showed the high- -1 treatments (T5-T8) either used alone or combined. Non- est value of US$ 121.0 for full NPK (120-90-60 kg ha ) fol- significant differences were observed among half NP and all lowed by BPF+full NP (US$ 89.7). The lowest expenses were GFC treatments. for half GFC (SC)+Biopower (US$ 37.5), half NP fertilizer (US$ 41.5) and half GFC (SC)+Biopower+EM (US$ 41.7). Rhizosphere microflora The maximum income (US$ 318.0) was also from full NPK Diazotrophic bacteria namely Azotobacter, Azospirillum, fertilizer as it yielded the highest fresh fodder biomass, and it Azoarcus and Zoogloea, and phosphate solubilizing bacte- was followed by BPF+full NP (US$ 305.7). The lowest income ria Bacillus and Pseudomonas were enumerated from the was obtained from full GFC (SC) treatment (US$ 231.7). The maize plants rhizosphere at active growth stage viz. 60 net profit was maximum with Biopower+EM+half NP (US$ days after sowing. Data on the count of Azotobacter (Table 235.9) and Biopower+BPF+half NP (US$ 231.9), whereas, -1 3) showed that their population was highest in the treat- full GFC (SC) (500 kg ha ) gave the lowest net profit (US$ ment of half GFC (SC)+Biopower+EM, which differed not 165.0). The value (total income) to cost (total expenditure) significantly with that of half GFC (SC)+Biopower. The low- ratio (VCR) was also determined to compare various treat- est count was observed in NP and NPK fertilizer treatments. ments (Table 4). Data showed that full NPK fertilizer gave the The treatments receiving any of the biofertilizer Biopower, lowest VCR (2.63), which was followed by BPF+full NP (3.41). BPF or EM singly or in combination with each other caused The highest VCR values were obtained from half GFC the establishment of higher populations of Azotobater in (SC)+Biopower (6.67) and half GFC (SC)+Biopower+EM maize rhizosphere. Azospirillum count in the rhizosphere (6.42). All the biofertilizer treatments had intermediate VCR was also maximum with Half GFC (SC)+Biopower+EM, figures showing better economic impact than the chemical which had no statistical difference with that in fertilizers used at full or half application rate. Ann. Microbiol., 57 (2), 177-183 (2007) 181 TABLE 3 - Comparison of organic, bio and chemical fertilizers for count of N-fixing and P-solubilizing microflora in maize rhizosphere -1 (log CFU g dry soil). Treatments Azotobacter Azospirillum Azoarcu Zoogloea Bacillus Pseudomonas -1 f d de d de d T1 Full NP (120-90 kg ha)5.27 4.41 3.44 4.09 3.64 3.58 -1 f d d d cd cd T2 Full NPK (120-90-60 kg ha)5.18 4.47 3.46 4.13 3.69 3.64 -1 ef d d cd ef e T3 Half NP (60-45 kg ha)5.38 4.51 3.34 4.16 3.54 3.42 e d cd cd ab c T4 BPF+full NP 5.40 4.44 3.55 4.14 3.87 3.69 c b b b c d T5 Biopower+1/2 NP 5.61 4.74 3.69 4.61 3.73 3.57 bc b ab b a ab T6 Biopower+BPF+1/2 NP 5.66 4.76 3.72 4.64 3.89 3.79 d c cd c b b T7 EM+half NP 5.50 4.66 3.56 4.28 3.81 3.76 b ab ab ab b a T8 Biopower+EM+1/2 NP 5.68 4.80 3.74 4.69 3.83 3.83 -1 e d d cd f e T9 Full GFC (NPK, 250 kg ha)5.39 4.50 3.50 4.21 3.42 3.33 -1 d cd c c e e T10 Full GFC (SC, 500 kg ha)5.51 4.60 3.58 4.29 3.55 3.40 ab b a ab ef d T11 1/2 GFC (SC)+Biopower 5.69 4.79 3.75 4.67 3.53 3.58 a a a a d cd T12 1/2 GFC (SC)+Biopower+EM 5.73 4.84 3.77 4.70 3.65 3.64 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05 TABLE 4 - Economic comparison of organic, bio and chemical fertilizers use for maize fodder production 1 2 3 Treatments Expenditure Fodder Yield Income Net Return VCR -1 -1 -1 -1 (US$ ha )(t ha )(US$ ha )(US$ ha ) -1 T1 Full NP (120-90 kg ha ) 83.0 8.73 291.1 208.1 3.51 -1 T2 Full NPK (120-90-60 kg ha ) 121.0 9.54 318.0 197.0 2.63 -1 T3 Half NP (60-45 kg ha ) 41.5 7.53 251.1 209.6 6.05 T4 BPF+full NP 89.7 9.17 305.7 216.0 3.41 T5 Biopower+1/2 NP 45.7 8.16 271.9 226.2 5.95 T6 Biopower+BPF+1/2 NP 52. 3 8.53 284.3 231.9 5.43 T7 EM+half NP 45. 7 8.26 275.5 229.8 6.03 T8 Biopower+EM+1/2 NP 49.8 8.57 285.7 235.9 5.73 -1 T9 Full GFC (NPK, 250 kg ha ) 62.5 7.62 254.0 191.5 4.06 -1 T10 Full GFC (SC, 500 kg ha ) 66. 7 6.95 231.7 165.0 3.48 T11 1/2 GFC (SC)+Biopower 37.5 7.51 250.3 212.8 6.67 T12 1/2 GFC (SC)+Biopower + EM 41. 7 8.03 267.5 225.9 6.42 1 -1 -1 Expenditure on fertilizer sources: N, P, K (US$ kg ) = 0.32, 0.50, 0.63, respectively (NFDC, 2004); BPF (US$ 0.67 kg ); Biopower -1 -1 -1 -1 (US$ 1.67 kg ); EM (US$ 0.33 L ; GFC (NPK, US$ 0.25 kg ); GFC (SC, US$ 0.13 kg ). Income of fresh fodder (US$ 33.3 per tonne). VCR, Value cost ratio. DISCUSSION microbes with major groups as lactic acid bacteria, photo- synthetic bacteria, ray fungi and yeast (Higa and This field study was envisaged with the view to find out Wididana, 1991). These microorganisms are well known to some combination of biofertilizers with half dose of chem- have beneficial effect on plant growth and their nutrient ical fertilizer to have the same effect on crop growth as accumulation. Panwar et al. (2000) observed that biofer- with the full dose of NPK chemical fertilizers. The results tilizers increased leaf area, chlorophyll concentration and on fodder yield depicted that BPF+full NP treatment per- total biomass production in wheat. Recently, the concept formed equally good as did the full dose of NPK fertilizers. of microbial consortium in the production and use of Biopower+BPF+half NP and Biopower+EM+half NP treat- biofertilizers is emerging strongly. This is due to the rea- ments gave statistically equal yield as with full dose of NP son that some beneficial microorganism having similar fertilizers, and better than as with half dose of NP fertiliz- ecological requirements flourish in an associative manner er. Khaliq et al. (2006) reported that integrated use of OM (Vessey, 2003). Ponnuswamy et al. (2002) reported that (farm yard manure)+EM with 1/2 mineral NPK yielded a combination of phosphobacteria+Azospirillum had posi- similar to the yield obtained from full recommended NPK, tive effect on yield and yield characters compared to no indicating that this combination can substitute for 85 kg N biofertilizer application and with phosphorus solubilizing -1 ha . The BPF contains potassium solubilizing bacteria bacteria (PSB) or Azospirillum alone. These findings coin- Bacillus mucilaginous, Biopower includes four types of N- cide with results of Nanda et al. (1995) who observed that fixing bacteria, and EM is having a large number of green fodder yield and benefit-cost ratio (VCR) were high- 182 G. Jilani et al. -1 Biopower, as it contained all the four enumerated bacteria est with a combination of 75 kg N ha and seed inocula- -1 which were colonized in the rhizosphere due to their inoc- tion with Azospirillum (22.0 t ha yield and 1.37 VCR) and -1 ulation on the seed. The results exactly coincide with find- lowest in the control (7.6 t ha yield and 0.07 VCR). The ings of Khokar (2004) who conducted research on interac- results of present study are also supported by Rout et al. tion of indigenous Azospirillum with maize and observed (2001) who found that Azotobacter, Azospirillum and their that the highest root colonization was under inoculum combinations gave more yield than untreated maize. They treatments. The application of EM and BPF increased the concluded that biofertilizers in combination with inorganic number of diazotrophs further, although their inoculum did nitrogen fertilizers can substitute up to 20 % nitrogenous not contain any of the enumerated bacteria. This might be fertilizers and can increase maize yield. Similarly, Mokhova due to the positive interactive effect of the microbes in et al. (2000) reported that application of Rhizobacterin these biofertilizers with N-fixing bacteria. These results (inoculum containing mixed culture of N-fixing and P-solu- were in conformity with Mehnaz et al. (1998) who con- bilizing bacteria) resulted in yield increases and required ducted research on detection of inoculated plant growth lower nitrogen fertilizer rates. promoting rhizobacteria in rice and isolated strains belong- The uptake or accumulation of the macronutrients like ing to Azospirillum, Azoarcus, Pseudomonas and Zoogloea. N, P and K is the direct reflection of the biomass produc- They found that the populations of these bacteria as well as tion. The result of this experiment revealed that full dose of that of indigenous ones were more under inoculum treat- NPK caused the maximum uptake of N, P and K nutrients ments as compared to uninoculated. The organic fertilizers by maize fodder, and it was followed by BPF+full NP, even better P uptake than from the chemical fertilizers. were better than chemical fertilizer in terms of increasing Biofertilizer treatments especially those with Biopower the number of N-fixing bacteria in the rhizosphere. These results indicate that microbial population of soil can be improved the NPK uptake over half dose of NP fertilizer. increased by applying organic matter and their effective- This was due to the fact that Biopower contained four dif- ness can be increased by inoculation with biofertilizer. In ferent species of N-fixing bacteria, which resulted into increased availability of nitrogen to the crop. Wu et al. this way availability of nutrients can be increased to plant (2005) indicated that half the amount of biofertilizer appli- and maximum yield can be achieved (Elshanshorey, 1995). cation had similar effects when compared with organic fer- In a previous study by Ali et al. (1998) on the bacterial tilizer or chemical fertilizer treatments. Microbial inoculum counts under farm yard manure and chemical fertilizers treatments, they found that diazotrophs, in presence or not only increased the nutritional assimilation of plants (total N, P and K), but also improved soil properties, such absence of NP fertilizer did not respond significantly to inoc- as organic matter content and total N in soil. Elshanshorey ulation in terms of N content. The count of P solubilizers (1995) also reported that biofertilizers increased nutrient especially that of Bacillus was higher under BPF treatment, concentration and uptake by cereal crops, which lead which was due to the reason that BPF contained Bacillus towards luxurious growth and better crop development. mucilaginous already in the inoculum. Application of EM Combination of N fertilizer with EM also increased the con- also had positive effect to increase the number of phos- centrations of NPK in plants (Khaliq et al., 2006). phorus solubilizing bacteria in the maize rhizosphere. This Phosphorus uptake was highest under BPF+full NP treat- was probably due to the reason that the yeast and photo- ment, even better than full dose of NP or NPK treatments. synthetic bacteria contained in EM released some biochem- In fact BPF has Bacillus mucilaginous bacteria which have ical compounds which enhanced the growth and population solubilizing effect on fixed phosphorus in the soil. Son et al. of N-fixing as well as P-solubilizing bacteria in rhizosphere. (2006) enlisted a number of microorganisms which have Similarly, Ren et al. (2006) reported that a bacterium strain the ability to solubilize phosphorus in the soil and make it B-916 of Bacillus subtilis, caused growth promotion in roots available to plants. The organic fertilizers viz. GFC either and leaves of two rice varieties in addition to inducing dis- with or without NPK and microbial inoculants gave very lit- ease resistance. tle P uptake, it was due to the reason that the amount of The economic analysis of data revealed highest expens- GFC was so little that it could not provide phosphorus as es and total income with NPK fertilizer due to the reason much needed by the crop. Potassium uptake was also high- that it produced the highest yield of fodder. However, est under full NPK treatment having statistical difference chemical fertilizer treatment couldn’t give a better net even with BPF+full NP which gave significantly better K return and VCR, rather it was among the lowest. In this uptake than with full NP fertilizers. As NP alone treatment regard, the organic and biofertilizers yielded better eco- did not contain K in it, so the uptake was lower. The BPF as nomic returns due to their less cost as compared with apparent from its name i.e. Biological Potassium Fertilizer chemical fertilizers. Biopower+BPF or EM+half NP were contained bacteria Bacillus mucilaginous which had a proved to be the best treatments in terms of economics. strong ability to make fixed potassium available from the This was due to low cost of these biofertilizers and their soil minerals to crops. The results are supported by efficiency on plant growth. Wu et al. (2005) indicated that Duraisami and Mani (2000) who stated that the inorganic, half the amount of biofertilizer application had similar organic and biological nitrogen sources also enhance the effects when compared with organic fertilizer or chemical uptake of P and K under maize. Khaliq et al. (2006) fertilizer treatments. observed that combination of both N sources with EM increased the NPK concentrations in cotton crop. The populations of diazotrophs as well as P solubilizers CONCLUSIONS were affected significantly with the application of biofertil- izers, and they were also variable with the types and com- The use of BPF+full NP gave fodder maize growth, yield and binations of the applied biofertilizers. Generally, the num- nutrients uptake equal to that with full dose of NPK fertiliz- ber of diazotrophic bacteria was enhanced more with ers. However, Biopower and EM along with half dose of NP Ann. Microbiol., 57 (2), 177-183 (2007) 183 also showed results similar to that with full dose of NPK. Hafeez F.Y., Hameed S., Zaidi A.H., Malik K.A. (2002). Biofertilizers for sustainable agriculture. In: Azam F., Iqbal Therefore, the study concluded that the beneficial microor- M.M., Inayatullah C., Malik K.A., Eds, Techniques for ganisms / biofertilizers applied in combination were a bet- Sustainable Agricultural, NIAB, Faisalabad, Pakistan, pp. 67- ter choice for farmers to reduce the use of chemical fertil- izers for sustainable crop production. The combined use of Hameed S., Yasmin S., Malik K.A., Zafar Y., Hafeez F.Y. (2004). Biopower+EM+half NP, was also found to give more eco- Rhizobium, Bradyrhizobium and Agrobacterium strains iso- nomic return. Further, the organic fertilizers or composts lated from cultivated legumes. Biol. Fertil. Soil, 39 (3): 179- applied in low quantity are insufficient to meet the nutrients requirement of the crop even if with biofertilizers, so they Haqqani A.M., Ali Z., Shafique S., Zahid S., Bakhsh A. (2003). A fodder of winter lean period. Agridigest – An In-house need to be applied in bulk (tonnes) quantities. The com- Journal of Zari Taraqiati Bank Limited, Pakistan, 23: 15-16. bined use of bio and organic fertilizers along with chemical Hegde D.M., Dwived B.S., Sudhakara S.N. (1999). Biofertilizers fertilizers is more economical in terms of crop yields per for cereal production in India - a review. Indian J. Agric. Sci., unit area, and it is also a sustainable crop production tech- 69: 73-83. nology. Higa T., Wididana G.N. (1991). The concept and theories of effective microorganisms. In: Parr J.F., Hornick S.B., Acknowledgements st Whitman C.E., Eds, Proceedings of 1 International This study was undertaken with the cooperation of Dr. J.N. Conference on Kyusei Nature Farming, USDA, Washington, Usmani, from STEDEC Technology Commercialization D.C., pp. 118-124. Cooperation of Pakistan, Ministry of Science and Higa T., Parr J.F. (1994). Beneficial and Effective Microorganisms Technology Karachi, Dr. Fauzia Yusuf Hafeez, DCS/Head for a Sustainable Agriculture and Environment. International Nature Farming Research Center, Atami, Japan. Plant Microbiology Division, NIBGE, Faisalabad, and Col (R) Shah Sadiq, from EM-Technologies (Pvt) Ltd., Lahore, Hussan W.U., Haqqani A.M., Shafeeq S. (2003). Knocking the Pakistan. The technical help rendered from these men- doors of Balochistan for fodder crops production. Agridigest - An In-house Journal of Zari Taraqiati Bank Limited, Pakistan, tioned persons and the biofertilizer materials provided by 23: 24-30. their organizations are highly acknowledged. Khaliq A., Abbasi M.K., Hussain T. (2006). Effects of integrated use of organic and inorganic nutrient sources with effective microorganisms (EM) on seed cotton yield in Pakistan. REFERENCES Bioresour. Tech., 97 (8): 967-972. Khokar S.N. (2004). Interaction of Azospirillum spp. with maize th and wheat. In: Abstracts 10 Int. Congress of Soil Sci. Soci. Ali S., Hamid N., Rasul G., Mehnaz S., Malik K.A. (1998). Pak. Tandojam, Pakistan. pp. 20. Contribution of non leguminous biofertilizer to rice biomass, nitrogen fixation and fertilizer N use efficiency under flood Laheurte F., Berthelin J. (1988). Effect of a phosphate solubiliz- soil condition. In: Malik K.A., Mirza M.S., Ladha J.K., Eds, ing bacteria on maize growth and root exudation over four Nitrogen Fixation with Non-Legumes, Kluwer Academic Pub., levels of labile phosphorus. Plant Soil, 105: 11-17. London, pp. 61-73. Mehnaz S., Mirza M.S., Malik K.A. (1998). Detection of inoculat- Bisen P.S., Verma K. (1994). Biochemical tests. 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A single medium for isolation of acetylene reducing (dinitrogen fixing) bacteria from soil. Can. J. Varsha N., Patel H.H. (2000). Aspergillus aculeatus as a rock Microbiol., 27: 8-14. phosphate solubilizer. Soil Biol. Biochem., 32: 559-565. Rout D., Satapathy M.R., Mohapatra B.K. (2001). Effect of bio- Vessey J.K. (2003). Plant growth promoting rhizobacteria as fertilizers on nitrogen economy in maize. Madras Agric. J., 88 biofertilizers. Plant Soil, 255: 571-586. (7-9): 530-532. Wu S.C., Cao Z.H., Li Z.G., Cheung K.C., Wong M.H. (2005). Sokal R.R., Rohlf F.J. (1997). Biometry. The Principles and Effects of biofertilizer containing N-fixer, P and K solubilizers Practice of Statistic in Biological Research, WH Freeman, New and AM fungi on maize growth: a greenhouse trial. York, USA. Geoderma, 125 (1-2): 155-166. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers

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Springer Journals
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Copyright © 2007 by University of Milan and Springer
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Annals of Microbiology, 57 (2) 177-183 (2007) Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers 1 1 1 2 3 1 1 Ghulam JILANI *, Abida AKRAM , Raja M. ALI , Fauzia Y. HAFEEZ , Imran H. SHAMSI , Arshad N. CHAUDHRY , Abid G. CHAUDHRY 1 2 University of Arid Agriculture, Rawalpindi 46300, Pakistan; National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan; College of Agriculture & Biotechnology, Zhejiang University, Hangzhou, China Received 5 December 2006 / Accepted 16 April 2007 Abstract - Field experiment was conducted on fodder maize to explore the potential of integrated use of chemical, organic and biofer- tilizers for improving maize growth, beneficial microflora in the rhizosphere and the economic returns. The treatments were designed to make comparison of NPK fertilizer with different combinations of half dose of NP with organic and biofertilizers viz. biological potas- sium fertilizer (BPF), Biopower, effective microorganisms (EM) and green force compost (GFC). Data reflected maximum crop growth in terms of plant height, leaf area and fresh biomass with the treatment of full NPK; and it was followed by BPF+full NP. The highest uptake of NPK nutrients by crop was recorded as: N under half NP+Biopower; P in BPF+full NP; and K from full NPK. The rhizosphere microflora enumeration revealed that Biopower+EM applied along with half dose of GFC soil conditioner (SC) or NP fertilizer gave the highest count of N-fixing bacteria (Azotobacter, Azospirillum, Azoarcus and Zoogloea). Regarding the P-solubilizing bacteria, Bacillus was having maximum population with Biopower+BPF+half NP, and Pseudomonas under Biopower+EM+half NP treatment. It was con- cluded that integration of half dose of NP fertilizer with Biopower+BPF / EM can give similar crop yield as with full rate of NP fertiliz- er; and through reduced use of fertilizers the production cost is minimized and the net return maximized. However, the integration of half dose of NP fertilizer with biofertilizers and compost did not give maize fodder growth and yield comparable to that from full dose of NPK fertilizers. Key words: N-fixation, diazotrophs, P-solubilizing bacteria, nutrients uptake, economics, biofertilizers. INTRODUCTION Organic materials hold great promise due to their local availability as a source of multiple nutrients and ability to Maize (Zea mays L.) is the most important cereal fodder improve soil characteristics (Khaliq et al., 2006). The and grain crop under both irrigated and rainfed agricultur- improvement of fertility and quality of soil, especially under al systems in the semi-arid and arid tropics (Hussan et al., low input agricultural systems, requires the input of organ- 2003). Green fodder has an immense importance for ani- ic materials (Soumare et al., 2003; Naureen et al., 2005). mal production and development. At present for 21 million Biofertilizers contain different types of microorganisms, heads of livestock in Pakistan, only 51% feed requirements which have an ability to convert nutritionally important ele- of these animals can be met from green fodder. There is ments from unavailable to available form through biologi- still a shortage of 26 million tonne of total digestible nutri- cal processes (Hedge et al., 1999; Vessey, 2003). They ents, and in terms of digestible protein shortage is 1.58 have emerged as an important component of the integrat- million tonne. So, there is a need of 150 million tonne ed nutrient supply system and hold a great promise to green fodder to feed the animals and meet their nutrients improve crop yields. Biofertilizers containing N-fixer requirement (Haqqani et al., 2003). (Azotobacter chroococcum), P-solubilizer (Bacillus mega- Traditional use of chemicals fertilizers in agricultural terium) and K-solubilizer (Bacillus mucilaginous) and production cannot be over-emphasized, but with fertilizer arbuscular mycorrhizal fungi (Glomus mosseae and Glomus costs going up, these need to be supplemented or substi- intraradices) have been developed (Hafeez et al., 2002; tuted with available organic or biofertilizers. Thus integrat- Wu et al., 2005). The P-solubilizing microorganisms (PSM) ed plant nutrients system was introduced which includes render insoluble phosphate into soluble form through the application of organic and biofertilizers to supplement process of acidification, chelation and exchange reactions chemical fertilizers to maintain and increase the soil fertili- (Gull et al., 2004; Son et al., 2006). Therefore, many ty for sustaining increased crop production. It helps for researchers have tried to increase the plant-available phos- making the highly productive cereals mono cropping sys- phate-fraction by means of PSM such as Bacillus (Raj et al., tems more sustainable (Rajendra et al., 1998). 1981), Enterobacter (Laheurte and Berthelin, 1988), Pseudomonas (Suh et al., 1995a, 1995b), Agrobacterium, Aspergillus (Varsha and Patel, 2000; Hameed et al., 2004) * Corresponding author. Phone: 0092-51-9062241; and Pantoea agglomerans (Son et al., 2006). Fax: 0092-51-9290160; E-mail: jilani62@yahoo.com 178 G. Jilani et al. The product of EM (effective microorganisms) contains T BPF as seed coating + full dose of NP fertilizer selected species of microbes including predominant popu- T Biopower as seed coating + half dose of NP fertilizer lations of lactic acid bacteria and yeasts, and smaller num- T Biopower + BPF + half dose of NP fertilizer bers of photosynthetic bacteria, actinomycetes and other T EM (1% solution) for seed soaking, and broadcasted -1 types of organisms (Higa and Wididana, 1991). All of these as soil mixed to 10 L EM with 100 kg soil ha + half are mutually compatible with one another and can coexist dose of NP fertilizer in liquid culture (Higa and Parr, 1994). A latest study indi- T Biopower + EM as in T + half dose of NP fertilizer 8 7 -1 cated that application of EM to cotton increased the effi- T Full dose of GFC (NPK), 250 kg ha -1 ciency of both organic and mineral nutrient sources (Khaliq T Full dose of GFC (SC), 500 kg ha -1 et al., 2006). Economic analysis suggested that the use of T Half dose of GFC (SC), 250 kg ha + Biopower as in 1/2 mineral NPK with EM+OM (organic matter) saved the T -1 mineral N fertilizer by almost 50% compared to a system T Half dose of GFC (SC), 250 kg ha + Biopower + EM with only mineral NPK application. as in T Currently, the need is to get maximum output with min- Each treatment plot size was 7 m x 5 m, and maize vari- imum cost, which is possible only if chemical fertilizers are ety Agaiti-85 (local name meaning the variety to be grown supplemented with organic and biofertilizers. The present in early season) was grown as test crop. The seed was -1 study was, therefore, envisaged with the objective to eval- sown at the rate of 50 kg ha with the help of hand drill on st uate the comparative effect of chemical, organic and 1 August, 2004. The treatment effect was recorded on the th biofertilizers on the growth, yield and nutrients uptake of growth parameters and fodder yield of crop at 60 day maize fodder. The interactions among the microorganisms after sowing. of different microbial products and fertilizers were also Physical and chemical properties of composite soil sam- investigated. ples taken before sowing from the experimental field were determined. The soil was sandy loam in texture, had pH -1 7.3, electrical conductivity 0.30 dS m , organic matter -1 -1 MATERIALS AND METHODS 0.43%, NO -N 1.2 mg kg , available-P 5.2 mg kg , and -1 extractable-K 78.5 mg kg . The plant samples taken at Comparative study of organic and biofertilizers with chem- harvest were analysed for NPK contents and their uptake ical fertilizer was carried out for maize fodder production was calculated by employing the following formula: -1 under rainfed conditions at the research farm of University Nutrients uptake (kg ha ) = -1 of Arid Agriculture, Rawalpindi, Pakistan during the sum- (Nutrient content in plants as %) (Dry yield as kg ha ) mer season of year 2003. The biofertilizers used for seed 100 dressing were: Biological Potassium Fertilizer (BPF, 10 kg –1 -1 ha ), Biopower (2.5 kg ha ) and Effective Microorganisms Isolation of microorganisms. Rhizosphere microorgan- -1 (EM, 2.5 L ha ). While the organic fertilizers were Green isms were isolated from each sample by serial dilution and Force Compost (GFC) enriched with NPK viz. GFC (NPK, spread plate method. One gram soil was dispersed in 10 mL -1 250 kg ha ) and Green Force Compost as soil conditioner of sterile distilled water and thoroughly shaken. One mL of -1 GFC (SC, 500 kg ha ). The nature and composition of above dilution was again transferred to 9 mL of sterile dis- -2 -3 -4 -5 these experimental materials is given under: tilled water to form 10 dilution. Similarly, 10 , 10 , 10 BPF is a biofertilizer inoculum containing K-solubilizing bac- dilutions were made for each soil sample. An aliquot of 0.1 teria Bacillus mucilaginous. mL from each dilution was taken with micropipette (0.01- Biopower is a biofertilizer containing four N-fixing bacteria 0.1 mL range) and plated on agar medium specific for each viz. Azotobacter, Azospirillum, Azoarcus and Zoogloea. microorganism to be isolated. After incubation total num- EM is a mixed inoculum containing mainly Lactobacillus, ber of colonies formed was counted with the help of digital Rhodopseudomonas, Actinomycetes and yeast. colony counter. Total count was determined as colony GFC (NPK) is composted municipal waste enriched with forming units (CFU): -1 NPK fertilizer to have 15-15-15 percent NPK contents. CFU g of moist soil = Mean plate count / (Volume of Green Force Compost (GFC) is a commercial product. sample plated) (Dilution factor) -1 -1 GFC (SC) is composted municipal waste having NPK con- CFU g of dry soil = CFU g of moist soil (100 + percent tents as 3.0-1.2-1.8 percent. This GFC is a soil conditioner moisture in sample / 100) (SC) product. The following N -fixing and P-solubilizing bacteria main- The experiment was laid out in a randomized complete ly found in the rhizosphere were enumerated by using spe- block design with four replications. Urea, single super phos- cific growth media for each species as suggested by phate and sulphate of potash were used as source of NPK, authors quoted against each microorganism. respectively. All the fertilizers were applied in the field Azotobacter: Burk’s medium (Cornish and Page, 2000) -1 o before maize sowing. Soil mixed with EM (10 L ha ) was incubated at 26 C for three days. broadcasted in T , T , and T . The experiment consisted of Azospirillum: Okon medium (Okon et al., 1976) incubated 7 8 12 the following treatments: at 35 C for 2 days. -1 T Full dose of NP fertilizers, 120-90 kg ha , Azoarcus: Jenson nitrogen free biotin medium (Döbereiner, respectively 1995) incubated at 30 C for three days until colour of -1 T Full dose of NPK fertilizers, 120-90-60 kg ha , media changed to blue then these were counted. respectively Zoogloea: Combined carbon medium formulated by Rennie -1 o T Half dose of NP fertilizers, 60-45 kg ha , (1981) incubated at 30 C for 2 days. respectively Bacillus and Pseudomonas: Both these species of P-solubi- lizing bacteria were isolated on Pikovskaya’s agar medium Ann. Microbiol., 57 (2), 177-183 (2007) 179 (Pikovskaya, 1948) containing insoluble tricalcium phos- niques including analysis of variance (ANOVA), and the phate. The plates were incubated at 26 C for 7 days. After treatments means were compared by application of the growth, the colonies of Bacillus and Pseudomonas were Duncan’s multiple range test (Sokal and Rohlf, 1997). isolated and re-cultured on nutrient agar media. The Pseudomonas was incubated at 20 C for 24 hours, while Bacillus at 37 C for 48 hours. RESULTS Purification of microorganisms. Colonies developed on Biometric parameters each specified medium were picked with the help of ster- Vegetative plant growth parameters like plant height, leaf ilised inoculating wire loop and streaked on separate plate. area and biomass yield were measured before harvesting All plates were then incubated at same temperature and for the maize fodder as given in Table 1. Data show that NPK time period as before indicated for each microorganism. fertilizers gave the highest values for these parameters, which had statistically significant difference with the plants Morphological characterisation of microorganisms. from other treatments. It was followed by BPF+full NP treat- Microorganisms of purified colonies were studied for 5 dif- ment which had statistically non significant difference with ferent morphological characters stereomicroscopic feature, full dose of NP fertilizer. The biofertilizers when combined colour, margin of colony, surface form, surface texture, ele- individually with half dose of NP fertilizer could not give vation (Cruickshank et al., 1975). Microorganisms were plant growth similar to full NP or NPK fertilizer. All these identified by Gram staining (Murray et al., 1994). biofertilizers were similar in their effect on plant growth. However, when two biofertilizers were applied together with Biochemical tests for microorganisms. Biochemical half NP fertilizer, they could produce plants with height sta- tests for microorganisms like catalase production, oxidase tistically similar to full dose of NP fertilizer. Thus treatments production, nitrate reduction, triple sugar iron test, and of Biopower+BPF+half NP and Biopower+EM+half NP were sugar fermentation were also performed. All procedures for not significantly different from full NP. Biopower or EM in biochemical tests were taken from Handbook of combination with half NP fertilizer was statistically superior Microbiology (Bisen and Verma, 1994). to only half dose of NP fertilizer. All GFC treatments had lower values than other treatments. Half GFC Economic analysis. Procedures for economic analysis (SC)+Biopower+EM used in combination had not signifi- were adopted from CIMMYT (1988). Expenditures incurred cantly higher value than other GFC treatments. Full GFC -1 on various fertilizing materials and incomes from the (SC) (500 kg ha ) gave the lowest fresh fodder yield among respective treatments were calculated on the basis of mar- all the bio, organic and chemical fertilizer treatments. The ket rates at the time of experiment. Further, the net return plant growth with half GFC (SC)+Biopower+EM was, how- was obtained by finding the difference between total ever, statistically equal to that with half dose of NP fertiliz- income and total expenditure of each treatment. Finally, er. the value to cost ratio (VCR) was calculated for each treat- ment through this formula: Nutrients uptake Expenditure on plant nutrients Nitrogen uptake by crop is shown in Table 2; it had statis- Value cost ratio = ————————————————— tically different values for various treatments. The highest Total income N uptake was observed in full NPK fertilizer treatment -1 (45.0 kg ha ) which was followed by BPF+full NP (40.7 kg -1 Statistical analysis. The data collected for various char- ha ) with a significant difference. Full NP fertilizer occu- acteristics were analysed by application of statistical tech- pied the third position; however it had statistically non sig- TABLE 1 - Comparison of organic, bio and chemical fertilizers for growth and yield of maize fodder Treatments description Plant height Leaf area Fodder yield 2 -1 (cm) (cm ) (kg ha ) -1 c b b T Full NP (120-90 kg ha ) 163 * 456 * 8732 * -1 a a a T Full NPK (120-90-60 kg ha ) 175 472 9540 -1 ef e d T Half NP (60-45 kg ha ) 150 420 7532 b b ab T BPF+full NP 167 461 9170 de d c T Biopower+half NP 155 441 8156 cd bc bc T Biopower+BPF+half NP 158 451 8528 de e c T EM+half NP 155 428 8264 cd cd bc T Biopower+EM+half NP 159 445 8572 -1 g f d T Full GFC (NPK) (250 kg ha ) 143 395 7620 -1 h h e T Full GFC (SC) (500 kg ha ) 132 370 6950 g g d T Half GFC (SC)+Biopower 140 384 7508 fg fg cd T Half GFC (SC)+Biopower+EM 145 389 8026 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05. 180 G. Jilani et al. TABLE 2 - Comparison of organic, bio and chemical fertilizers for nutrients uptake by maize fodder -1 Treatments description Nutrients uptake (kg ha ) Nitrogen Phosphorus Potassium -1 c b d T Full NP (120-90 kg ha ) 36.6*7.29 * 30.9 * -1 a a a T Full NPK (120-90-60 kg ha ) 45.0 7.99 45.2 -1 e e de T Half NP (60-45 kg ha ) 26.7 4.42 28.6 b a b T BPF+full NP 40.7 8.28 40.5 d d cd T Biopower+half NP 30.6 5.28 31.5 cd c cd T Biopower+BPF+half NP 33.7 6.32 34.9 d cd cd T EM+half NP 31.8 5.73 32.9 cd c c T Biopower+EM+half NP 34.2 6.15 35.4 -1 e d d T Full GFC (NPK, 250 kg ha ) 25.9 5.21 30.4 -1 f f e T Full GFC (SC, 500 kg ha ) 22.0 3.61 24.5 ef ef de T Half GFC (SC)+Biopower 24.8 4.11 27.4 de de cd T Half GFC (SC)+Biopower+EM 29.2 4.81 31.3 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05 nificant difference with Biopower+EM+half NP and Biopower+EM+half NP. These were also having the lowest Biopower+BPF+half NP. Generally, non-significant differ- population in chemical fertilizer treatments. The number of ences were observed among the GFC treatments except other two diazotrophs Azoarcus and Zoogloea was also half GFC (SC)+Biopower+EM under which the N uptake higher in the treatments receiving Biopower alone or in -1 was 29.2 kg ha being superior to other GFC treatments. combination especially with EM. The biofertilizers gave The highest values of phosphorus uptake (8.28 and higher number of rhizosphere microflora in the treatments -1 7.99 kg ha ) were recorded in BPF+full NP and full NPK where organic fertilizers were also applied. Regarding the treatment, respectively with non significant difference phosphate solubilizing bacteria Bacillus and Pseudomonas, (Table 2). These were followed by full NP treatment show- their count was statistically higher with the treatments -1 ing 7.29 kg ha P uptake. The treatments (T and T ) in Biopower+BPF+half NP, and Biopower+EM+half NP, 6 8 which bio-fertilizers were used in combination showed non- respectively. The application of BPF either alone or in com- significant difference between themselves. They had signif- bination with other biofertilizers resulted into higher num- icantly higher values than all the GFC treatments. Among ber of Bacillus in the rhizosphere of maize. Contrasting to all GFC treatments, half GFC (SC)+Biopower+EM treat- diazotrophs, the populations of P solubilizers were higher in ment had higher value of phosphorus uptake than others. chemical fertilizer treatments as compared to organic ones. The maximum potassium uptake in maize fodder crop -1 was 45.2 kg ha , obtained from full NPK (Table 2). The Economic analysis BPF+full NP treatment showed significantly lower value The economic analysis on the performance of organic and -1 (40.5 kg ha ) than full NPK but higher than full NP. Full biofertilizers in comparison with chemical fertilizers for maize GFC (NPK) treatment resulted in similar K uptake as with fodder production was performed as in Table 4. The per full NP, however they were inferior to Biopower and EM hectare expenditure on various treatments showed the high- -1 treatments (T5-T8) either used alone or combined. Non- est value of US$ 121.0 for full NPK (120-90-60 kg ha ) fol- significant differences were observed among half NP and all lowed by BPF+full NP (US$ 89.7). The lowest expenses were GFC treatments. for half GFC (SC)+Biopower (US$ 37.5), half NP fertilizer (US$ 41.5) and half GFC (SC)+Biopower+EM (US$ 41.7). Rhizosphere microflora The maximum income (US$ 318.0) was also from full NPK Diazotrophic bacteria namely Azotobacter, Azospirillum, fertilizer as it yielded the highest fresh fodder biomass, and it Azoarcus and Zoogloea, and phosphate solubilizing bacte- was followed by BPF+full NP (US$ 305.7). The lowest income ria Bacillus and Pseudomonas were enumerated from the was obtained from full GFC (SC) treatment (US$ 231.7). The maize plants rhizosphere at active growth stage viz. 60 net profit was maximum with Biopower+EM+half NP (US$ days after sowing. Data on the count of Azotobacter (Table 235.9) and Biopower+BPF+half NP (US$ 231.9), whereas, -1 3) showed that their population was highest in the treat- full GFC (SC) (500 kg ha ) gave the lowest net profit (US$ ment of half GFC (SC)+Biopower+EM, which differed not 165.0). The value (total income) to cost (total expenditure) significantly with that of half GFC (SC)+Biopower. The low- ratio (VCR) was also determined to compare various treat- est count was observed in NP and NPK fertilizer treatments. ments (Table 4). Data showed that full NPK fertilizer gave the The treatments receiving any of the biofertilizer Biopower, lowest VCR (2.63), which was followed by BPF+full NP (3.41). BPF or EM singly or in combination with each other caused The highest VCR values were obtained from half GFC the establishment of higher populations of Azotobater in (SC)+Biopower (6.67) and half GFC (SC)+Biopower+EM maize rhizosphere. Azospirillum count in the rhizosphere (6.42). All the biofertilizer treatments had intermediate VCR was also maximum with Half GFC (SC)+Biopower+EM, figures showing better economic impact than the chemical which had no statistical difference with that in fertilizers used at full or half application rate. Ann. Microbiol., 57 (2), 177-183 (2007) 181 TABLE 3 - Comparison of organic, bio and chemical fertilizers for count of N-fixing and P-solubilizing microflora in maize rhizosphere -1 (log CFU g dry soil). Treatments Azotobacter Azospirillum Azoarcu Zoogloea Bacillus Pseudomonas -1 f d de d de d T1 Full NP (120-90 kg ha)5.27 4.41 3.44 4.09 3.64 3.58 -1 f d d d cd cd T2 Full NPK (120-90-60 kg ha)5.18 4.47 3.46 4.13 3.69 3.64 -1 ef d d cd ef e T3 Half NP (60-45 kg ha)5.38 4.51 3.34 4.16 3.54 3.42 e d cd cd ab c T4 BPF+full NP 5.40 4.44 3.55 4.14 3.87 3.69 c b b b c d T5 Biopower+1/2 NP 5.61 4.74 3.69 4.61 3.73 3.57 bc b ab b a ab T6 Biopower+BPF+1/2 NP 5.66 4.76 3.72 4.64 3.89 3.79 d c cd c b b T7 EM+half NP 5.50 4.66 3.56 4.28 3.81 3.76 b ab ab ab b a T8 Biopower+EM+1/2 NP 5.68 4.80 3.74 4.69 3.83 3.83 -1 e d d cd f e T9 Full GFC (NPK, 250 kg ha)5.39 4.50 3.50 4.21 3.42 3.33 -1 d cd c c e e T10 Full GFC (SC, 500 kg ha)5.51 4.60 3.58 4.29 3.55 3.40 ab b a ab ef d T11 1/2 GFC (SC)+Biopower 5.69 4.79 3.75 4.67 3.53 3.58 a a a a d cd T12 1/2 GFC (SC)+Biopower+EM 5.73 4.84 3.77 4.70 3.65 3.64 * Means not sharing a common letter in a column differ significantly at P ≤ 0.05 TABLE 4 - Economic comparison of organic, bio and chemical fertilizers use for maize fodder production 1 2 3 Treatments Expenditure Fodder Yield Income Net Return VCR -1 -1 -1 -1 (US$ ha )(t ha )(US$ ha )(US$ ha ) -1 T1 Full NP (120-90 kg ha ) 83.0 8.73 291.1 208.1 3.51 -1 T2 Full NPK (120-90-60 kg ha ) 121.0 9.54 318.0 197.0 2.63 -1 T3 Half NP (60-45 kg ha ) 41.5 7.53 251.1 209.6 6.05 T4 BPF+full NP 89.7 9.17 305.7 216.0 3.41 T5 Biopower+1/2 NP 45.7 8.16 271.9 226.2 5.95 T6 Biopower+BPF+1/2 NP 52. 3 8.53 284.3 231.9 5.43 T7 EM+half NP 45. 7 8.26 275.5 229.8 6.03 T8 Biopower+EM+1/2 NP 49.8 8.57 285.7 235.9 5.73 -1 T9 Full GFC (NPK, 250 kg ha ) 62.5 7.62 254.0 191.5 4.06 -1 T10 Full GFC (SC, 500 kg ha ) 66. 7 6.95 231.7 165.0 3.48 T11 1/2 GFC (SC)+Biopower 37.5 7.51 250.3 212.8 6.67 T12 1/2 GFC (SC)+Biopower + EM 41. 7 8.03 267.5 225.9 6.42 1 -1 -1 Expenditure on fertilizer sources: N, P, K (US$ kg ) = 0.32, 0.50, 0.63, respectively (NFDC, 2004); BPF (US$ 0.67 kg ); Biopower -1 -1 -1 -1 (US$ 1.67 kg ); EM (US$ 0.33 L ; GFC (NPK, US$ 0.25 kg ); GFC (SC, US$ 0.13 kg ). Income of fresh fodder (US$ 33.3 per tonne). VCR, Value cost ratio. DISCUSSION microbes with major groups as lactic acid bacteria, photo- synthetic bacteria, ray fungi and yeast (Higa and This field study was envisaged with the view to find out Wididana, 1991). These microorganisms are well known to some combination of biofertilizers with half dose of chem- have beneficial effect on plant growth and their nutrient ical fertilizer to have the same effect on crop growth as accumulation. Panwar et al. (2000) observed that biofer- with the full dose of NPK chemical fertilizers. The results tilizers increased leaf area, chlorophyll concentration and on fodder yield depicted that BPF+full NP treatment per- total biomass production in wheat. Recently, the concept formed equally good as did the full dose of NPK fertilizers. of microbial consortium in the production and use of Biopower+BPF+half NP and Biopower+EM+half NP treat- biofertilizers is emerging strongly. This is due to the rea- ments gave statistically equal yield as with full dose of NP son that some beneficial microorganism having similar fertilizers, and better than as with half dose of NP fertiliz- ecological requirements flourish in an associative manner er. Khaliq et al. (2006) reported that integrated use of OM (Vessey, 2003). Ponnuswamy et al. (2002) reported that (farm yard manure)+EM with 1/2 mineral NPK yielded a combination of phosphobacteria+Azospirillum had posi- similar to the yield obtained from full recommended NPK, tive effect on yield and yield characters compared to no indicating that this combination can substitute for 85 kg N biofertilizer application and with phosphorus solubilizing -1 ha . The BPF contains potassium solubilizing bacteria bacteria (PSB) or Azospirillum alone. These findings coin- Bacillus mucilaginous, Biopower includes four types of N- cide with results of Nanda et al. (1995) who observed that fixing bacteria, and EM is having a large number of green fodder yield and benefit-cost ratio (VCR) were high- 182 G. Jilani et al. -1 Biopower, as it contained all the four enumerated bacteria est with a combination of 75 kg N ha and seed inocula- -1 which were colonized in the rhizosphere due to their inoc- tion with Azospirillum (22.0 t ha yield and 1.37 VCR) and -1 ulation on the seed. The results exactly coincide with find- lowest in the control (7.6 t ha yield and 0.07 VCR). The ings of Khokar (2004) who conducted research on interac- results of present study are also supported by Rout et al. tion of indigenous Azospirillum with maize and observed (2001) who found that Azotobacter, Azospirillum and their that the highest root colonization was under inoculum combinations gave more yield than untreated maize. They treatments. The application of EM and BPF increased the concluded that biofertilizers in combination with inorganic number of diazotrophs further, although their inoculum did nitrogen fertilizers can substitute up to 20 % nitrogenous not contain any of the enumerated bacteria. This might be fertilizers and can increase maize yield. Similarly, Mokhova due to the positive interactive effect of the microbes in et al. (2000) reported that application of Rhizobacterin these biofertilizers with N-fixing bacteria. These results (inoculum containing mixed culture of N-fixing and P-solu- were in conformity with Mehnaz et al. (1998) who con- bilizing bacteria) resulted in yield increases and required ducted research on detection of inoculated plant growth lower nitrogen fertilizer rates. promoting rhizobacteria in rice and isolated strains belong- The uptake or accumulation of the macronutrients like ing to Azospirillum, Azoarcus, Pseudomonas and Zoogloea. N, P and K is the direct reflection of the biomass produc- They found that the populations of these bacteria as well as tion. The result of this experiment revealed that full dose of that of indigenous ones were more under inoculum treat- NPK caused the maximum uptake of N, P and K nutrients ments as compared to uninoculated. The organic fertilizers by maize fodder, and it was followed by BPF+full NP, even better P uptake than from the chemical fertilizers. were better than chemical fertilizer in terms of increasing Biofertilizer treatments especially those with Biopower the number of N-fixing bacteria in the rhizosphere. These results indicate that microbial population of soil can be improved the NPK uptake over half dose of NP fertilizer. increased by applying organic matter and their effective- This was due to the fact that Biopower contained four dif- ness can be increased by inoculation with biofertilizer. In ferent species of N-fixing bacteria, which resulted into increased availability of nitrogen to the crop. Wu et al. this way availability of nutrients can be increased to plant (2005) indicated that half the amount of biofertilizer appli- and maximum yield can be achieved (Elshanshorey, 1995). cation had similar effects when compared with organic fer- In a previous study by Ali et al. (1998) on the bacterial tilizer or chemical fertilizer treatments. Microbial inoculum counts under farm yard manure and chemical fertilizers treatments, they found that diazotrophs, in presence or not only increased the nutritional assimilation of plants (total N, P and K), but also improved soil properties, such absence of NP fertilizer did not respond significantly to inoc- as organic matter content and total N in soil. Elshanshorey ulation in terms of N content. The count of P solubilizers (1995) also reported that biofertilizers increased nutrient especially that of Bacillus was higher under BPF treatment, concentration and uptake by cereal crops, which lead which was due to the reason that BPF contained Bacillus towards luxurious growth and better crop development. mucilaginous already in the inoculum. Application of EM Combination of N fertilizer with EM also increased the con- also had positive effect to increase the number of phos- centrations of NPK in plants (Khaliq et al., 2006). phorus solubilizing bacteria in the maize rhizosphere. This Phosphorus uptake was highest under BPF+full NP treat- was probably due to the reason that the yeast and photo- ment, even better than full dose of NP or NPK treatments. synthetic bacteria contained in EM released some biochem- In fact BPF has Bacillus mucilaginous bacteria which have ical compounds which enhanced the growth and population solubilizing effect on fixed phosphorus in the soil. Son et al. of N-fixing as well as P-solubilizing bacteria in rhizosphere. (2006) enlisted a number of microorganisms which have Similarly, Ren et al. (2006) reported that a bacterium strain the ability to solubilize phosphorus in the soil and make it B-916 of Bacillus subtilis, caused growth promotion in roots available to plants. The organic fertilizers viz. GFC either and leaves of two rice varieties in addition to inducing dis- with or without NPK and microbial inoculants gave very lit- ease resistance. tle P uptake, it was due to the reason that the amount of The economic analysis of data revealed highest expens- GFC was so little that it could not provide phosphorus as es and total income with NPK fertilizer due to the reason much needed by the crop. Potassium uptake was also high- that it produced the highest yield of fodder. However, est under full NPK treatment having statistical difference chemical fertilizer treatment couldn’t give a better net even with BPF+full NP which gave significantly better K return and VCR, rather it was among the lowest. In this uptake than with full NP fertilizers. As NP alone treatment regard, the organic and biofertilizers yielded better eco- did not contain K in it, so the uptake was lower. The BPF as nomic returns due to their less cost as compared with apparent from its name i.e. Biological Potassium Fertilizer chemical fertilizers. Biopower+BPF or EM+half NP were contained bacteria Bacillus mucilaginous which had a proved to be the best treatments in terms of economics. strong ability to make fixed potassium available from the This was due to low cost of these biofertilizers and their soil minerals to crops. The results are supported by efficiency on plant growth. Wu et al. (2005) indicated that Duraisami and Mani (2000) who stated that the inorganic, half the amount of biofertilizer application had similar organic and biological nitrogen sources also enhance the effects when compared with organic fertilizer or chemical uptake of P and K under maize. Khaliq et al. (2006) fertilizer treatments. observed that combination of both N sources with EM increased the NPK concentrations in cotton crop. The populations of diazotrophs as well as P solubilizers CONCLUSIONS were affected significantly with the application of biofertil- izers, and they were also variable with the types and com- The use of BPF+full NP gave fodder maize growth, yield and binations of the applied biofertilizers. Generally, the num- nutrients uptake equal to that with full dose of NPK fertiliz- ber of diazotrophic bacteria was enhanced more with ers. However, Biopower and EM along with half dose of NP Ann. Microbiol., 57 (2), 177-183 (2007) 183 also showed results similar to that with full dose of NPK. Hafeez F.Y., Hameed S., Zaidi A.H., Malik K.A. (2002). Biofertilizers for sustainable agriculture. In: Azam F., Iqbal Therefore, the study concluded that the beneficial microor- M.M., Inayatullah C., Malik K.A., Eds, Techniques for ganisms / biofertilizers applied in combination were a bet- Sustainable Agricultural, NIAB, Faisalabad, Pakistan, pp. 67- ter choice for farmers to reduce the use of chemical fertil- izers for sustainable crop production. The combined use of Hameed S., Yasmin S., Malik K.A., Zafar Y., Hafeez F.Y. (2004). Biopower+EM+half NP, was also found to give more eco- Rhizobium, Bradyrhizobium and Agrobacterium strains iso- nomic return. Further, the organic fertilizers or composts lated from cultivated legumes. Biol. Fertil. Soil, 39 (3): 179- applied in low quantity are insufficient to meet the nutrients requirement of the crop even if with biofertilizers, so they Haqqani A.M., Ali Z., Shafique S., Zahid S., Bakhsh A. (2003). A fodder of winter lean period. Agridigest – An In-house need to be applied in bulk (tonnes) quantities. The com- Journal of Zari Taraqiati Bank Limited, Pakistan, 23: 15-16. bined use of bio and organic fertilizers along with chemical Hegde D.M., Dwived B.S., Sudhakara S.N. (1999). Biofertilizers fertilizers is more economical in terms of crop yields per for cereal production in India - a review. Indian J. Agric. Sci., unit area, and it is also a sustainable crop production tech- 69: 73-83. nology. Higa T., Wididana G.N. (1991). The concept and theories of effective microorganisms. In: Parr J.F., Hornick S.B., Acknowledgements st Whitman C.E., Eds, Proceedings of 1 International This study was undertaken with the cooperation of Dr. J.N. Conference on Kyusei Nature Farming, USDA, Washington, Usmani, from STEDEC Technology Commercialization D.C., pp. 118-124. Cooperation of Pakistan, Ministry of Science and Higa T., Parr J.F. (1994). Beneficial and Effective Microorganisms Technology Karachi, Dr. Fauzia Yusuf Hafeez, DCS/Head for a Sustainable Agriculture and Environment. International Nature Farming Research Center, Atami, Japan. Plant Microbiology Division, NIBGE, Faisalabad, and Col (R) Shah Sadiq, from EM-Technologies (Pvt) Ltd., Lahore, Hussan W.U., Haqqani A.M., Shafeeq S. (2003). Knocking the Pakistan. The technical help rendered from these men- doors of Balochistan for fodder crops production. Agridigest - An In-house Journal of Zari Taraqiati Bank Limited, Pakistan, tioned persons and the biofertilizer materials provided by 23: 24-30. their organizations are highly acknowledged. Khaliq A., Abbasi M.K., Hussain T. (2006). 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