Microsoft Word - 11-Agra_32289_.doc 670 Original Article Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 DEVELOPMENT AND FORMULATION OF Azospirillum lipoferum AND Pseudomonas fluorescens AS EFFECTIVE BIOLOGICAL AGENTS FOR ENHANCED AGRO-PRODUCTIVITY DESENVOLVIMENTO E FORMULAÇÃO DE Azospirillum lipoferum E Pseudomonas fluorescens ENQUANTO AGENTES BIOLÓGICOS EFICAZES PARA UMA AGRO- PRODUTIVIDADE MELHORADA Saravanakumar TAMILSELVI 1 ; Asmita DUTTA 1 ; M SINDHU 1 , G subbayan MURUGESAN 1 ; Rengaraju BALAKRISHNARAJA 1 1. Departament of Biotecnology, Bannari Amman Instituto de Tecnologia,Sathyamangalam, TN, Índia. balakrishnarajar@bitsathy.ac.in ABSTRACT: Biofertilizer is a group of beneficial microorganisms used for improving the productivity of soil by fixing atmospheric nitrogen or by solubilizing soil phosphorus. They also stimulate plant growth through synthesis of growth promoting substances. In this present study, Azospirillum lipoferum is grown in Nitrogen free Bromothymol blue (Nfb) medium and Pseudomonas fluorescens in King’s B medium. Bioprocess condition was optimized for both of the culture and found that Pseudomonas fluorescens has shown highest growth at 300C in pH 8 after 72 hours of incubation where as Azospirillum lipoferum showed highest cell concentration at 310C in pH 7, with incubation period of 72 hours. The optimized culture is mixed with different formulations of powder and liquid carrier such as Saw dust, Rice husk, Date seed powder, Matka khad, Jiwamrit and Beejamrit respectively. Shelf life study for 0, 30, 60, 90 and 120 days by cell counting and spread plate method showed that shelf life of the biofertilizer produced from Powder and liquid carriers had high amount of viable microbial population up to 120 days storage. Among biofertilizer based bio inoculants, Saw dust showed maximum population of 77x109cfu/ml for Azospirillum lipoferum and 72 x 109 CFU/ml for Pseudomonas strain on 120th day and the liquid carrier Matka khad showed 85x109 cfu/ml for Azospirillum lipoferum and 78 x 109 CFU/ml for Pseudomonas fluorescens. KEYWORDS: Biofertilizers. Azospirillum lipoferum. Pseudomonas fluorescens. Powder carriers and liquid formulations. INTRODUCTION Biofertilizers are the bioinoculants of specific beneficial microorganisms that promote the growth of plant crops by converting the unavailable form of nutrients into available form. These biofertilizers also induce resistance in plants against pests, to improve soil fertility, to help plant growth by increasing the number and biological activity of desired microorganisms in the root surface (SIVASAKTHIVELAN et al., 2013). Azospirillum is a nitrogen fixing biofertilizer that colonizes in the root. Bacteria produces growth-promoting substances like indole acetic acid (IAA), gibberellins, pantothenic acid, thiamine and niacin and it also increases the rootlet density and root branching resulting in the increased uptake of mineral and water (VIJENDRAKUMAR et al., 2014). Azospirillum belongs to the family of Rhodospirillaceae and order Rhodospirillales associated with roots of monocots and can fix Nitrogen of 20-40 kg/ha, in addition to growth regulating substances. The Azospirillum form relationship with many plants mostly with those having the C4-dicarboxylic pathway of photosynthesis and Slack pathway, because they grow and fix nitrogen on salts of organic acids such as malic, aspartic acid (KAUSHAL et al.,2013). Azospirillum under stress conditions enhance plant growth by fixing atmospheric nitrogen. Production of growth promoting substances influence root development by increased uptake of nutrients from the land, and inhibiting pathogenic fungi and bacteria in the rhizosphere. (HOSSAIN ET al., 2015). Pseudomonas fluorescence is a very common gram negative bacteria. It has antagonistic activity. It can produce some secondary metabolite or some antifungal compound such as fluorescent pigments, siderophores, hydrocyanic acid (HCN) and more important lytic enzymes. These lytic enzymes can degrade the chitin, β-1,3-glucan and protein components present on the fungal cell wall (RAMYASMRUTHI et al.,2012). It produces IAA and Promote enhancement of root length, shoot length, or number of lateral root (WAHYUDI et al., 2011). It is reported that Pseudomonas fluorescence is able to produce an antifungal metabolite called pyrrolnitrin, mainly used against Rhizoctonia Sp. and Fusarium Sp. (ANBUSELVI et al., 2010). It Received: 18/11/15 Accepted: 20/03/16 671 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 also has a potential of phosphate solubilisation, if it is placed on Pikovskaya media it forms a clear halo zone (FEKADU et al., 2013). So Pseudomonas acts as both biofertilizer and biopesticide. A Bioinoculant can improve product stability, shelf life and also protect bacteria against different environmental conditions and provide initial food source. Application of PGPR either to increase crop health or to manage plant diseases depending on the development of bioformulations with suitable carriers that maintain the survival of bacteria for a considerable length of time (JAMBHULKAR et al., 2014).They can be applied for seed treatment, bio priming, seedling, foliar spray, fruit spray and sucker treatment (RITIKA et al., 2014). Therefore, liquid inoculants formulation with good field performance characteristics that uses low cost materials and are easily attainable by small producers who could overcome many problems associated with processing solid carriers (SIVASAKTHIVELAN et al., 2012). In this study, Azospirillum lipoferum and Pseudomonas fluorescence were optimized for bioprocess conditions such as pH, temperature, incubation period and effective formulation were made by using liquid and powder carriers that promote the growth of the bacteria for the usage as an effective biological agent. MATERIAL AND METHODS Microorganisms Azospirillum lipoferum (Nitrogen Fixer) and Pseudomonas fluorescence were procured from the Bannari Amman Sugars-BIOLAB, Sathyamangalam. Azospirillum lipoferum was maintained on Nitrogen Free Bromothymol blue Malate (Nfb) (Deshwal et al., 2013). Optimization of the growth condition of bacterial strains The growth conditions such as pH, temperature and incubation period of Azospirillum lipoferum and Pseudomonas fluorescence were optimized and the data were analyzed using MAT lab Version-8.1.0.604 (Prema et al., 2013). Microbial analysis of different carriers Formulations of Powder carriers Various organic materials and agricultural wastes such as Saw dust, Rice husk and Date seeds powder were used for the mass multiplication of Azospirillum and Pseudomonas using the methodology of Marjan et al., 2011. Formulation of Liquid Carriers Vedic krishi inputs such as Matkakhad, Beejamrit, Jiwamrit were the liquid carriers formulated using the methodology of Sanjay et al., 2012. Shelf life of bioinoculants The shelf lives of bioinoculants were checked for the different carriers’viz. powder and liquid carriers by spread plate technique. MATLAB Version-8.1.0.604 is used to analyse the shelf life of carriers.(Jorjani et al., 2011). Field study Field performance of each formulations both liquid and powder has checked using Shallot (small onion, collected from Sathyamangalam). Different morphological characteristics for onion has scrutinized by ANOVA Agres Stat Version 3.1. Least Significant Difference test (LSD) at 1 % probability level was applied to compare the differences among treatment mean values. RESULTS AND DISCUSSION The culture of Azopirllum lipoferum and Pseudomonas fluorescens were maintained in Nitrogen free Bromothymol blue (Nfb) medium and King’s B medium respectively Optimization of growth condition for Bacterial strains Azospirillum lipoferum maintained in Nfb medium showed maximum growth at pH 7, in 31oC after 74 hours of incubation (Figure 1A, 1B, 1C). Motiur et al., 2006 studied that the isolates of Azospirillum,MR-3, MR-4 and MR-8 showed maximum growth at 41°C and rest of the isolates showed maximum growth between 35° and 37°C. The isolates MR-1, MR-3, MR-4, MR-7, MR-8 and MR-13 showed optimum growth at pH 8.0 and the isolates MR-6, MR-11, MR-14, MR-15 and MR-16 showed optimum growth at pH 7.0, A.amazonense (MR-5) showed optimal growth at pH 6.5. The optimal incubation period, pH, temperature for Pseudomonas fluorescens was found to be 8, 300 C and 72 hours (Figure 2A, 2B, 2C) respectively. Prema et al., (2013) optimised Pseudomonas culture for the maximum production of siderophore with optimal medium composition such as 0.5 µM iron, 55 µM glucose, 30oC, pH 7.0andincubation time of 72 hrs. 672 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 1. Optimization of pH, Temperature and Incubation period of Azospirillum lipoferum Figure 2. Optimization of pH, Temperature and incubation period of Pseudomonas fluorescens Shelf life study for Bioinoculants Shelf life study of 120 days for Azospirillum lipoferum and Pseudomonas fluorescence in powder carrier’s material is given in Figure 3-A, B. Among the three powder carriers used saw dust was proved to have highest cell count of 109cfu/ml for both the organisms. Figure 3. Shelf life study for powder carriers using MATLAB A) Azospirillum lipoferum B) Pseudomonas flouroscences 2C - Incubation period (Pseudomonas) 2B - Temperature (Pseudomonas) 2A - pH (Pseudomonas) 1C- Incubation period (Azospirillum) 1A–pH (Azospirillum) 1B–Temperature (Azospirillum) 673 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Gandhi et al., 2009 reported the effect of vermicompost in maintaining the shelf life of bioinoculant such as Azospirillum lipoferum, Bacillus megaterium and Pseudomonas fluorescens after 12 months in comparison with lignite carrier. Among Vermicompost based bioinoculants, B.megaterium showed maximum population of 7.60 x 108 cfu/g dry weight on 360th day followed by Pseudomonas 108 cfu/g dry weight respectively. Liquid Formulations Shelf life study of 120 days for Azospirillum lipoferum and Pseudomonas fluorescence in liquid carrier material is given in Figure 4-A, B. Among the three liquid carriers used Matka khad was proved to have highest cell count of 109cfu/ml for both the organisms. Figure 4. Shelf life study for liquid carriers using MATLAB Kavi et al., 2014 reported the survival of these three PGPR strains in liquid formulations amended with additives PVP, trehalose and glycerol for a period of six months storage. The results revealed that the required population (1×108 cells /ml) of saline tolerant strains was maintained both in carriers and in liquid based formulation. Morphological characters for onion Powder carriers for Azospirillum lipoferum and Pseudomonas fluorescence (soil treatment). The analysis of variance is presented in Table 1. Table 1. Analysis of variance for powder carriers *Values are mean of the duplicate. P.C-Powder carriers, SD-Saw Dust, RH-Rice Husk, DSP-Date Seeds Powder; S-soil, C- Control. Leaf length Among the carriers, Rice husk has shown the best carrier treatment than saw dust and date seeds powder (Figure 5). Leaf number There is no significant difference among all the carriers and the concentration. Plant height Among the carriers, Rice husk has shown the best carrier treatment than saw dust and date seeds powder. C4 &C5 of the entire carrier has shown best treatmentsthanC1, C2 & C3. Root length There is no significant difference among all the carriers and the concentration (Table 2). Azospirillum lipoferum(S)+Pseudomonas fluorescens(S) Leaf length(cm) Leaf number Plant height(cm) Root length(cm) P.C/ Vol.(g) SD RH DSP SD RH DSP SD RH DSP SD RH DSP C 32.9000 32.9000 32.9000 7.0000 7.0000 7.0000 44.3000 44.3000 44.3000 9.0000 9.0000 9.0000 0.5(C1) 28.1500 32.1000 25.1500 7.5000 12.5000 9.5000 51.4500 51.5500 42.3000 21.1000 17.2000 15.1500 1.0 (C2) 27.5500 35.0000 33.2000 10.0000 12.5000 13.5000 51.5500 55.7500 52.6000 21.4000 18.3500 17.1000 1.5 (C3) 30.3500 36.0000 34.8000 11.5000 13.0000 14.0000 54.8833 57.5000 58.6000 22.1500 19.0000 21.2000 2.0 (C4) 30.6500 36.9500 34.9000 12.0000 14.0000 16.0000 56.4000 59.9500 59.3500 23.2500 20.5000 23.2000 SED= 2.15896 CD(0.01)= 5.93714 SED= 2.88675 CD(0.01)= 7.93857 SED= 5.34653 CD(0.01)= 14.70295 SED= 3.31937 CD(0.01)= 9.12827 A) Azospirillum lipoferum B) Pseudomonas flouroscences 674 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 5. Plant growth parameters-A) Leaf length, B) Leaf number, C) Plant height & D) Root length Table 2. Analysis of variance for powder carriers *Values are mean of the duplicate. P.C-Powder carriers, SD-Saw Dust, RH-Rice Husk, DSP-Date Seeds Powder, S-soil, C-Control Bulb weight Among the carriers, Date seeds power has shown the best treatment than rice husk and saw dust.C4 & C5 have shown the best treatment than control (Figure 6). Bulb diameter There is no significant difference among all the carriers. C4 & C5 have shown best treatments than control. Neck diameter There is no significant difference among all the carriers. C4 & C5 have shown best treatments than control. Azospirillum lipoferum(S)+Pseudomonas fluorescens(S) Bulb weight(g) Bulb diameter(cm) Neck diameter(cm) P.C /Vol. (g). SD RH DSP SD RH DSP SD RH DSP C 2.2400 2.2400 2.2400 4.6000 4.6000 4.6000 3.0000 3.0000 3.0000 0.5 (C1) 5.6133 4.7333 3.1533 6.7500 6.2000 5.8500 3.7500 3.7000 2.6000 1.0 (C2) 5.7733 6.5300 9.9000 6.1333 6.8500 7.7000 3.8000 4.2000 4.6000 1.5 (C3) 10.0033 6.2100 10.9433 9.4000 7.4500 7.0500 5.8000 4.8500 4.9000 2.0 (C4) 10.4400 7.7933 11.2033 9.7000 7.7500 7.5433 6.7500 5.0500 5.0000 SED= 1.47656 CD(0.01)=4.06054 SED=0.84977 CD(0.01)=2.33686 SED=0.83240 CD(0.01)=2.28910 A) Leaf length B) Leaf number C) Plant height D) Root length 675 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 6. Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter Powder carriers for Azospirillum lipoferum and Pseudomonas fluorescence (foliar spray treatment) The results are summarized in Table 3. Table 3. ANOVA of growth parameter for powder carrier *Values are mean of the duplicate SD – Saw dust, RH – Rice husk, DSP – Date seed powder, FS-Foliar spray, C-Control Leaf length After analysis it has been found that among the carriers saw dust has shown the best treatment. C4, C5 (1.5 gm, 2 gm) of saw dust have shown the best result and C2,C3 (0.5 gm, 1 gm) have shown poor result. Date seed powder have shown the poor treatment among the entire carrier. C2,C1 (0.5 gm and control) of Date seed and control of rice husk have shown the poor treatment (Figure 7). Leaf number While considering leaf number among the carrier Rice husk and date seed powder have shown the best treatment and Saw dust has shown the poor treatment.C4,C5 (1.5 gm, 2 gm) of all the carrier have shown best result and control has shown the poor treatment. Plant height Similar to leaf length, plant height also high in Saw dust compared to rice husk and date seed carriers. Control of Rice husk, Date seed and Saw dust and C2 (0.5 gm) of Saw dust have shown the poor result. Azospirillum lipoferum(FS)+Pseudomonas fluorescens(FS) Leaf length(cm) Leaf number Plant height(cm) Root length(cm) P.C./Vol .(g) SD RH DSP SD RH DSP SD RH DSP SD RH DSP C 32.9000 32.9000 32.9000 7.0000 7.0000 7.0000 44.3000 44.3000 44.3000 9.0000 9.0000 9.0000 0.5 (C1) 28.8500 34.3167 25.1000 9.5000 9.5000 9.5000 49.7000 54.1000 44.1300 18.8000 20.2500 16.8800 1.0 (C2) 29.9500 33.1500 32.2500 10.5000 11.0000 14.0000 51.3000 56.3000 53.1700 18.9500 21.0500 18.4700 1.5 (C3) 30.3500 35.7500 35.0000 11.5000 13.5000 14.0000 54.7000 63.4000 58.1067 22.0500 25.0000 22.1733 2.0 (C4) 31.1833 38.6500 37.1000 14.0000 14.5000 16.0000 58.3500 67.8000 64.4500 23.4500 26.4500 24.0500 SED= 2.83713 CD(0.01)= 7.80210 SED= 2.24846 CD(0.01)= 6.18325 SED= 3.90456 CD(0.01)= 10.73754 SED= 1.98247 CD(0.01)= 5.45180 A) Bulb weight B) Bulb diameter C) Neck diameter 676 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Root length While considering root length there is no significant difference among all the carriers. But among the concentration, C4 and C5 (1.5 gm, 2 gm) of all carriers have shown the best result than that of control. Figure 7- Plant growth parameters-A) Leaf length, B) Leaf number, C) Plant height & D) Root length Bulb weight After analyzing it has found that among the carrier Saw dust and Date seed have shown the best result while Rice husk has shown the poor result. Among the volume compared to control, C3, C4 and C5 (1.0 gm, 1.5 gm, 2 gm) have shown best treatment (Table 4 and Figure 8). Bulb diameter While considering bulb diameter there is no significant difference in the carrier but among the volume C3, C4 and C5 (1 gm, 1.5 gm, 2 gm) have shown best result than that of C2 (0.5 gm) and control. Neck diameter Date seed powder and Rice husk have shown highest neck diameter compared to saw dust carrier. Among the volume C5 (2 gm) of all carriers shown better result while control and C2 (0.5 gm) have reported to be poor treatment. Table 4. ANOVA of growth parameter for powder carrier *Values are mean of the duplicate. P.C-Powder carriers, SD-Saw Dust, RH-Rice Husk, DSP-Date Seeds Powder, FS- Foliar Spray , C- Control Azospirillum lipoferum(FS)+Pseudomonas fluorescens(FS) Bulb weight(g) Bulb diameter(cm) Neck diameter(cm) P.C / Vol. (g). SD RH DSP SD RH DSP SD RH DSP C 2.2400 2.2400 2.2400 4.6000 4.6000 4.6000 3.0000 3.0000 3.0000 0.5 (C1) 4.1833 5.0633 4.8300 5.2500 7.4500 5.6500 4.0000 4.1000 2.8500 1.0 (C2) 4.7300 6.3833 10.2433 5.3500 8.2000 8.0500 4.5500 4.0000 4.4500 1.5 (C3) 5.4300 7.2833 10.2400 6.4000 8.3000 8.2000 4.6000 4.4000 4.6000 2.0 (C4) 6.7500 8.2233 12.9700 6.6000 9.3500 9.1000 5.9000 5.3167 4.7000 SED= 1.77646 CD(0.01)= 4.88527 SED= 1.01336 CD(0.01)= 2.78673 SED= 0.96686 CD(0.01)= 2.65886 A) Leaf length B) Leaf number C) Plant height D) Root length 677 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 8. Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter Liquid Carriers for Azospirillum lipoferum and Pseudomonas fluorescence (soil treatment) The results are presented in Table 5 and Figure 9. Leaf length Among the carriers, Rice husk has shown the best carrier treatment than saw dust and date seed powder. Leaf number There is no significant difference among all the carriers and the concentration. Plant height Among the carriers, Rice husk has shown the best carrier treatment than saw dust and date seed powder. C4 & C5 of all the carrier have shown best treatments than C1, C2 & C3. Root length There is no significant difference among all the carriers and the concentration. Table 5. Analysis of variance for liquid carriers *Values are mean of the duplicate. L.C.-Liquid carriers, MK-Matka khad, BM-Beejamrit, JM-Jiwamrit, C-Control, S-Soil Azospirillum lipoferum(S)+Pseudomonas fluorescens(S) Leaf length(cm) Leaf number Plant height(cm) Root length(cm) L.C./ Vol.(ml) MK BM JM MK BM JM MK BM JM MK BM JM C 31.8000 31.8000 31.8000 11.0000 11.0000 11.0000 50.9000 50.9000 50.9000 16.9000 16.9000 16.9000 0.5 (C1) 28.3500 32.4000 25.6500 12.5000 12.0000 7.5000 47.9500 51.4500 42.0500 17.3000 17.2000 14.5000 1.0 (C2) 28.5500 34.4000 26.7500 13.0000 13.5000 11.0000 48.5167 57.7333 44.4000 17.1000 21.1000 15.5000 1.5 (C3) 28.7000 37.1000 30.8500 15.5000 14.5000 12.0000 50.5500 62.6500 51.4500 19.9500 23.1500 18.3000 2.0 (C4) 31.6500 37.3500 33.4000 10.0000 15.0000 14.0000 53.8500 64.8000 58.7667 20.2000 18.6167 23.3800 SED= 2.93096 CD(0.01)= 8.06015 SED= 2.00278 CD(0.01)= 5.50763 SED= 4.33611 CD(0.01)= 11.92431 SED= 3.83832 CD(0.01)= 10.55538 A) Bulb weight B) Bulb diameter C) Neck diameter 678 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 9. Plant growth parameters-A) leaf length, B) leaf number, C) plant height & D) Root length Bulb weight Among the carriers, C2, C3, C4 & C5 of saw dust and C4 & C5 of Date seeds power have shown the best treatment.C2 & C3of date seeds powder have shown the poor treatment (Table 6 and Figure 10). Bulb diameter C1, C3, C4 & C5 of saw dust and Control of Date seeds power and rice husk have shown the best treatment. C2 of rice huskand C2& C3 of date seeds powder have shown the poor treatment. Neck diameter C4 & C5 of saw dust have shown best treatments than C2 of date seeds powder. Table 6. Analysis of variance for liquid carriers *Values are mean of the duplicate. P.C-Powder carriers, SD-Saw Dust, RH-Rice Husk, DSP-Date Seeds Powder,C-Control Azospirillum lipoferum(S)+Pseudomonas fluorescens(S) Bulb weight(g) Bulb diameter(cm) Neck diameter(cm) L.C./Vol .(ml) MK BM JM MK BM JM MK BM JM C 7.3500 7.3500 7.3500 8.5000 8.5000 8.5000 3.6000 3.6000 3.6000 0.5 (C1) 9.6733 4.7200 1.4500 7.3000 4.5333 3.3000 4.0500 2.7500 1.7000 1.0 (C2) 10.0300 5.3700 3.6833 7.4333 5.9500 4.4000 5.1500 3.2000 2.3500 1.5 (C3) 10.3733 6.2133 7.7300 7.7500 6.2500 7.0000 5.7000 3.3000 3.4000 2.0 (C4) 10.4100 6.2300 8.1800 8.6000 6.3000 7.0500 6.0500 3.6000 3.7500 SED= 1.39437 CD(0.01)= 3.83450 SED= 0.63584 CD(0.01)= 1.74857 SED= 0.17575 CD(0.01)= 0.48332 A) Leaf length B) Leaf number C) Plant height D) Root length 679 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 10. Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter Liquid Carriers for Azospirillum lipoferum and Pseudomonas fluorescence (foliar spray treatment) Leaf length There is no significant difference among the different liquid formulation. All the carriers shown similar results in this parameter. But among the volume C3,C4,C5 (1.0 ml, 1.5 ml, 2.0 ml) shows the best result and C2 (0.5 ml) have shown the poor result (Table 7 and Figure 11). Leaf number While considering this parameter, among the carrier Beejamrit has shown high significance and best result while Jeewamrit and Matkakhad have shown the poor result. Among the concentration C2, C3 that is 0.5 ml and 1.0 ml have shown poor treatment and C4, C5 (1.5 ml, 2.0 ml) has reported to be the best treatment. Plant height According to the analysis of the data it has been found that there is no significant difference among the carrier but among the concentration C4, C5 (1.0 ml, and 2.0 ml) have shown the best treatment and C2 (0.5 ml) has shown the poor treatment. Root length While considering root length, there is no significant difference among the carrier, but among the concentration C1,C2,C3 have (control, 0.5 ml, 1.0 ml) have shown poor treatment and C4, C5 (1.5 ml, 2.0 ml) has shown the best treatment. Table 7. Analysis of variance for liquid carriers *Values are mean of the duplicate. L.C.-Liquid carriers, MK-Matka khad, BM-Beejamrit, JM-Jiwamrit, C-Control, FS-Foliar spray Azospirillum lipoferum(FS)+Pseudomonas fluorescens(FS) Leaf length(cm) Leaf number Plant height(cm) Root length(cm) L.C./ Vol.(ml) MK BM JM MK BM JM MK BM JM MK BM JM C 31.8000 31.8000 31.8000 11.0000 11.0000 11.0000 50.9000 50.9000 50.9000 16.9000 16.9000 16.9000 0.5 (C1) 30.8500 30.8000 26.9333 7.0000 7.5000 10.0000 130.8500 45.7000 49.2000 17.4000 13.1000 20.5000 1.0 (C2) 31.0500 32.4000 29.2500 9.5000 13.0000 10.5000 31.0500 51.1000 54.4000 17.7000 16.4500 23.5500 1.5 (C3) 31.9500 33.6500 29.4500 10.5000 13.0000 11.6667 31.9500 51.5500 55.6000 18.0500 17.8000 23.6500 2.0 (C4) 33.1000 43.8500 35.1500 12.5000 20.0000 12.0000 33.1000 66.2000 66.1500 19.2667 19.9500 28.4500 SED= 3.24135 CD(0.01)= 8.91372 SED= 3.10972 CD(0.01)= 8.55174 SED= 37.91621 CD(0.01)= 104.26957 SED= 2.40137 CD(0.01)= 6.60378 680 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 11. Plant growth parameters-A) leaf length, B) Leaf number, C) Plant height & D) Root length Bulb weight Among the carrier Beejamrit has shown significant difference and C5 (2ml) of Beejamrit has shown the best result. C2 and C3 (0.5 ml, and 1.0 ml) of Matka khad and C2 (0.5 ml) of jeewamrit has shown the poor result (Table 8 and Figure 12) Bulb diameter After the analysis it is found that among the carriers Beejamrit has shown good results. C3, C4 and C5 (1.0 ml, 1.5 ml, 2.0 ml) of Beejamrit have shown the best treatment and Matkakhad’s C3 and C2 (1.0 ml, 0.5 ml) have shown the poor result. Neck diameter While considering this parameter, among the carrier Matkakhad has shown the poor treatment but Jeewamrit and Beejamrit have shown the best treatment. Among the volume C4 and C5 (1.5 ml, 2.0 ml) has shown good result and the C2 and C3 (0.5 ml, 1.0 ml) have shown poor result. It is been reported that Pseudomonas fluorescens and other PGPRs induced a significant increase in root and shoot length, nodules, weight and even protein content in Mungbean plant (Dhanya et al., 2014, Heidari et al., 2014, Maiyappan et al., 2010 ). It is also found that Strains of Pseudomonas putida and Pseudomonas fluorescens have increased root and shoot elongation in canola, lettuce, tomato and also yields in potato, radishes, rice, sugar beet, tomato, lettuce, apple, citrus, beans, ornamental plants, and wheat (Brahmaprakash et al.,2012). Table 8. ANOVA of yield parameters for liquid carrier *Values are mean of the duplicate. L.C.-Liquid carriers, MK-Matka khad, BM-Beejamrit, JM-Jiwamrit, C-Control, FS- Foliar spray Azospirillum lipoferum(FS)+Pseudomonas fluorescens(FS) Bulb weight(g) Bulb diameter(cm) Neck diameter(cm) L.C./Vol.(ml) MK BM JM MK BM JM MK BM JM C 7.3500 7.3500 7.3500 8.5000 8.5000 8.5000 3.6000 3.6000 3.6000 0.5 (C1) 2.5400 4.1933 3.2133 3.6000 3.8500 4.4333 3.0000 3.0000 3.3000 1.0 (C2) 3.3100 8.9833 3.7833 5.9500 6.2500 5.9000 3.1500 4.3500 3.4333 1.5 (C3) 5.9200 9.2200 3.8400 6.7500 6.5500 5.9333 4.0000 4.5500 3.5000 2.0 (C4) 8.0267 12.1700 9.0833 7.3500 6.7000 7.1000 4.4833 4.9000 3.7500 SED= 2.00266 CD(0.01)= 5.50730 SED= 0.91364 CD(0.01)= 2.51251 SED= 0.67245 CD(0.01)= 1.84923 A) Leaf length B) Leaf number C) Plant height D) Root length 681 Development and formulation of Azospirillum lipoferum… TAMILSELVI, S. Biosci. J., Uberlândia, v. 32, n. 3, p. 670-683, May/June. 2016 Figure 12. Analysis of yield parameter for liquid carrier CONCLUSIONS The highest cell concentration of 109 CFU was obtained for A.lipoferum at pH 7, in 31oC after 74 hours of incubation and for Pseudomonas fluorescens maximum cell concentration is obtained at pH 8, temperature 30 ̊ C and 72 hours of incubation respectively. Different effective formulation of both powder and liquid carriers such as saw dust, rice husk, and date seed powder, Matka khad, Jiwamrit and Beejamrit respectively, were mixed with the optimised culture, which promotes the growth of the bacteria and produce an effective biofertilizer. Among the carriers (powder and liquid), highest cell viability was obtained in saw dust and Matka khad even after 120 days of storage. For powder formulations, most of the carriers shown same result than control plant. Rice husk and date seeds powder have shown best result than saw dust.. Saw dust and Matka khad were the effective powder and liquid carrier formulations respectively with highest cell viability after 120 days of storage. With respect to field performance such as yield and growth parameters, Saw dust and Beejamrit have proved be the best powder and liquid carriers. Thus biofertilizers has proven to be effective source than chemical fertilizers and was considered to be safe for practising agriculture naturally. RESUMO: Biofertilizante é um grupo de microorganismos benéficos utilizados para melhorar a produtividade do solo através da fixação de azoto atmosférico ou por solubilização de fósforo no solo. Eles também estimulam o crescimento vegetal através de síntese de substâncias promotoras do crescimento. No presente estudo, Azospirillum lipoferum é cultivado em um meio de azul de bromotimol sem nitrogênio (Nfb) e Pseudomonas fluorescens num meio de King's B. A condição de bioprocesso foi optimizada para ambas as culturas e descobriram que Pseudomonas fluorescens mostraram maior crescimento a 300ºC em pH 8 após 72 horas de incubação, enquanto que Azospirillum lipoferum mostraram maior concentração de células a 310ºC em pH 7, com um período de incubação de 72 horas. A cultura optimizada é misturada com diferentes formulações de pó e veículo líquido tal como serragem, casca de arroz, pó de semente de tâmaras, Matka khad, Jiwamrit e Beejamrit respectivamente. O estudo do prazo de validade para 0, 30, 60, 90 e 120 dias por contagem celular e método de espalhamento em placa mostrou que o prazo de validade do biofertilizante produzido a partir do pó e veículos líquidos teve grande quantidade de população microbiana viável até 120 dias de armazenamento. 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