DEVELOPMENT AND FORMULATION OF Azospirillum lipoferum AND Pseudomonas fluorescens AS EFFECTIVE BIOLOGICAL AGENTS FOR ENHANCED AGRO-PRODUCTIVITY DESENVOLVIMENTO

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 30C in pH 8 after 72 hours of incubation where as Azospirillum lipoferum showed highest cell concentration at 31C 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 77x10cfu/ml for Azospirillum lipoferum and 72 x 10 CFU/ml for Pseudomonas strain on 120 day and the liquid carrier Matka khad showed 85x10 cfu/ml for Azospirillum lipoferum and 78 x 10 CFU/ml for Pseudomonas fluorescens.


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) (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.

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
The optimal incubation period, pH, temperature for Pseudomonas fluorescens was found to be 8, 30 0 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, 30 o C, pH 7.0andincubation time of 72 hrs.

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 10 9 cfu/ml for both the organisms.1B-Temperature (Azospirillum) 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 10 8 cfu/g dry weight on 360 th day followed by Pseudomonas 10 8 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 10 9 cfu/ml for both the organisms.The analysis of variance is presented in Table 1.

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).

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.The results are summarized in Table 3.

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.

Leaf length(cm)
Leaf

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.

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.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.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.

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.

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).

CONCLUSIONS
The highest cell concentration of 10 9 CFU was obtained for A.lipoferum at pH 7, in 31 o C after 74 hours of incubation and for Pseudomonas fluorescens maximum cell concentration is obtained at pH 8, temperature 30̊ 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.

Figure 3 .
Figure 3. Shelf life study for powder carriers using MATLAB

Figure 4 .
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 (1×10 8 cells /ml) of saline tolerant strains was maintained both in carriers and in liquid based formulation.

Figure 6 .
Figure 6.Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter

Figure 8 .
Figure 8. Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter

Figure 9 .
Figure 9. Plant growth parameters-A) leaf length, B) leaf number, C) plant height & D) Root length

Figure 10 .
Figure 10.Yield parameters are a) Bulb weight, b) Bulb diameter & c) Neck diameter

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.

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

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

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