Isolation and Screening of Multifunctional Plant Growth Promoting Rhizobacteria (PGPR) from Onion Rhizosphere (Allium cepa)

Plant Growth Promoting Rhizobacteria (PGPR) can enhance the growth and productivity of plants through direct and indirect mechanisms. In this study, rhizobacteria were isolated from onion rhizosphere and their plant growth promoting activities were studied. The investigations of activities include: nitrogen fixation, phosphate solubilization, zinc mobilization, hydrogen cyanide and indole-3-acetic acid production abilities. Plant growth promoting rhizobacteria (PGPR) influence plant health and productivity by various ways. It can be summarized that the isolated multifunctional PGPR strains may have potential to be successful bio-inoculants for onion plant.


Introduction
Plant roots in the soil show profuse activity relative to the bulk soil. pH, nutrients and exudates gradients changing as distance from the root increases [1]. This region of gradients in chemical and physical factors strongly influenced by the presence of plant roots and characterized by high rates of microbial population and activity is increasingly referred to as the rhizosphere. In fact, in 1904, Hiltner first defined the rhizosphere as that zone of soil in which the micro flora is influenced by plant roots [2].
Microorganisms are essential for maintenance of sustainable ecosystems and bio-diversity. Colonization of the plant root system is the very first step in nearly all interactions between plants and soilborne microbes. Plant growth promoting rhizobacteria (PGPR) influences plant health and productivity by various ways. A good selection of a PGPR strain requires understanding of bacterial communities colonizing the rhizosphere. To date, only limited information exists on microbial diversity and dynamic of population in agricultural soil [3]. The properties related to the PGP character are auxin production, nitrogen fixation, phytopathogen antagonism, cyanogenesis, phosphate solubilization and amino cyclopropane carboxylic acid (ACC) deaminase activity [4]. Indian economy is agricultural based. But the use of chemical fertilizer has led to a considerable increase in agricultural productivity. The use of PGPR gives better way to replace chemical fertilizer, pesticides. PGPR help in the disease control in plants. PGPR especially if they are inoculated on the seed before planting, can establish themselves on the crop roots this is possible with onion crop as it is re-planted. Beneficial soil microbes are studied and exploited for the development of sustainable agriculture. The purpose of manipulating crop rhizosphere microbial populations by inoculation of beneficial bacteria to increase plant growth. It has shown considerable promise in laboratory and greenhouse studies. The important environmental benefits of this approach, leading to a reduction in the use of agricultural chemicals and the fit with sustainable management practices, are driving force of this project.
The objectives of this study are to isolate bacteria from onion root rhizosphere, to isolate plant growth promoting rhizobacteria with multifunctional ability and to evaluate and analyze growth promoting ability under in-vitro conditions.

Sampling
The sample used to isolate Rhizobacteria is rhizosphere soil sample around Onion roots. Onion root washed with water to remove adhering soil particles. Roots washed in saline suspension taken in 5 sterile tubes one after the another aseptically. Washing collected and mixed thoroughly with the help of Vortex mixture.

Isolation
10% Nutrient Agar, Pikovaskaya medium, Solid Extract Agar, Norris broth were used for isolation. Loop full of each washing aseptically spread on the sterile 10% Nutrient agar plates i.e., 10% Salt added to Nutrient Agar and on 10% Soil Extract Agar plates. Incubated these plates for 48 hours at Room temperature. Growth observed in the form of number of different colonies on both the medium (Figure 1). Then transferred those colonies on Nutrient Agar slant with appropriate label and stored them in refrigerator for further tests.

Screening
Phosphate solubilizing capacity of each isolate checked by making grids on the Pikovskaya's agar medium (Table 1), the activity was checked by spot inoculating the isolates. Zone of clearance taken to be the positive test (Figure 2).  Zn solubilizing property of isolates checked by adding zinc oxide in Nutrient Agar as per above procedure ( Figure 3).

Gms/Litre
Test for nitrogen fixation was done by inoculating isolates in Nitrogen free medium (  Then isolate was tested for Indole Acetic Acid Production. IAA or Indole acetic acid is plant growth promoting hormone. For the experiment, each isolate was inoculated in Tryptone broth. After incubation 1 ml of Broth was taken in Eppendorf tube and allowed to centrifuge for 2 min at about 15000 rpm. The supernatant was then taken and to it Salkowaskys Reagent was added. The reaction between reagent and IAA produced by isolate produced the characteristic red color which is taken as confirm Positive test. The last test performed was to detect whether the Isolate can produce HCN gas or not. For the test the organism was grown on Glycerin containing medium and then on the upper part of test tube the Picric acid paper was placed. The tubes were then incubated at Room Temperature for 48 hours. The reaction of HCN Gas with picric acid imparts red color to the Paper. This was taken as positive test (Figure 4).
However, the cultures 14, 15, 22, 26, 27, 44 are of no use because they show property of production of toxic HCN gas which can inhibit the growth of beneficial organisms in the vicinity.

Conclusion
Undoubtedly there are numerous and complicated interaction that occur in the rhizosphere. High degrees of activity by both the plant roots and the microbial community are often difficult to conclusively characterize. Does the microbial community drive the structure and function of the plant community, or vice versa? Is there a constant coevolution and simultaneous succession of both communities? Further research is clearly necessary.