Innoculant technology in Populus deltoides rhizosphere for effective bioremediation of Polyaromatic hydrocarbons (PAHs) in contaminated soil, Northern India

Four bacterial strains viz. Kurthia sp. SBA4, Micrococcus varians SBA8, Deinococcus radiodurans SBA6 and Bacillus circulans SBA12 identified as Polyaromatic hydrocarbons (PAH) degrader, isolated form the rhizospheric soil of Populus deltoides growing in non-contaminated sites of Uttarakhand Himalyan region, India. Out of these four isolates, M. varians SBA8 degraded appreciable amount of PAH along with some plant growth promoting properties viz. indole acetic acid (IAA) 19 μg ml, phosphate solubilization 1.8 μg ml, produced siderophore and possessed ACC deaminase activity. Along with these traits, M. varians SBA8 also exhibited biocontrol activities against phytopathogenic fungi Rhizoctonia solanii, Macrophomina phaseolina and Fusarium oxysporum with 50, 43.3 and 34.6 percent growth inhibition (PGI). Moreover, mycelial deformities were also observed in the test fungal spp. The M. varians SBA8 proved to be competent rhizobacteria in rhizosphere niche in treatments T1 (Sterile soil + plant cuttings + bacterization) and T2 (sterile soil + anthracene + plant cuttings + bacterization). Rhizoremediation potential of M. varians SBA8 was also determined in PAH amended soil model system. Significant enhancement in shoot, root length, root and shoot biomass including stem girth of P. deltoides with respect to control was recorded and concurrently the bacterium degraded 43.6 % of PAH as determined by HPLC analysis in soil model system after 120 days of inoculation. Multipurpose inoculant was also formulated using M. varians SBA8 strain immobilized in different lignocellulosic waste material used as carriers. Rock phosphate, cocoa peat and wheat bran based bioinoculant were found suitable for rhizoremediation of PAH.


Introduction
Over the last century, industrialized global economy has led to dramatically increase in production of toxic anthropogenic pollutants into the environment, causing widespread contamination of soil and water. Such anthropogenic activities which release huge amounts of petroleum hydrocarbons (PHC), polyaromatic hydrocarbons (PAH), halogenated hydrocarbons, pesticides, solvents, metals, and salt, havecaused deleterious demage on human and ecosystems (Meagher, 2000). Out of these contaminants, polyaromatic hydrocarbons (PAHs) are major hazardous environmental pollutants that possess carcinogenic and mutagenic properties (Menzie and Potokib, 1992;Wilson and Jones, 1993). Basically, PAH in the ecosystem spread through various ways viz. petrogenesis, fossil fuel combustion, waste incineration and by products of industrial processes that include coal gasification, production of metals, petroleum refining, component of wood preservatives, smokehouses, vehicles and wood stoves (Shuttleworth and Cerniglia, 1995;Wingfors et al., 2001).
Remedial options using physico-chemical treatments are expensive and environmentally invasive, being inadequate for the treatment of large contaminated sites (Cunningham and Ow, 1996). However, bioremediation is the perfect tool for hazardous compounds with less input of chemicals, energy and time, using suitable microbes (Yuan et al., 2002). Recently, the synergistic use of plants and microbes to clean up the PAH-contaminated soil generated encouraging results (Glick, 2003;Huang et al., 2004), as the rhizosphere of plant provides better environment for bacteria to survive and proliferate (Yee et al., 1998). Very few studies have been reported regarding the directed introduction of a microbial strain for PAH degrading activities on plant seed to colonize the root and proliferate on the root system (Kuiper et al., 2001;Spirang et al., 2002). Rhizoremediation treatment can be successfully exploited by using plant growth-promoting and bioprotecting rhizobacteria. Along with bioremediation plant growth promoting bacteria are known to participate in many other important ecosystem interventions, such as the biological control of plant pathogens, nutrient cycling and/or seedling growth (Zahir et al., 2004). Rhizobacteria that produce indole acetic acid (IAA), siderophores, hydrogen cyanic acid (HCN), 1-Aminocyclopropane-1-carboxylate (ACC) deaminase and solubilize phosphate activity which are capable of stimulating plant growth (Glicket al., 1998;Rajkumar et al., 2006) are suitable biological agents for plant growth as well as bioremediation.
To take advantage of the plant-bacterium relationship for degrading PAH compounds in rhizosphere Populus deltoides (deciduous plant) was used as bio-injector of pollutant degrading bacteria. P. deltoides has a number of advantages such as a highly branched and deep root system; therefore it can be used as a vector/ bio-injectior for root-colonizing bacteria, for the penetration of impermeable soil layer and transport of bacteria. Moreover, its roots can exude up to 35 % of their photosynthate as exudate carbon (Gregory and Atwell, 1991) release oxygen which provides better redox conditions and has been widely used for phytoremediation purposes. However, little work has been done on PGPR activities of forest plants in PAH contaminated soil by using selected rhizosphere-competent PAH degrading strains (Bisht et al., 2014). In the light of above facts, the present study was envisaged to utilize a beneficial plant-microbe pair for effective rhizoremediation of PAH in soil model system using P. deltoides and previously studied selected soil bacterial strains.
Finally a carrier based multipurpose bioinoculant was formulated for proper dissemination in agriculture system especially for PAH contaminated sites.

Materials and Methods
In vitro plant growth promoting activities of PAH degrading soil isolates Four PAH degrading strains were isolated from the rhizosphere of P. deltoides growing in noncontaminated sites of Garhwal Himalayan regions (Bisht et al., 2010). These bacteria were further checked for plant growth promoting activities viz., indole-3-acetic acid (IAA) production, phosphate solubilization, siderophore, HCN (Hydrogen cyanic acid) and 1-Aminocyclopropane-1-carboxylate (ACC) deaminase production. Along with biocontrol activity against selected soil borne phytopathogenic fungal strains were also checked. These plant growth promoting activities were studied to screen among thesefour PAH degrading strains to act as potential rhizospheric competitor and beneficial plant growth promoter so that plant can be efficiently act as remediator agent for the pollutant used in our study.. IAA production was determined by Salkowski's method (Rodriguez et al., 2008). The potential to solubilize relatively insoluble tricalcium phosphate was checked by the method of Kumar and Narula (1999). HCN production was observed by following Miller and Higgins (1970) method. Siderophore production was detected qualitatively as described by Schwyn and Neilands (1987) and for ACC deaminase activity determination, Honma and Shimomura (1978) method was used.

In vitro antifungal activity of selected soil isolates
The test fungal strains Fusarium oxysporum and Fusarium solanii were procured from Microbial Type Culture Collection center (MTCC), Chandigarh, India (accession numbers-MTCC 284 and 350 respectively) while Macrophomina phaseolina, Rhizoctinia solanii and Sclerotinia sclerotium were provided by Forest Research Institute, Dehradun, India. In vitro fungal mycelium growth inhibition was observed by dual culture technique (Verma et al., 2001). The percentage growth inhibition (PGI) was calculated using the following formula: PGI = [(R -r)/R ×100] Where, r is the radius of the fungal growth opposite the bacterial colony and R is the maximum radius of the fungus growth away from the bacterial colony. Furthermore, fungal mycelia growing towards the zone of inhibition were processed for morphological studies. The mycelia were picked up from zone of inhibition and control plates, thereafter, transferred to a drop of lacto-phenol on a clean glass slide. Deformities, if any occurred due to antagonism were observed under the fluorescent microscope.

Survival of potential PAH degrading isolates under different osmotic stress regime
The efficient strains which had the ability to metabolize PAH, and further narrowed down for the plant growth promoting attributes were chosen for stresses of different salt concentrations i.e. 1, 1.5, 1.7, 1.9 and 2.1 percent using the method of Elsheikh and Wood (1990).

Determination of antibiotic susceptibility and cell surface hydrophobicity of PAH degrading isolates
Antibiotic resistance of selected PAH degrading isolates was determined by disc diffusion assay on Mueller Hinton agar using Himedia octodiscs. Results were recorded by observing and monitoring the zone of inhibition (if any), around the antibiotic discs. Cell surface hydrophobicity of isolates was determined by their adherence to hydrocarbons which is based on the partitioning of cells in a two phase system with a slight modification from Zhang and Miller (1994) method. For, this isolates were grown in nutrient broth and cells of exponential phase were collected by centrifugation at 5000xg for 15 min at 4 ºC, washed twice with sterile distilled water (SDW) to remove any interfering solutes and then resuspended in a 0.2 M phosphate buffer. The bacterial suspension (4.0 ml) with n-Heptanol (2 ml) was vortexed vigorously for 3 min in a test tube and left at room temperature for 30min. The aqueous phase was recovered with the help of pipette. The optical density was measured at 600nm and hydrophobicity was calculated by the formula: Percentage of adherence to hydrocarbon = 100 x 1-OD of aqueous phase OD of initial cell Field trial application of efficient PAH degrading isolates P. deltoides cutting bacterization The P. deltoides cuttings with uniform shape and size were selected and soaked in water overnight for pre-sowing treatment. Cuttings were surface-sterilized with 70% alcohol for 30 sec followed by treating with 0.1% HgCl 2 for one minute and then washings with sterile distilled water 5-6 times. Sterilized dried cuttings were bacterized with a slight modification as described by Shim et al. (2000).
All the pots were watered as and when required routinely and were kept outdoors and partially covered to protect the plants from rainfall. During cultivation, minimal and maximal temperature ranged from 24 to 38°C respectively and the experiment was conducted from 5 May 2010 to 5 September 2010, as described previously by Bisht et al. (2014). Observations on various growth parameters of P. deltoides were recorded such as shoot and root length, fresh and dried shoot and root biomass, stem girth and number of leaves after every 30 days. The data was analyzed using ANOVA, where the means of studied treatments were compared using LSD at P = 0.05 significant level. MSTAT-C software was used for computing the data.

Root colonization studies
Root colonization study by selected soil isolate SBA8 was determined by its intrinsic antibiotic resistant pattern. Bacterial strain was inoculated onto cutting of P. deltoides by bacterization method with 10 8 CFU/ml of target strain. To enumerate the viable cells, plants were carefully uprooted from their respective pots after 30, 60, 90 and 120 d of inoculation, and all root segments 5 mm below stem remnants were excised. This was done to ensure that only the bacteria that colonized the roots and not the bacteria that remained on the stem were assayed (Nautiyal, 1997). Roots were washed thoroughly with 0.85% sterile saline water, to remove the soil particles. The associated rhizosphere soil was studied to determine the population density of target strain with its antibiotic-resistant marker system along with other normal indigenous bacteria (NIB) in rhizosphere soil using serial dilution plate technique on nutrient Agar (NA) medium containing resistant antibiotic (100 µg/ml). A rhizosphere or root segment was considered to be colonized when the target microbe was detected on to NA plates after 24 -48 h of incubation at 27°C.

Quantitative removal of hydrocarbon in soil model system
For determination of residual anthracene content in soil, P. deltoides were uprooted and anthracene amended soils attached to roots were carefully collected from respective pots. The soil samples were air dried in the dark and the anthracene was extracted from rhizospheric soil as described by Gao and Zhu (2004) and anthracene biodegradation in soil was determined by HPLC (Shimadzu) analysis (Filonov et al., 2006).

Bioinoculant formulation
Saw dust, wheat bran, cocoa peat, sugar bagasse, rock phosphate and dried leaves of P. deltoides were used as solid lignocellulosic waste carrier material, although in present investigation the dried leaves of P. deltoides were also used as solid carrier material. For bioinoculant formulation, the test culture was grown in nutrient broth for 24 h at 150 rpm, the cells ≈ 10 6 CFU/ml was used for inoculants preparation (Somasegaran and Hoben, 1994). For preparation of carrier materials autoclavable, grinded and air dried material i.e. ≈40 g of materials were packed in polythene bags made up of 50-75 mm thick, low density and flexible sheets. The polythene bags were sealed leaving about 2/3 vacant space to give proper aeration to the inoculant. 1 ml of bacterial suspension was introduced aseptically with a hollow needle directly through the wall of the bag and the small hole was immediately covered with the help of self-adhesive tape. Immediately, after sealing, the inoculum and carriers were hand mixed by shaking or kneading between the fingers. The materials inside the sealed polythene bags were spread and kept at 27°C for 2 days for curing so as to attain maximum number of cells in the formulations. After curing, the polythene bags having inoculants were stored at room temperature in dark to prevent from U.V radiation and heat. Three replicates of each treatment were done. Physiochemical properties of carrier material such as inherent moisture content, water holding capacity (WHC), pH, total phosphorous, C/N ratio and total potassium were also measured in accordance to standard procedure (Page et al., 1982;Jimenez and Ladha, 1993).

Population dynamics of PAH degrading isolates in different substrate carrier based formulation
For inoculant preparation, selected isolates were grown in Nutrient broth at 27°C and when cell concentration exceeded 10 7 colony forming unit (CFU)/ml, it was inoculated into the carrier material (Somasegaran and Hoben, 1994). Samples were collected from bio-formulation after different time intervals under aseptic conditions and viable cells were determined by inoculating suitable dilutions on NA medium having antibiotic (100 µg/ml) and colony forming unit (CFU) were enumerated (Pandey and Maheshwari, 2007a,b). The CFU/g of inoculants was monitored after every 30 days for a period of 180 days.

Results
Four bacterial strains isolated from the rhizosphere of P. deltoides have already been identified as Kurthia sp. SBA4, Micrococcus varians SBA8, Deinococcus radiodurans SBA6 and Bacillus circulans SBA12 which have the ability to metabolize PAH and are chemotactically active against PAH (Bisht et al., 2010), were subjected for further rhizormediation studies. Among these four isolates, only one soil isolate M. varians SBA8 exhibited higher PGP and biocontrol activity, therefore, anthracene biodegradation studies was conducted with this bacterium only in PAH contaminated soil-model system.

Plant growth promoting attributes
On the basis of appreciable biodegradation abilities of PAH in solution, M. varians SBA8 was selected for further studies. IAA production and phosphate solubilization were quantified for this selected isolate. M. varians SBA8 produced maximum amount of IAA after 96h of incubation i.e. 19 µg.ml -1 (Figure 1). The IAA production profile of all the strains had a declining trend after 96h and kept falling subsequently. Apparently highest amount of phosphorous liberated by this efficient isolate was 1.8µg.ml -1 after 120h ( Figure  2). The pH of the broth also dipped downfrom 7.2 ± 0.5 to 4.4 ± 0.5 after 192 h respectively.

In vitro antagonism test and cell surface hydrophobicity
The M. varians SBA8 inhibited the growth of M. phaseolina, R. solani and F. oxysporum, by 43.3%, 50% and 34.6%, respectively ( Table 1). The mycelia picked from the interaction zone showed morphological deformities like lysis of hyphal cell, halo cell formation, loss of cytoplasm and granulation in hyphae (Figure 3). However, relative cell surface hydrophobicity of M. varians SBA8 isolate was observed in n-Heptanol and it was calculated as 20% of the percentage of the cells adsorbed to Heptanol phase.

Field study for rhizoremediation
Increase of 6.6% and 23.5% and 4.7% and 33.5% in shoot and root length was observed in T 1 and T 2 treatments after 60 days whereas, it remained 1.8% and 8.4% and 2.2% and 4.7% in the same treatments after 120 days. An increase of 6.6%, and 33.1% and 1.6% and 30.5% in fresh and dry shoot biomass was recorded in T 1 and T 2 treatments while 10.7% and 48.2% and 8.7% and 51.6% increase was obtained in fresh and dry root biomass in T 1 and T 2, respectively. Moreover, an increase of 2.8% and 10.5% in stem girth was observed in T 1 and T 2 treatments, respectively, after 120 days (Table 2). However, in treatment T 3 all the plants growth parameters were relatively less than control and other treatments due to application of PAH. -

Root colonization assay
For root colonization antibiotic marker strategies were performed for evaluation of CFU/ml count. Based on its intrinsic antibiotic resistance profile, M. varians SBA8 isolate was found to be resistant against a large number of antibiotics (Table 3) therefore; it was further screened to find out the tolerance limit to the maximum concentration of individual antibiotic. It was established that M. varians SBA8 showed highest resistance to erythromycin (100 µg/ml), hence abbreviated as M. varians SBA8 Erth+ . The result of root colonization assay of M. varians SBA8 suggested it as a good colonizer, which is a desirable property in a suitable PGPR. It was observed that increase in population count was maintained after 60 days in T 1 treatment then started decline whereas, continuous decrease in cell population was observed in T 2 treatment. The normal indigenous bacterial population (NIB) was comparatively higher till 90 days study (Table 4).

Viability of M. varians SBA8 in different solid lignocellulosic waste as a carrier Biodegradation of anthracene in soil
Rhizoremediation of anthracene with M. varians SBA8 was analyzed by HPLC analysis. It was noticed that the presence of several intermediate compounds were eluted at different retention times ranging from 1.2 to 28 min. The residual concentration of anthracene was determined by calculating the peak area relative to Anthracene concentration 10mg/2.5kg soil Isolate Percent Degradation standard with pure anthracene. M. varians SBA8 resulted in 43.6% degradation of anthracene after 120 days in treatment T 2 (Figure 4).
Under in vivo conditions, M. varians SBA8 showed an appreciable biodegradation activity and also possessed PGP attributes including antagonistic activity against soil borne phytopathogens. Therefore, suitable system was provided for proper reproduction and dispensing of this isolate for agriculture and other industrial uses. According to Indian standard: specification for rhizobial inoculants (1997), the survival of these potential isolates in different carriers were checked for 6 months, to find out the possibilities of effective carrier material for these multidimensional isolates for formulation. Analysis of bagasse for viable count showed that initial population was started with 10 8 CFU/g, then there was continuous ≈10 times decreases in population count in the first 60 days, after that it remain constant for next month and then a ≈10 4 times decline in population count were observed after 180 days. Similar trend was followed with wheat bran as it showed approx. constant cell density as the initial count i.e. ≈10 8 for 60 days after that it reduced to ≈10 3 times after 180 days respectively. Cocoa peat and rock phosphate proved out to be better carrier material as they gives ≈10 times increase in cell density for 60 days and then declined to ≈10 3 and ≈10 2 times respectively after 180 days. Saw dust was considered a moderate carrier material comparatively to saw dust and bagasse as it continued to ≈10 times decline after 4 months and then reduced to ≈10 3 times after 180 days. Dried leaves of P. deltoides showed an evidence of poor carrier material as there was a continuous reduction in CFU/g and after 120 days it showed no CFU/g (Table 5).

Discussion
Present investigation assessed the potential of a selected soil isolate to be used for rhizoremediation of PAH contaminated soil model system. However, this study tried to establish a perfect 'plant-microbe pair' for bioremediation of PAH in soil. Bisht et al. (2010) found that soil isolates Kurthia sp. SBA4, M. varians SBA8, D. radiodurans SBA6 and B. circulans SBA12 have promising characteristics for rhizoremediation of PAH using P. deltoides rhizosphere system in soil. To explore this, the potential of the best soil isolate i.e. M. varians SBA8 was evaluated as it possessed all the PGP traits and rhizospheric bioremediation activity. Various research studies from rhizobacterial isolates were focused on the plant growth promoting agents, because these improve plant growth by synthesizing phytohormones precursors (Ahmad et al., 2008), solubilize inorganic P and nutrient cycling (Khan et al., 2002). In remediation context, rhizobacterial system has been proved to be more effective in minimizing the bioavailability and biotoxicity of pollutants (Khan 2005a,b;Wani et al., 2008). Previously, Bacillus sp, Micrococcus sp. and Pseudomonas sp. have been reported to be efficient candidates for phosphate solubilization and IAA application in agriculture systems (Lucas Garcia et al., 2004;Antoun et al., 2004;Rajkumar et al., 2006). Therefore, the rhizobacterium used in this study was a promising plant growth stimulator and a biocontrol agent for P. deltoides root rot disease. Soil isolate M. varians SBA8 used in current study caused significant inhibition of mycelial growth of M. phaseolina, R. solanii and F. oxysporum. Several workers have reported the inhibition of soil borne phytopathogen by rhizobacteria (Barnard, 1994) but the reports for Micrococcus sp. are scanty.
In the dual culture assay, M. varians SBA8 not only inhibited the mycelial growth but also caused deformities of hyphae cell as clearly depicted in Figure 3. In some of these processes bacterial cells attached to the mycelia and penetrated into the hyphal wall resulting in lysis in both F. oxysporum and R. solani (Chung et al., 1998). On the other hand, M. varians SBA8 used in present study did not show mycelial or conidial attachment but showed the involvement of diffusible secondary metabolites responsible for inhibition of fungal pathogens. Earlier also, Lee and Kobayashi (1989) showed the deformities in the mycelial morphology of Rhizoctonia solani caused by the action of antifungal metabolites of B. cepacia.
However, in present study we have evaluated the M. varians SBA8 not only for plant growth promoting and biocontrol activities but also for bioremediation potential. Previously, Glicket al. (1998) has reported the production of ACC deaminase from rhizobacteria that results in improved root growth by lowering the concentration of plant ethylene. The effect on different plant-root systems under the influence of ACC deaminase from rhizobacteria is well explained (Belimov et al., 2005). Glick (2003) reported the development of plant seedlings in presence of PGPR having ACC deaminase activity under stress conditions like salt stress. M. varians SBA8 used in this study has the ability to produce ACC deaminase (Table 1).
Additionally, M. varians SBA8 also produced IAA and solubilized P with reduction in broth pH which may be due to organic acids production (Kumar and Narula, 1999) after 96 h and 120 h in culture condition (Figure 1 and 2). Similarly, Pandey and Maheshwari (2007b) observed that a rhizobacterial strain S. meliloti PP3 produces maximum 80 µg/ml IAA after 7 days of incubation. Leinhos and Bergmann (1995) reported that the addition of IAA to soil enhanced the uptake of iron and other elements (e.g. zinc, calcium etc.) by plant roots which lead to an increase in plant growth. Very few reports are available for phosphate solubilizing microorganisms from rhizosphere of Populus sp. growing in hilly regions. The present study showed the potential of rhizobacteria as plant growth promoter as well as P solubilizer, suggesting it can be used as a suitable biofertilizers for agricultural improvement (Kumar et al., 2001;2011). The PGP attributes provided by efficient soil isolate like M. varians SBA8 enhanced the growth of host plant in rhizosphere and also leads to PAH degradation in rhizospheric soil due to perfect plant-microbe interaction. Appreciable surface hydrophobicity was observed with M. varian SBA8 i.e. 20% against the hydrocarbon. This is a desirable property responsible for the increase in the aqueous PAH solubility by facilitating transport of anthracene in soil and efficient substrate-to-cell contact mechanisms. Earlier, Glick (2003) suggested that PAH increasingly bound in soil particles as the time passes. So, it is necessary to increase the movement of tightly bound hydrophobic PAH to the microbes where they can be effectively metabolized.
A key concern for the safety, efficacy, and commercial potential for any environmental application of an isolate is the ability of the microbe to survive in target habitats. Henceforth, the efficient isolate M. varians SBA8 with ability to biodegrade PAH along with PGP traits was subjected for its ability to compete for an ecological niche in the rhizosphere of P. deltoides for 90 days in hydrocarbon contaminated soil as well as in noncontaminated soil. Though, resistance against antibiotic was also utilized for colonization studies (Table 4). Similarly, Nautiyal (1997) established a procedure for selecting a rhizosphere-competent biocontrol bacterium Pseudomonas fluorescens NBRI1303 with its antibiotic marker system and checked its effect on plant growth and its ability to compete with other indigenous population of rhizosphere in chick pea. Earlier, Pandey et al. (2005) also monitored the survival of an antibioticresistant marker strain Pseudomonas GRC 1 which exhibited efficient root colonization after different time intervals of observation (i.e., 30, 60, and 90 days) and enhanced plant growth and growth yield. In current study the population density of M. varians SBA8 increased up to 60 days of inoculation, which became almost stable thereafter. It has been suggested, that bacteria that attain colony-forming units of about 10 3 per gram or higher on root mass can be considered a good colonizers (Meyer and Abdallah, 1978) and in this study we have obtained the population density higher than 10 3 per gram of soil. Successful application of plant-microbe systems for rhizoremediation relies on in situ establishment of a high level of competence of introduced bacteria (Liu et al., 2007).
Rhizoremediation study demonstrated that the PAH-degrading and growth-promoting M. varians SBA8 exhibited a significant enhancement in shoot and root length, root and shoot biomass including stem girth of Populus plant in hydrocarbon contaminated as well as non-contaminated soil with respect to control. Previous studies indicated that inoculation of barley seeds with phenanthrene degrading bacteria improved the growth of plants in the peat mixture containing (5 mg/g) phenanthrene (Anokhina et al., 2004), while in our work Populus sp. were planted with 10 mg/kg anthracene contaminated soil. Additionally, with respect to other treatment i.e. T 0 and T 3, maximum plant growth was achieved in treatment T 2 (Table 2). Therefore, it clearly indicate that PAH amended in soil act as carbon source and it might accelerate the growth rate of our target strain i.e. M. varians SBA8 so that plant growth promoting substance can be secreted at higher amount which conferred the remarkable change in plant growth parameters. Present results demonstrated that M. varians SBA8 substantially degraded 43.6% anthracene respectively after 120 days in soil model system as determined by HPLC analysis (Figure 4). Earlier, Wischmann and Steinhart (1997) reported substantial removal of benzo(a)anthracene, chrysene, and benzo(a)pyrene, with 11%, 19%, and 54% respectively after 15 weeks whereas in our study remediation with suitable plant microbe system i.e. P. deltoides and its rhizobacterial system were used for efficient removal of anthracene within 120 days only. Reports on Micrococcus sp. as good root colonizers are scanty.
The viability of the inoculum in an appropriate formulation for a certain length of time is of utmost importance as contribution to provide adequate nutrition. Effectively in agriculture plant nutrients may be limiting factors, particularly as regards nitrogen and phosphorus which are major elements required for plant growth and higher seeds production (Pandey et al., 2007a). Materials having high organic content, high water holding capacity and good aeration has been considered as good carrier for bioinoculants (Brockwell and Bottomley, 1995). In this study also, all the physical parameters measured were found optimum for bagasse, cocoa peat, saw dust, wheat bran and rock phosphate were determined (data not shown). All the carrier materials used in this study were also a good source of P and K and inorganic content. Our target strain M. varians SBA8 was studied to check the viability in six different solid carrier materials (Table 5). Based on its viability study, rock phosphate, cocoa peat and wheat bran were most suitable materials as its final population was found in the range of ≈10 5 CFU/g after 6 months of incubation. Earlier also peat is supposed to be an excellent carrier for bioinoculant and has been accepted worldwide for this purpose (Smith, 1995). However, unavailability of peat in tropical countries including India raised a concern for a suitable replacement. In view of this, we studied cocoa peat along with other carrier for the bioinoculant formulation. Similarly, wheat bran was successfully used as a carrier for microbial strains (Jackson et al., 1991). Sawdust was found to be a moderate carrier material as the viability of isolate in sawdust was not comparable with that in other lignocellulosic carrier materials. The addition of rock phosphate with bacterial cells has been suggested to serve as a good replacement for a chemical mixture of fertilizers (Bashan, 1986).

Conclusion
Conclusively, potential soil isolate M. varians SBA8 observed with rhizosphere competence, PGP and biocontrol activities qualifies to be an appropriate candidate for rhizoremediation of PAH. The strategy for using plant-microbe interactions for PAH degradation instead of direct amendment of contaminated soil with bacteria was adapted because in the latter strategy inoculation with bacteria is readily removed by indigenous microbial population. Also its formulation in wheat bran, cocoa peat rock phosphate based carrier materials presents immense possibilities of environmental restoration especially PAH contaminated sites. However it offers a multidimensional benefit for application on PAH contaminated sites. The potential of these formulations applied through Populus rhizosphere should be exploited for environmental and commercial purpose.