Plant species affect colonization patterns and ...

Plant species affect colonization patterns and metabolic activity of associated endophytes during phytoremediation of crude oil-contaminated soil K. Fatima, A. Imran, I. Amin, Q. M. Khan & M. Afzal

Environmental Science and Pollution Research ISSN 0944-1344 Volume 23 Number 7 Environ Sci Pollut Res (2016) 23:6188-6196 DOI 10.1007/s11356-015-5845-0

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Author's personal copy Environ Sci Pollut Res (2016) 23:6188–6196 DOI 10.1007/s11356-015-5845-0

RESEARCH ARTICLE

Plant species affect colonization patterns and metabolic activity of associated endophytes during phytoremediation of crude oil-contaminated soil K. Fatima 1,2 & A. Imran 1 & I. Amin 1 & Q. M. Khan 1 & M. Afzal 1

Received: 23 September 2015 / Accepted: 18 November 2015 / Published online: 25 November 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Plants coupled with endophytic bacteria hold great potential for the remediation of polluted environment. The colonization patterns and activity of inoculated endophytes in rhizosphere and endosphere of host plant are among the primary factors that may influence the phytoremediation process. However, these colonization patterns and metabolic activity of the inoculated endophytes are in turn controlled by none other than the host plant itself. The present study aims to determine such an interaction specifically for plant-endophyte systems remediating crude oil-contaminated soil. A consortium (AP) of two oil-degrading endophytic bacteria (Acinetobacter sp. strain BRSI56 and Pseudomonas aeruginosa strain BRRI54) was inoculated to two grasses, Brachiaria mutica and Leptochloa fusca, vegetated in crude oil-contaminated soil. Colonization patterns and metabolic activity of the endophytes were monitored in the rhizosphere and endosphere of the plants. Bacterial augmentation enhanced plant growth and crude oil degradation. Maximum crude oil degradation (78 %) was achieved with B. mutica plants inoculated with AP consortium. This degradation was significantly higher than those treatments, where plants and bacteria were used individually or L. fusca and endophytes were used in combination. Moreover, colonization and

Responsible editor: Zhihong Xu * M. Afzal [email protected]; [email protected] 1

Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P. O. Box 577, Faisalabad, Pakistan

2

Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan

metabolic activity of the endophytes were higher in the rhizosphere and endosphere of B. mutica than L. fusca. The plant species affected not only colonization pattern and biofilm formation of the inoculated bacteria in the rhizosphere and endosphere of the host plant but also affected the expression of alkane hydroxylase gene, alkB. Hence, the investigation revealed that plant species can affect colonization patterns and metabolic activity of inoculated endophytic bacteria and ultimately the phytoremediation process. Keywords Phytoremediation . Plant-endophyte partnerships . Crude oil . Bacterial colonization . Biofilm formation . Gene abundance . Gene expression

Introduction The insatiable reliance of the progressing world on oil and petrochemicals has increased during the past few decades that has resulted in extensive release of hydrocarbon pollutants in the environment. The adverse ecological and socioeconomic effects of oil pollution demand that eco-friendly and proficient remediation technologies be devised as countermeasures. The synergistic use of plants and endophytes has gained acknowledgment as one of these efficient green technologies (Jidere et al. 2012; Afzal et al. 2014; Auta et al. 2014). Plants provide a niche and essential nutrients for habitation and survival of endophytes while endophytic bacteria improve plant growth directly by producing beneficial metabolites or indirectly by reducing the amount of pollutants (Moore et al. 2006; Zhu et al. 2014; Ijaz et al. 2015). The association of inoculated bacteria with the host plant can occur in multiple ways, one of which is the biofilm formation, which is an important mode of microbial colonization in certain environments, ultimately affecting overall

Author's personal copy Environ Sci Pollut Res (2016) 23:6188–6196

functioning of microbes (Kearns et al. 2005; Dasgupta et al. 2013; Das et al. 2014). The presence of oildegrading microorganisms in biofilm enhances the degradation of toxic hydrocarbon pollutants (Singh et al. 2006; Pamp and Nielsen, 2007; Tribelli et al. 2012). In addition, colonization, survival, and activity of inoculated oil-degrading bacterial strains in rhizosphere and endosphere of host plants are crucial for sustainable growth of the host and mitigation of recalcitrant hydrocarbon contaminants in its vicinity (Khan et al. 2013; Hassanshahian et al. 2014; Sun et al. 2014a, b). The plant hosts employed in this investigation are grasses, B. mutica and L. fusca, that are well-known for salt tolerance, thus are used for the rehabilitation of saline soil (Ashraf et al., 2012; Ijaz et al., 2015). However, the potential of B. mutica and L. fusca for remediation of crude oil-contaminated soil has been rarely evaluated in association with endophytes. Although several studies showed that plant species affect the community structure in rhizosphere and endosphere, information on the effect that plants species may incur on biofilm formation, colonization, and metabolic activity of the inoculated endophytic bacteria is scarce in scientific literature. Therefore, this study evaluates the effect of plant species on colonization pattern and metabolic activity of the inoculated endophytes during the phytoremediation of crude oil-contaminated soil. Occurrence of inoculated endophytes as biofilm in various compartments (rhizoplane, root, and shoot) of the plants was studied by confocal laser scanning microscope (CLSM). The persistence and metabolic activity of the inoculated endophytic bacteria in rhizosphere and endosphere were evaluated by culture-dependent and -independent approaches.

Material and methods Bacterial strains The bacterial strains used in this study were selected from a wide collection of hydrocarbon-utilizing endophytic bacteria previously isolated from plants vegetated in crude oilcontaminated soil (Fatima et al. 2015). The strains included Pseudomonas aeruginosa BRRI54 (isolated from the root of B. mutica) and Acinetobacter sp. strain BRSI56 (isolated from the shoot of B. mutica). Both strains were able to degrade a variety of straight chain alkanes as well as aromatic compounds and showed the presence of alkane hydroxylase gene (alkB). These strains were also positive for plant growthpromoting traits e.g., phosphate solubilization, siderophore production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity.

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Tagging of bacterial strains with yellow fluorescent protein (yfp) and formulation of bacterial consortium To monitor the colonization patterns, both strains were tagged with yfp gene as described earlier (Wu et al. 2010; Shahid et al. 2012). These labeled strains were used as inoculum in the form of consortium AP, which was prepared by mixing equal portions of pure bacterial cultures (108 cells/ml). In vitro biofilm formation BRRI54-yfp and BRSI56-yfp were first tested for biofilm formation on 22-mm thin cover slips immersed in 50-mL sterile culture tubes containing 15-mL minimal medium (M9) with 1 % crude oil or glucose. The bacterial strains were inoculated and incubated at 37 °C for 4 days. The cover slips were removed from the culture tubes carefully, washed thoroughly with 1× phosphate buffer saline solution aseptically, and airdried (Dasgupta et al. 2013). Formation of biofilm was viewed under 100× oil immersion objective lens using CLSM (Fluo view, FV 1000, Olympus). Experimental design Crude oil-contaminated soil (crude oil 25.6 g kg−1 soil) was collected from oil-pumping site located in Chakwal, Pakistan. Soil was sieved through stainless 2-mm screen and thoroughly mixed. Equal amount (1.5 kg) of soil was transferred into 7×3 pots, for seven treatments to be performed in triplicates. Before sowing, the soil was amended with 50-mL AP consortium (≈ 108 cfu/ml). This ex situ experiment was designed to study (1) the effect of oil contamination on plant growth, (2) the effect of vegetation on oil-degradation;, (3) the effect of bacterial augmentation on oil-degradation, (4) the effect of plant species on colonization pattern and activity of the inoculated bacterial strains, (5) the effect of colonization pattern and activity on plant growth and oil degradation. The seven treatments were as follows: 1. 2. 3. 4. 5.

Agricultural (uncontaminated) soil with vegetation Crude oil-contaminated soil without vegetation Crude oil-contaminated soil with AP augmentation Crude oil-contaminated soil with L. fusca vegetation Crude oil-contaminated soil with L. fusca vegetation and AP inoculation 6. Crude oil-contaminated soil with B. mutica vegetation 7. Crude oil-contaminated soil with B. mutica vegetation and AP inoculation Thirty cuttings of L. fusca or B. mutica with similar weight and size were vegetated in each pot depending upon the treatment. The plants were grown in greenhouse and given equal

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amounts of water when needed. Plants were harvested after 3 months of planting, and data on growth parameters was recorded. Rhizosphere soil was obtained by sampling the soil loosely attached to roots while bulk soil (non-rhizospheric soil) was collected by thoroughly mixing the rest of the soil in pot. Root and shoot length and dry weight were recorded. Samples were then stored at −80 °C until further analysis. The colonization pattern of inoculated bacteria was also determined in the rhizosphere and endosphere of the plants using CLSM as described earlier (Dasgupta et al. 2013). Analysis of crude oil residues and biostimulant efficiency After plant harvesting, the level of residual crude oil in the soil samples was determined gravimetrically as described previously (Shankar et al. 2014; Das et al. 2014, Varjani et al. 2015), and biostimulant efficiency (BE%) was calculated as reported by Burghal et al. (2015). Persistence of the inoculated endophytes The abundance of inoculated strains in rhizosphere soil, shoot, and root interior of L. fusca and B. mutica was checked by plate count method (Lin et al. 2010; Afzal et al. 2012). Quantification of abundance and expression of alkB gene The abundance and expression of catabolic genes encoding alkane hydroxylase (alkB) were estimated by real-time PCR as explained earlier (Andria et al. 2009; Afzal et al. 2011).

Results

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Colonization and distribution of endophytes within host plants Fluorescence under CLSM confirmed that the inoculated endophytes were efficient plant colonizers indicating their capacity to inhabit host plants (L. fusca and B. mutica). Endophytes first colonized the rhizoplane followed by establishment of aggregates/biofilms on the entire root surface which indicated the possible entry of these bacteria into roots through these points (Fig. 4a). Following rhizoplane colonization, strains entered the roots and after translocation localized as single cell in the stems and leaves of the host plants (Fig. 4b–d). Maximum colonization of inoculated endophytes was observed in rhizoplane and inside the root as compared to the aerial tissues of the plant. No fluorescence was detected in uninoculated control plants. Plant growth responses to endophytes Growth parameters (root length, shoot length, root dry weight, and shoot dry weight) were determined to evaluate the influence of endophyte-inoculation on plant growth (Table 1). In soil contaminated with crude oil, endophytes inoculation improved root length (21–26 %), shoot length (11–18 %), root dry weight (25–38 %), and shoot dry weight (11–18 %) of both L. fusca and B. mutica. Among the two types of endophytes inoculated plants, B. mutica showed more increase in root length (19 %), shoot length (39 %), root dry weight (34 %), and shoot dry weight (38 %) than L. fusca plants. However, oil contamination in uninoculated soil significantly reduced root length (32–40 %), shoot length (29–32 %), root dry weight (37–46 %), and shoot dry weight (26–36 %). Of the two grasses, B. mutica plants were least affected under crude oil contamination than L. fusca plants.

In vitro attachment and colonization of bacteria on solid substratum

Effect of plant species on crude oil degradation

The ability of bacterial strains BRRI54-yfp and BRSI56-yfp to attach and colonize to solid substrate (glass) in the presence of crude oil (1 %) was studied by biofilm formation. The biofilm formation was observed under CLSM (Figs. 1, 2 and 3). The initial event of bacterial attachment occurred in the first 24 h of growth (Fig. 2a), and bacterial cells subsequently assembled on the solid substratum. At about 48 h of growth, cells were cemented in the form of clusters near oil-water interface (Fig. 2b). As the cells grew, the bacteria utilized the oil as energy source and oil contents reduced with time. After 72 and 96 h of incubation (Fig. 2c and d), cell clusters were fully grown and more aggregated to form a well-defined biofilm with very few oil droplets visible on the surface of glass. Biofilm formation of strain Acinetobacter sp. BRSI56-yfp was more obvious as compared to that of BRRI54-yfp strain under these experimental conditions (Fig. 3a and b).

B. mutica exhibited 9 % more crude oil degradation than L. fusca while synergistic action of B. mutica and endophytes exhibited maximum (78 %) crude oil-degradation. Maximum crude oil degradation (71–78 %) was observed in treatments having plants inoculated with endophytes; it was significantly higher than what was observed for plants and bacteria individually (Fig. 5). Vegetated soil exhibited more crude oil degradation (60–66 %) than unvegetated soil, whereas the augmentation of unvegetated soil with endophytes consortium resulted in 52 % crude oil degradation. On the other hand, least degradation (40 %) of crude oil was detected in the control soil that was uninoculated and unvegetated. The biostimulants efficiency (BE) analysis showed that the highest BE value (95 %) was observed with B. mutica augmented with AP consortium while the lowest BE value (30 %)


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Fig 1 Biofilm formation on thin cover slip (22 mm) by Acinetobacter sp. strain BRSI56yfp after 12 h (a) and 96 h (b) of growth in minimal medium amended with glucose as sole energy source

a was observed in the unvegetated soil augmented with hydrocarbon-degrading consortium (Fig. 6). Effect of plant species on persistence and metabolic activity of inoculated endophytes The ability of inoculated endophytes to colonize unvegetated soil as well as the rhizosphere and endosphere of B. mutica and L. fusca was estimated. Among the two types of grasses in this investigation, B. mutica hosted more numbers of the inoculated endophytes than L. fusca: both in the rhizosphere and endosphere (Table 2). Furthermore, bacterial cell count in the roots of both plants was significantly higher than those in rhizosphere and shoots. However, relatively lower numbers of the inoculated endophytes were recovered from unvegetated soil than the rhizosphere soil. Similarly, higher levels of alkB gene abundance and expression were seen in the rhizosphere and endosphere of

b B. mutica than that of L. fusca. Maximum alkB gene abundance and expression were observed within the root tissue of B. mutica; which was significantly higher than gene abundance and expression in the rhizosphere and shoot interior (Table 2). Moreover, the inoculated AP consortium showed higher levels of alkB gene abundance and expression in the rhizosphere and endosphere of the inoculated plants than in the unvegetated soil.

Discussion Due to rapid industrialization and increasing anthropogenic activities, contamination of soil and groundwater with hydrocarbons poses a greater threat not only to microflora but also to humans and animals (Arslan et al. 2015; Hong et al. 2015). Microbial biofilms are highly efficient and successful ecological communities that may contribute in remediation of oil-

Fig 2 Biofilm formation on thin cover slip (22 mm) by Acinetobacter sp. strain BRSI56yfp after 24 h (a), 48 h (b), 72 h (c), and 96 h (d) of growth in minimal medium amended with 1 % crude oil as sole energy source

a

b

c

d


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Fig 3 Biofilm formation on thin cover slip (22 mm) by Pseudomonas aeruoginosa strain BRRI54-yfp after 12 h (a) and 96 h of growth in minimal medium amended with 1 % crude oil as sole energy source

a contaminated soils (Deppe et al. 2005; McGenity et al. 2012; Dasgupta et al. 2013). In this study, we have evaluated the ex situ potential of specially designed consortium, AP, of two endophytes in association with two grass species for the remediation of crude oil-contaminated soil. CLSM analysis indicated that the inoculated endophytes were able to form biofilm on solid substratum; the capability appeared to be maximum in the close proximity of oil water interface (Figs. 1, 2, 3 and 4). Several earlier studies have demonstrated that natural oil-degrading bacteria can accumulate in the vicinity of oil and other pollutants and efficiently evade the limitation of low bioavailability of hydrocarbons in remediation process (AlAwadhi et al. 2003; Stewart and Franklin, 2008). Successful root colonization by hydrocarbon-degrading bacteria is an essential criterion to ensure beneficial effects

b of endophytes on phytoremediation process (Benizri et al. 2001; Compant et al. 2010; Sun et al. 2014a, b). In this study, bacterial cells were visualized as single cell as well as aggregated colonies on the rhizoplane that later on colonized the interior of the root and stem cells. Earlier reports corroborate this translocation of bacteria by indicating that endophytic bacteria are able to translocate through the xylem vessels from roots to aerial parts of the host plant via transpiration flow or by colonizing intercellular spaces (Newman et al. 2003; Compant et al. 2005, 2008). In phytoremediation, the foremost issue is development of plant tissue, which reflects the adaptation of plants to various stresses. In this study, the presence of crude oil in soil inhibited plant growth and development. This can be attributed to the toxic components of the crude oil (Luqueño et al. 2011; Tara

Fig 4 Endophytic bacteria, tagged with yfp, colonization on the rhizoplane (a), on the outline of some rhizodermal cells (b and c), and inside the cortex (d) of Brachiaria mutica

a

b

c

d

Author's personal copy Environ Sci Pollut Res (2016) 23:6188–6196 Table 1 Effect of endophytes consortium (AP) inoculation on root and shoot length and weight (dry) of Leptochloa fusca and Brachiaria mutica vegetated in the crude oil contaminated soil

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Treatments

Elongation (cm)

Plant biomass (g)

Root

Shoot

Root

Shoot

28.1c (1.3) 19.3e (1.5) 22.6d (1.6)

65b (3.5) 46d (4.0) 68ab (5.1)

16.3b (1.8) 10.8d (1.5) 12.9c (1.2)

28.4ab (2.4) 20.9c (5.2) 25.3b (5.2)

42.6a (2.7) 25.3cd (1.5) 33.2b (2.6)

77a (4.9) 52c (4.5) 58bc (3.2)

20.3a (2.5) 12.4c (1.3) 16.2b (1.4)

32.3a (2.2) 19.9c (5.2) 26.5b (2.6)

Leptochloa fusca Agricultural soil Crude oil-contaminated soil Crude oil-contaminated soil with AP inoculation Brachiaria mutica Agricultural soil Crude oil-contaminated soil Crude oil-contaminated soil with AP inoculation

Means in the same column followed by the same letter are not different at a 5 % level of significance, the standard error of three replicates is presented in parentheses. Comparisons between treatments were carried out by oneway analysis of variance (ANOVA). Plants were harvested after 93 days of experiment

et al. 2014). However, AP inoculation improved root and shoot length and tissue development of both grass species (L. fusca and B. mutica). It might be due to the hydrocarbon-degrading and ACC deaminase activity of the inoculated endophytes. ACC deaminase activity of bacteria reduces the ethylene level in plants during stress conditions, such as crude oil contamination in soil, thus improving plant’s adaptation and growth (Sheng et al. 2008; Mitter et al. 2013; Afzal et al. 2014). Uninoculated and unvegetated soil showed lower reduction (40 %) of crude oil (Fig. 5) as compared to inoculated and vegetated soil. This degradation of crude oil is performed by native microbes and/or natural physicochemical processes (e.g., volatilization or photo-oxidation) (Joner et al. 2002; Sun et al. 2014a, b). The augmentation of AP consortium in the unvegetated soil resulted in 52 % reduction in oil concentration. Several earlier studies demonstrated the same trend, whereby augmentation of crude oil-contaminated soil with hydrocarbon-degrading bacteria resulted in reduction in the 90 80

Crude oil degradation (%)

Fig 5 Effect of bacterial consortia AP (Acinetobacter sp. strain BRSI56 and Pseudomonas aeruginosa strain BRRI54) inoculation on crude oil degradation after 93 days of vegetation. Error bars indicate standard error among three replicates

contaminant concentrations (Shabir et al. 2008). Moreover, vegetation enhanced the mineralization of hydrocarbons up to 60–66 %. Instead of using either plant or bacteria alone, their bipartite association i.e., augmentation of plants with bacterial inoculation is superior strategy in the degradation process. Plants provide nutrients to indigenous microbial population hence improving their capability to degrade organic pollutants (Yousaf et al. 2011; Khan et al. 2013). The augmentation of AP consortium in combination with L. fusca and B. mutica resulted in 71 and 78 % of crude oil degradation, respectively, and this degradation was significantly higher than either sole bacterial augmentation or vegetation. Maximum (78 %) crude oil degradation was exhibited by B. mutica and the AP consortium. These results are in agreement with earlier reports that plant-endophyte association is a more effective methodology for the cleanup of soil polluted with petroleum hydrocarbons than vegetation or microbial augmentation alone (Chaudhry et al. 2005; Andria et al., 2009; Yousaf et al., 2011; Afzal et al. 2013).

70 60 50 40 30 20 Control

AP augmentaon

Leptochloa fusca

L. fusca + AP

Treatments

Brachiaria muca

B. muca + AP


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Biosmulaon eﬀeciency (%)

Fig 6 Percentage of biostimulant efficiency of treated soil samples after 93 days of bioremediation process

80

60

40

20

0

Treaatments

Different plants host different microorganisms in their rhizosphere and endosphere, but little is known about the colonization patterns and metabolic activities of the inoculated endophytes in the rhizosphere and endosphere of plants. Inoculated bacteria showed not only colonization but also expressed alkane-degrading genes demonstrating their dynamic character in the mineralization of crude oil (Table 2). Contrary to the high abundance and activity of the endophytes in vegetated soil, rather less persistence and activity were seen in unvegetated soil. This reveals that vegetation improved the persistence and activity of the endophytes in the soil. Vegetation offers nutrients and habitat to associated microorganisms and ultimately the numbers of pollutant-degrading microorganisms increases in the vicinity of the root as well as the plant interior (Andria et al. 2009; Weyens et al. 2009). Furthermore, among the two tested plants, higher persistence of inoculated endophytes and better abundance and

expression of alkB gene were observed in the rhizosphere and endosphere of B. mutica than that of L. fusca. This shows that different plants host different numbers of microorganisms in their rhizosphere and aerial tissues and also stimulate their activity differently. It might be due to the fact that different plants release a variety of nutrients in different amounts in their rhizosphere and endosphere and consequently stimulate a variety of microorganisms in their components (Compant et al. 2010; Yousaf et al. 2011; Afzal et al. 2014). Moreover, higher levels of alkB gene abundance and expression were observed in the roots of both plants as compared to the rhizosphere soil and shoot interior. Previous studies corroborate that inoculated endophytes exhibit better persistence and activity within the root than in the rhizosphere soil or shoot interior (Andria et al., 2009; Afzal et al. 2011; Yousaf et al. 2011). In our investigation, the abundance of alkB gene exhibited positive relationships with gene expression (r=0.74)

Table 2 Colony forming unit (CFU), gene abundance and expression of alkane hydroxylase genes (alkB) in the non-rhizosphere soil and rhizosphere soil and endosphere of Leptochloa fusca and Brachiaria mutica inoculated with endophytic consortium Treatments

Cfu g−1 dry weight×103

Gene abundance (copies g−1 dry weight×103)

Gene expression (copies g−1 dry weight×103)

Soil (unvegetated) Rhizosphere Brachiaria mutica Leptochloa fusca Root B. mutica

12g (1.5)

0.79g (0.27)

0.31f (0.11)

910b (24) 327f (14)

23.6b (0.40) 10.8d (0.16)

2.1c (0.34) 1.4d (0.18)

1272a (54)

91.8a (1.46)

7.4a (0.15)

d

c

L. fusca

523 (21)

12.3 (1.05)

2.9b (0.37)

Shoot B. mutica L. fusca

656c (34) 460e (10)

6.3e (0.64) 4.5f (0.50)

1.5d (0.34) 1.0e (0.16)

Means in the same column followed by the same letter are not significantly different at a 5 % level of significance, the standard error of three replicates is presented in parentheses. Comparisons between treatments were carried out by one-way analysis of variance (ANOVA)


and hydrocarbon removal (r = 0.87). During the phytoremediation of hydrocarbon-contaminated soil, strong positive relationships have been previously reported between hydrocarbon degradation, gene abundance, and gene expression (Yousaf et al. 2011; Khan et al. 2013). This represents that the occurrence and activity of catabolic genes is directly associated with hydrocarbon degradation.

Conclusions The grasses, L. fusca and B. mutica, showed potential to remediate soil contaminated with crude oil and their remediation potential was further enhanced by inoculation of bacterial endophytes. Endophytes showed high levels of colonization in the rhizosphere and endosphere of the plants, where the colonization patterns and metabolic activity were affected by the type of plant host. Higher levels of colonization and metabolic activities of the inoculated endophytes were observed in the rhizosphere and endosphere of B. mutica than in the rhizosphere and endosphere of L. fusca. Similarly, more crude oil degradation was exhibited by the combined use of B. mutica and endophytes than that of L. fusca and endophytes. Conclusively, the combined use of B. mutica and endophytes is a more promising approach for the remediation of crude oilcontaminated soil. Acknowledgments The authors thank the Higher Education Commission (HEC), Pakistan, for the financial support (grant number HEC-20111997) for this work.

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