Lolium multiflorum

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Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3 ab

ab

a

a

Hina Jabeen , Samina Iqbal , Fiaz Ahmad , Muhammad Afzal & Sadiqa Firdous a

Soil and Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), PO Box 577, Jhang Road, Faisalabad 38000, Pakistan b

Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan Accepted author version posted online: 06 Aug 2015.

Click for updates To cite this article: Hina Jabeen, Samina Iqbal, Fiaz Ahmad, Muhammad Afzal & Sadiqa Firdous (2015): Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3, International Journal of Phytoremediation, DOI: 10.1080/15226514.2015.1073666 To link to this article: http://dx.doi.org/10.1080/15226514.2015.1073666

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ACCEPTED MANUSCRIPT Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3 Hina Jabeena, b, Samina Iqbala, b*, Fiaz Ahmada, Muhammad Afzala, Sadiqa Firdous a

Soil and Environmental Biotechnology Division, National Institute for Biotechnology and

Downloaded by [National Library of Biological Sciences] at 03:09 13 August 2015

Genetic Engineering (NIBGE), PO Box 577, Jhang Road, Faisalabad 38000, Pakistan. b

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

*Corresponding

Author:

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[email protected]; [email protected] ABSTRACT For effective remediation of contaminants, plant-endophyte partnership is a promising field to be explored. Generally endophytic bacteria assist their host plant by withstanding the stress induced by the contaminants. The objective of this study was to explore the suitability of plant-bacterial partnership for chlorpyrifos (CP) remediation using ryegrass and a CP degrading endophyte, Mesorhizobium sp. HN3 which belongs to plant growth promoting rhizobia. The inoculated yfp-tagged Mesorhizobium sp. HN3 efficiently colonized in the rhizosphere, enhanced plant growth and degradation of CP and its metabolite 3,5,6 trichloro-2-pyridinol (TCP). Significantly lower CP residues were observed in the roots and shoots of plants vegetated in inoculated soil which might be attributed to the efficient root colonization of HN3yfp. These results suggest the involvement of Mesorhizobium sp. HN3yfp in CP degradation inside the roots and rhizosphere of plants and further emphasize on the effectiveness of endophytic bacteria in stimulating the remediation of pesticide contaminants.

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This is the first report which

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ACCEPTED MANUSCRIPT demonstrates the efficacy of bacterial endophyte for degradation of CP residues taken up by the plant and enhanced remediation of chlorpyrifos contaminated soil. Key words Biodegradation;

Mesorhizobium

sp.;

Chlorpyrifos;

3,5,6-trichloro-2-pyridinol;

Plant-


endophyte partnership

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ACCEPTED MANUSCRIPT Introduction Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) (CP), an organophosphate pesticide is used worldwide to control a wide range of economically significant crop pests and domestic insect pests including termites. Besides the target pests, CP is also


known to adversely affect or kill other organisms including ecologically important insects like beetles, bees, and wasps as well as aquatic organisms e.g. protozoans, fish and tadpoles etc. Moreover, exposure to this compound is related to broad-spectrum effects including a variety of nerve disorders and interruption of many vital functions in higher animals and humans (Eaton et al. 2008; Farag et al. 2010; Ventura et al. 2012). CP has low water solubility (2 mg L-1) and strong affinity for organic matter and soil particles as indicated by the high soil adsorption coefficient (log Koc = 1.61-4.72), hence, its residues remain in the environment for undefined period of time (Gavrilescu, 2005; Gebremariam et al. 2012). The absorption and translocation of CP by wheat and oil seed rape roots and other crop systems have been reported (Wang et al. 2007) and its residues have been detected in vegetables, fruits and meat (Parveen et al. 2004). Chlorpyrifos exceeds Stockholm Convention criteria for bioaccumulation with reported values of log Kow from 4.7 to 5.11 (Gebremariam et al. 2012). The environmental and health problems associated with CP contamination arouse attention towards its remediation in eco-friendly and cost-effective way. There are extensive reports on microbial degradation as an effective and environmentally prudent technology for the degradation of chlorpyrifos and its metabolites (Anwar et al. 2009; Chishti et al. 2013). Phytoremediation, i.e., use of plants for the detoxification of pollutants in the environment, is getting a significant level of

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ACCEPTED MANUSCRIPT public attention and becoming a rapidly expanding field owing to its „green‟ approach (Huang et al. 2011). In this regard, bioavailability and phytotoxicity of the contaminant and biodegradation/biotransformation capability of the plant are important parameters to be considered (Abhilash et al. 2013). However, one of the limitations is that pollutants adversely


affect the plant growth resulting in reduced biomass which in turn could influence the phytoremediation process (Weyens et al. 2009). This limitation has been compensated by the combined use of plants and microorganisms for the remediation of contaminated sites (Khan et al. 2013). Term rhizo-remediation has been employed for microbe assisted phytoremediation which involves mutual interactions of plant and rhizospheric microorganisms exhibiting contaminant degradative activities. More recently, enhanced pollutant removal by plants in combination with bacterial endophytes capable of degrading a certain contaminant has been demonstrated as a promising approach to alleviate contaminant induced stress on the plant and increase remediation efficiency (Ho, Hsieh and Huang 2013). In some cases, it has been demonstrated that plants can stimulate the pesticide degradation by microbial communities (Sun et al. 2004). Phytoremediation of CP and involvement of associated microbes is also limited to few studies (Dubey and Fulekar 2012; Lee, Strand and Doty 2012). The present study describes the inoculation of a chlorpyrifos degrading endophyte, Mesorhizobium sp. HN3 in the rhizosphere of ryegrass for enhancing the degradation of the pesticide. Ryegrass is a good choice to exploit plant-microbe interactions for degradation of contaminants because of its extensive root system that helps in improving the growth of microbes in its rhizosphere and in turn the remediation potential of the system is enhanced (Korade and Fulekar 2009a). Mesorhizobium sp. HN3 is a CP degrading bacterium isolated and characterized

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ACCEPTED MANUSCRIPT in our lab (Jabeen, Iqbal and Anwar 2014). It belongs to plant growth promoting (PGP) rhizobia which survives in the plant rhizosphere as well as in the bulk soil. Moreover, its ability to live as plant endophyte was exploited for the degradation and removal of CP residues accumulated in plant roots and shoots hence rendering it a good candidate for detoxifying CP.


Materials and Methods Bacterial strains Mesorhizobium sp. HN3, a bacterial strain capable of degrading CP and its toxic metabolite TCP, reported previously by Jabeen et al. 2014, was used in this study. E. coli DH5α carrying a broad host range ampicillin resistant (Amp R) plasmid pBBRIMCS-4 (Kovach et al. 1995) containing YFP (yellow fluorescent protein) cassette was obtained from National Institute for Biotechnology and Genetic Engineering, NIBGE, Biotechnology Resource Centre (NBRC), Faisalabad. To monitor the colonization process, Mesorhizobium sp. HN3 was labelled with yfp gene as described earlier (Wu et al. 2010; Shahid et al. 2012). Inoculum of HN3yfp (107 CFU mL-1) was prepared as described by Anwar et al. (2009) and used for CP remediation studies. Soil collection and fortification with CP The experimental soil without background contamination of pesticides was collected from University of Agriculture, Faisalabad. Soil was air dried and sieved through a stainless steel sieve (to remove stones and plant materials etc.), ground and stored until further use. Technical grade CP (5% stock solution in acetonitrile) was used to spike 20 g sand and mixed with 25 % of the experimental soil. The solvent (acetonitrile) was allowed to disperse and

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ACCEPTED MANUSCRIPT evaporate completely at room temperature for about 24 hours. The spiked soil was mixed with the rest of the experimental soil to obtain a final concentration of 50 mg kg -1 (w/w) CP. Experimental design Ryegrass (Lolium multiflorum var Taurus) previously reported to tolerate CP (Ahmad et


al. 2012; Korade and Fulekar 2009a) was used in these studies. For the experiment, plastic pots (1.5 kg soil each) were filled with agricultural soil spiked with CP (50 mg kg-1). The study included following treatments: 1. CP contaminated soil 2. CP contaminated soil inoculated with HN3yfp 3. Ryegrass planted in un-contaminated soil 4. Ryegrass planted in CP contaminated soil 5. Ryegrass planted in CP contaminated soil and inoculated with HN3yfp The seeds of the Lolium multiflorum were surface sterilized with 1% H2O2 and sown in each pot (150 seeds/pot). For the inoculated treatments, the soil was mixed with 50 mL bacterial suspension (107 CFU mL-1) and with 0.85% NaCl for the un-inoculated control treatments before sowing. The plants were grown in green house at 25±2 °C with 16 h light and 8 h dark. One week following seed germination, poor emerging seedlings were removed and 100 plants were maintained per pot. Plants were harvested after 15, 30 and 45 days of sowing, shoots were cut above ground and roots were separated from the bulk soil. Soil from each pot was mixed thoroughly to get homogenized samples for CP residue analysis. CP concentration in the soil and

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ACCEPTED MANUSCRIPT within plant tissues, plant growth parameters and bacterial population in soil and roots were observed. Whole experiment was performed in triplicate. Measurements of growth parameters After harvesting, representative samples (50 g fresh weight each of roots and shoots) were


separated for pesticide residue analysis. Growth parameters were measured for rest of the sample size and corrected for whole sample. Elongation of roots and shoots was measured using a ruler. Fresh weights were measured by directly weighing the freshly separated and cleaned roots and shoots with a physical balance. For assessing the dry weights, the roots and shoots were dried in oven at 65 °C for 8-12 hours until a constant weight was achieved. Detection and enumeration of the bacteria in the soil Rhizospheric soil was collected by removing soil adhered to roots and suspended in 10 mL 0.85 % saline solution. The suspension was agitated for 1h at 37 °C, the soil particles were allowed to settle down and 10 fold dilutions were prepared. Same procedure was adopted for bulk soil. By spreading these dilutions on LB-CP-ampicillin (50 µg mL-1) agar plates, HN3yfp were recovered and counted. The colonies morphologically similar to Mesorhizobium sp. HN3yfp were further confirmed by restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA intergenic spacer region using genomic DNA of the randomly selected bacterial colonies as described earlier (Ahmad et al. 2012). Root and shoot colonization by Mesorhizobium sp. HN3yfp For observing colonization of Mesorhizobium sp. HN3yfp in the plant tissues, 15, 30 and 45 days old roots and shoots of the ryegrass were washed separately with sterile distilled water as soon as they were harvested. The washed root and shoot samples were examined under Confocal

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ACCEPTED MANUSCRIPT Laser Scanning Microscope (CLSM; Olympus Fluoview Ver.1.3) at 4X and 10X magnifications for observing the colonization of Mesorhizobium sp. HN3yfp within ryegrass root hairs, root tips, root and shoot surfaces and inside the tissues. Extraction and analysis of chlorpyrifos residues in the soil and plant


CP and TCP residues were extracted from rhizospheric (planted) and bulk (un-planted) soil, roots and shoots following Essumang, Togoh and Chokky (2009) and Ahmad et al (2009) with slight modifications. Plant tissues were ground in liquid nitrogen using pestle and mortar. Ground fresh plant tissue (10 g) was added to a 100 mL conical flask followed by the addition of 10 g anhydrous sodium sulfate and 100 mL 1:1 (v/v) ethyl acetate/dichloromethane. The mixture was thoroughly mixed by shaking the flask on the rotary shaker for 3 hrs. The organic layer was then decanted, evaporated using the rotary evaporator at 40 °C and dissolved in 2 mL ethyl

acetate.

Extract

was

further

subjected

to

silica

gel

clean-up

using

ethyl

acetate/dichloromethane as eluent (Essumang et al. 2009). The estimation of CP and TCP in soil, root and shoot samples was carried out using High Performance Liquid Chromatography (HPLC) equipped with ODS2 C18 reversed-phase column. The gradient mobile phase consisting of acetonitrile, water and acetic acid was used with the flow rate of l mL min-1. Retention time for CP and TCP were 8 min and 4.1 min respectively and detection wavelength for both was 290 nm. Data analysis Statistical analyses of plant biomass and CP and TCP degradation in soil, roots and shoots were performed on three replicates of data obtained from all treatments. The significance of differences were treated statistically by the ANOVA and evaluated by post hoc comparison of

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ACCEPTED MANUSCRIPT means using Tukey‟s test in Statistica 6.0 software. The degradation rate constants and theoretical half-life values (DT50) were determined using algorithms Ct/C0= e-kt and from the (Cycoń, Wójcik and Piotrowska-Seget 2009),

linear regression equation between ln(Ct/C0)

where C0, Ct indicated the CP concentration at zero and t time while k and t represent the rate


constant (day-1) and degradation period in days respectively. Results and Discussion Biodegradation of CP in the rhizospheric and bulk soil Inoculation of pollutant degrading bacteria in the plant rhizosphere has recently been reported as a useful strategy for bioremediation of contaminants (McGuinness and Dowling 2009). Among other plants, ryegrass (Lolium multiflorum) has been revealed as a good candidate for such tasks because of its extensive root system and capability to support proliferation of microorganisms in its rhizosphere which in turn enhance pollutant degradation. In the present studies, the residual concentration of the pesticide in contaminated soil was monitored to explore the effect of ryegrass and Mesorhizobium sp. HN3yfp partnership on CP degradation. CP degradation and TCP accumulation and removal were compared between three soil treatments i.e., planted (un-inoculated), un-planted (inoculated), and planted+inoculated (Fig. 1a and 1b). After 15 days of sowing, 22% of the added CP was degraded in the planted soil (uninoculated) whereas in the inoculated (un-planted) soil, 36% degradation was achieved. On the other hand when the planted soil was inoculated with Mesorhizobium sp. HN3yfp, 44% of the total applied pesticide was degraded. At the end of the experiment (45 days), CP degradation was 79% and 91% in the planted (un-inoculated) and inoculated (un-planted) soil respectively, whereas complete degradation was achieved in the planted+inoculated soil. Hence inoculation of

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ACCEPTED MANUSCRIPT Mesorhizobium sp. HN3 in the planted soil significantly (P

Lolium multiflorum

Lolium multiflorum

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