Influence of plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 using Helianthus annuus on degradation of endosulfan from contaminated soil

Accepted Manuscript Influence of plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 using Helianthus annuus on degradation of endosulfan from contaminated soil

Rupa Rani, Vipin Kumar, Zeba Usmani, Pratishtha Gupta, Avantika Chandra PII:

S0045-6535(19)30473-4

DOI:

10.1016/j.chemosphere.2019.03.037

Reference:

CHEM 23351

To appear in:

Chemosphere

Received Date:

08 February 2019

Accepted Date:

07 March 2019

Please cite this article as: Rupa Rani, Vipin Kumar, Zeba Usmani, Pratishtha Gupta, Avantika Chandra, Influence of plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 using Helianthus annuus on degradation of endosulfan from contaminated soil, Chemosphere (2019), doi: 10.1016/j.chemosphere. 2019.03.037

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Influence of plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08,

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Bacillus sp. PRB77 and Bacillus sp. PRB101 using Helianthus annuus on degradation of

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endosulfan from contaminated soil

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Rupa Rania, Vipin Kumara*, Zeba Usmania, Pratishtha Guptaa, Avantika Chandraa

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a Laboratory

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Department of Environmental Science and Engineering,

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Indian Institute of Technology (Indian School of Mines), Dhanbad

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of Applied Microbiology

Dhanbad-826 004, Jharkhand, India

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*Corresponding Author; Email: [email protected];

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Tel: +91-9471191352 (M), +91-326-2235643 (O)

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ABSTRACT

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Endosulfan is a broad spectrum insecticide used in agriculture for protection of various food and

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non-food crops. It is persistent in nature and hence found in soil, air and water. The potential use

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of plants and microorganisms for the removal of endosulfan from soil was studied. Helianthus

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annuus plant was grown in soil spiked with 5, 10, 25 and 50 mg kg-1 concentrations of

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endosulfan and inoculated with plant growth promoting rhizobacterial strains Paenibacillus sp.

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IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 for 40, 80 and 120 days. Potential of

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plant for endosulfan uptake was evaluated by investigating the endosulfan levels in plant tissues

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(root and shoot). The results indicated that endosulfan accumulation followed the pattern of root

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> shoot as well as decrease in uptake of endosulfan in root and shoot of a plant grown in 1

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bacterial inoculated soil as compared to un-inoculated soil. Bacterial inoculation had a positive

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effect on endosulfan degradation. Maximum degradation of 92% at 5 mg kg-1 of endosulfan in

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soil was observed on inoculation with PRB101 after 120 days of inoculation. The results showed

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that plant growth promoting bacteria enhances plant biomass production. Lipid peroxidation was

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also estimated by determining the malondialdehyde (MDA) production, which is a biomarker of

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oxidative damage. Decrease in MDA formation by root and leaves of plants grown in the

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bacteria inoculated plant was also observed. The results suggested the effectiveness of plant

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growth promoting rhizobacteria to boost accumulation potential, biomass production and

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enhance remediation of endosulfan contaminated soil.

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Keywords:

Endosulfan

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Phytoremediation

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Helianthus annuus

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Endosulfan degrading bacteria

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Plant bacteria partnership

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1. Introduction

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Contamination of soil by pesticide residues is a major area of research. Endosulfan

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(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a hexahydro-6,9-methano-2,4,3-benzodio-3-oxide) is a

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broad-spectrum organochlorine insecticide and has been widely used as an insecticide for

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different crops throughout the world (U.S. EPA, 2002; Xie et al., 2011). Endosulfan consists of

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alpha- and beta- isomers (7:3) (Kong et al., 2018). Due to its indiscriminate use, endosulfan

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isomers have penetrated into almost all parts of the ecosystem (US-EPA, 2007, 2009) such as in

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humans (Arrebola et al., 2001), soil (Connolly et al., 2001) and rivers (Broomhall, 2002). 2

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Endosulfan is listed as a persistent toxic pollutant by the Agency for Toxic substances and

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Disease Registry (ATSDR) in 2001. In 2011, it was enlisted as a persistent organic pollutant

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(POPs) because of its bioaccumulation potential, toxicity and persistence (POPRC, 2009).

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Because of its persistence in nature, it undergoes bioaccumulation and biomagnification in the

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food chain and therefore, causes adverse effects to the environment and human beings (Singh

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and Singh, 2014a). The previous researchers have suggested that endosulfan is toxic to fishes

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(Shao et al., 2012) and human health (Dey et al., 2013; Nawaz et al., 2014; Syed et al., 2014).

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These health and environmental issues have led to developing of ecofriendly and efficient

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remediation techniques for the removal of endosulfan from contaminated soil.

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Phytoremediation is an economical and ecofriendly emerging technique to remediate organic

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pollutants, including pesticides from soil using various mechanisms and microbial interactions

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with pants (Gerhardt et al., 2009; Mitton et al., 2012). Plant considers several biologically active

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characteristics, such as rhizodegradation, stabilization, transformation and accumulation for the

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removal of pollutants (Ahmad et al., 2012). The major limitation of phytoremediation technique

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is that many plants do not survive under stress due to contaminants (Chaudhary et al., 2005;

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Romkens et al., 2002) and even plant that exhibited resistant to pollutants usually showed less

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biomass production without adding any remediation properties (Gaskin et al., 2008; Kao et al.,

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2004; Weyens et al., 2009a).

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In order to minimize this drawback, bacterial species were associated with plants, thus

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exhibiting the competence to degrade contaminants (Weyens et al., 2009b; McGuinness et al.,

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2009). Several researchers have reported the rhizobacteria-plant interactions in an efficient

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treatment of pollutants (Ahmad et al., 2012; Afzal et al., 2011; Korade and Fulekar, 2009).

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Several organic compounds had adverse effects on plants resulting in oxidative stress, as well as

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membrane lipid peroxidation (LPO). Exposure of DDT (Mitton et al., 2014) and endosulfan

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(Ramirez Sandoval et al., 2011; Mitton et al., 2016) increase lipid peroxidation (LPO) levels in

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several plant species indicating the efficacy of LPO to be measured as toxicity indicator.

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The study aimed to assess the effect of inoculation of endosulfan degrading bacterial strains

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on plant growth, uptake from the roots and shoots of the Helianthus annuus plant and

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degradation of endosulfan supplemented in soil. Lipid peroxidation was also evaluated as a

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toxicity biomarker.

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2. Materials and methods

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2.1 Bacterial strains

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Bacterial strains Paenibacillus sp. IITISM08 (Rani et al., 2018), Bacillus sp. PRB77 and

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Bacillus sp. PRB101 (Rani and Kumar, 2017) previously isolated from pesticide stressed soil

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were used in the present study. These bacterial strains have the potential to degrade endosulfan

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and demonstrated a substantial production of plant growth promoting (PGP) traits under

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endosulfan stress. In brief, Paenibacillus sp. IITISM08 degraded 51% of endosulfan (50 µg mL-

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1)

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PRB101 were able to degrade 70% and 74% of endosulfan in broth and 63% and 67% in

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sterilized soil, respectively at recommended doses (2 µg mL-1) in the lab scale study (Rani and

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Kumar, 2017).

in broth (Rani et al., 2018). Moreover, bacterial strains, Bacillus sp. PRB77 and Bacillus sp.

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Bacterial strains were cultured in 100 mL of Luria Bertani broth (LB) (tryptone g L-1 10.0,

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yeast extract 5.0, NaCl 10.0, pH 7.0-7.2) and incubated at 30 °C. The cells were then harvested

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by centrifugation for 20 min at 10,000 rpm and re-suspended in sterile NaCl (0.9%) solution to

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attain the final cell concentration of 108 CFU mL-1 prior to inoculation.

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2.2. Pot experiment

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An experiment was conducted using Helianthus annuus (sunflower) in an earthen pot (5

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kg soil) to evaluate the potential of Paenibacillus sp. IITISM08 (Rani et al., 2018), Bacillus sp.

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PRB77 and Bacillus sp. PRB101 (Rani and Kumar, 2017) for uptake of endosulfan in different

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parts (root and shoot) of the plant, endosulfan degradation and plant growth promotion.

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Agricultural soil collected from research garden of IIT (ISM) Dhanbad, India was used for the

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pot experiment. The physico-chemical properties of soil were characterized in previous studies

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(Rani et al., 2019) with following properties; pH (7.90), organic carbon (13.95 g kg-1), available

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N (16.58 mg kg-1), available P (15.99 mg kg-1), available K (87.44 mg kg-1) and endosulfan

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(0.0061 mg kg-1). The soil was sterilized in an autoclave at 121 °C and for 15 min.

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2.2.1. Fortification of soils

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Endosulfan (technical grade) used was procured from Dr. Ehrenstorfer GmbH, Germany

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(Lot no. 31218, purity 99.0%) to amend in soil following the method described by Brinch et al.

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(2002). Endosulfan was dissolved in acetone to attain a solution of 5 mg mL-1 concentration and

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was added with only 25% of the soil. The solvent was allowed to evaporate under fume-hood for

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24 h and the endosulfan amended soil was thoroughly mixed with the rest of non-spiked soil

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using metal spatula to give a final endosulfan concentration of 5, 10, 25 and 50 mg kg-1 of soil.

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The same procedure was used without endosulfan for control. Finally, the bell shaped earthen

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pots were filled with endosulfan amended soils for the study.

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2.2.2. Experimental design

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The seeds of Helianthus annuus were procured from the local market, the germination

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percentage was > 90%. The seeds were surface sterilized with 30% (v/v) H2O2 for 20 min and

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thoroughly washed several times with sterilized dH2O. Seeds were dried for 30 min in the shade 5

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and then 10 seeds were sown in each pot (19 cm upper diameter x 16 cm height x 11.5 cm

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bottom diameter). Before sowing, 5 kg of sterilized soil was treated with 50 mL suspension of

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the inoculant Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 (109

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CFU mL-1) and with sterile NaCl (0.9%) solution for control (Rani et al., 2019).

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After germination, seedlings were counted and maintained to three per pot. Plants were

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harvested at 40, 80 and 120 days after sowing. After the plants and roots were removed from the

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soil, and the soil was mixed to obtain homogenized soil subsamples for endosulfan extraction

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and collected at an interval of 40, 80 and 120 days after sowing.

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The experiment was performed in complete randomized block design with four

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treatments. The treatments included: E0 (soil without endosulfan), E5 (soil+ 5 mg kg−1

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endosulfan), E10 (soil+ 10 mg kg−1 endosulfan), E25 (soil+ 25 mg kg−1 endosulfan), E50 (soil+ 50

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mg kg−1 endosulfan). The Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp.

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PRB101 were inoculated in the treatments and the sterile 0.9% NaCl solution was inoculated in

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control. The experiment was performed in triplicates. This experiment includes positive control

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(endosulfan without bacterial strains) and negative control (E0) (bacterial strains without

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endosulfan).

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2.2.3. Extraction of endosulfan and cleanup

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Subsamples of air dried soil (5 g) and wet plant tissues (3 g) from plants of different

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treatments were homogenized with anhydrous sodium sulfate and supplemented with 1 mg kg-1

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of endosulfan as internal standard in a pestle and mortar; and subjected to Soxhlet extraction

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with hexane and methylene chloride (50:50) for 4 h. The extracts obtained were transferred to

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round bottom flask and were concentrated by using a rotary evaporator to 2 mL. Lipids were

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separated from the plant extracts using gel permeation chromatography in Bio Beads S-X3 (200-

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400 mesh; Bio-Rads Laboratory, Hercules, CA, USA) and then the extracts were dried under

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vacuum and nitrogen flow to a constant weight. Further sample clean-up/ subfractionation of all

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extracts containing pesticides was performed with silica gel chromatography. The extracts were

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then concentrated to 1 mL and stored in sealed vials prior to gas chromatography at -20 °C

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(Metcalfe and Metcalfe, 1997; Miglioranza et al., 2003; Mitton et al., 2016).

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2.2.4. Gas Chromatographic analysis

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The α-endosulfan, β-endosulfan and endosulfan sulfate were quantified according to

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Miglioranza et al. (2003), using Chemito CERES 800 Plus GC-ECD (Thermo Fischer) equipped

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with a fused silica capillary column of 30 m, DB-5 (0.32 mm i.d., 0.25 μm film thicknesses). The

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initial oven temperature was 100 °C, and held for 1 min, afterward an increase of 5 °C min−1 up

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to 150 °C, held for 1 min, then 1.5 °C min−1 up to 240 °C and then 10 °C min−1 up to 300 °C for

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3 min. The temperature of the injector and detector was set at 275 °C and 300 °C, respectively.

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Ultra-high purity Helium was used as the carrier gas at a flow rate of 1.5 mL min−1 and nitrogen

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was used as a make-up gas.

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Quantification of endosulfan (technical grade; procured from Dr. Ehrenstorfer GmbH,

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Germany Lot no. 31218, purity 99.0%) was performed using a standard curve. A standard curve

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of endosulfan was linear with a value of the regression coefficient (R2) > 0.971 over the

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concentration of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.1, 1, 10, 30 and 50 mg L-1. The retention

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time of alpha-endosulfan is 13.4 min, beta-endosulfan is 21.7 min and endosulfan sulfate is 24.2

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min, respectively (Rani et al., 2019).

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2.2.5. Degradation kinetics of endosulfan in soil

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The endosulfan degradation in soil can be described with the first order kinetic equation

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eq. (1). ln values of the residues of endosulfan (Ct/C0) were plotted against respective days,

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which showed a straight line for all the treatment.

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Ct = C0 x e-kt

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(Ct: concentration of endosulfan at time t, C0: concentration of endosulfan (mg kg-1) at time zero,

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k: degradation rate constant (day-1) and t: degradation time in days)

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Eq. (1)

The half-life (T1/2) of endosulfan in different treatments was evaluated using the

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algorithm as expressed as:

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T1/2 = 0.693/k

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2.2.6. Enumeration of bacteria inoculated in soil

Eq. (2)

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At the time of harvesting (40, 80 and 120 days after sowing), rhizospheric soil (1 g) was

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collected and mixed with 9 mL of NaCl solution (0.9% w/v) and agitated for 1 h at 30 °C. Serial

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dilutions were made using 0.9% (w/v) NaCl solution. Colony forming units (CFU mL-1) of the

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suspension were evaluated using the dilution plate method on LB agar plates amended with

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endosulfan (50 mg L-1). The plates were incubated for 7 days at 28 °C and bacterial colonies

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were calculated (Islam et al., 2014; Rani et al., 2019).

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2.2.7. Estimation of lipid peroxidation

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The thiobarbituric acid method was used to evaluate the changes in content of lipid

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peroxidation in roots and leaves of H. annuus plant by calculating the malondialdehyde (MDA)

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formation. Briefly, 1 g of fresh tissues were homogenized in 0.1% of trichloroacetic acid (TCA)

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(2.5 mL). The mixture was centrifuged at 10,000 rpm for 10 min and the supernatant (1 mL) was

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collected and mixed with 20% TCA (4 mL) containing 0.5% thiobarbituric acid (TBA). The

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mixture was heated at 95 °C for 30 min and after cooling on ice for 10 min, the mixture was

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centrifuged at 10,000 rpm for 15 min. The absorbance of the supernatant was taken at 532 nm

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and 600 nm (Demiral and Turkan, 2005).

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2.2.8. Analysis of plant growth parameters

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Plants were harvested at 40, 80 and 120 days after sowing and root length and shoot

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length were measured (Rani et al., 2019). Root and shoot dry weights were evaluated after drying

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in oven at 70 ˚C for 24 h (Ruiz et al., 2000).

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2.2.9. Estimation of Chlorophyll and protein content

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Chlorophyll and protein content were estimated according to the method of Arnon (1949) and Lowry et al. (1951), respectively. The percent of growth inhibition (%GI) (Rani et al., 2019; El-Nahhal and Hamaduna, 2017a) was calculated following the Eq. (3):

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%GI = 100* (Pc – Pt) / Pc

Eq. (3)

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Where Pc: root or shoot length or root or shoot weight or chlorophyll or protein content of

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the control (E0; without endosulfan), Pt: root or shoot length or root or shoot weight or

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chlorophyll or protein content of the treatments E5, E10, E25 and E50.

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2.3. Quality control and assurance

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Instrumental and laboratory blanks were examined throughout the protocol, which

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suggested that samples were not contaminated while laboratory handling. Limit of detection

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(LOD) was evaluated by multiplying the standard deviation by three (ADLG, 1996; Singh and

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Singh, 2014b). The LOD for alpha-endosulfan was 0.00010 mg L-1, beta-endosulfan was

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0.00012 mg L-1 and endosulfan sulfate was 0.00006 mg L-1. The limit of quantification for alpha-

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endosulfan was 0.00011 mg L-1, beta-endosulfan was 0.00016 mg L-1 and endosulfan sulfate was

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0.00007 mg L-1 (Rani et al., 2019).

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To check the accuracy of extraction efficiencies of endosulfan from contaminated soil and

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plant tissue, the experiments utilized different concentrations of 0.0001, 0.0005, 0.001, 0.005,

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0.01, 0.1, 1, 10, 30 and 50 mg kg-1. The recovery ranges for alpha- endosulfan, beta- endosulfan

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and endosulfan sulfate were 91.2-97.2%, 94.5-99.7% and 90.4-95.1%, respectively (Rani et al.,

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2019).

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2.4. Statistical analysis

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All the results were presented as mean values of three replicates ± Standard Deviation

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using the XLSTAT package of MS Excel 2010. The results were analysed using a statistical

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package, SPSS version 21.0 (SPSS Inc. Chicago, USA). One-way ANOVA (analysis of

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variance) was followed by Tukey's HSD (honest significant difference) post hoc test was

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performed between different treatments in a pot experiment to evaluate the significant difference

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between uptake of endosulfan by plant tissues of H. annuus, degradation of endosulfan in soil,

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production of MDA, plant growth, chlorophyll and protein content of H. annuus. The statistical

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significance level was p<0.05.

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3. Results and Discussion

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3.1.

Uptake of endosulfan by plant tissues

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Accumulation of endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate) in the plant

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tissues (root and shoot) of H. annuus as a function of their harvesting period (40, 80 and 120

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days after sowing) are presented in Fig. 1. Endosulfan concentration was low in all the

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treatments inoculated with bacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and

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Bacillus sp. PRB101 at 40, 80 and 120 days after sowing. Nevertheless, at 120 days after sowing,

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at 5 and 50 mg kg-1 concentration of endosulfan, roots of PRB101 inoculated H. annuus plants

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were the minimum accumulator of α-endosulfan (0.00022 and 0.00199 mg kg-1), β-endosulfan

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(0.00128 and 0.00279 mg kg-1) and endosulfan sulfate (0.00011 and 0.00123 mg kg-1) as

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compared to control (un-inoculated soil) of α-endosulfan (0.00084 and 0.00612 mg kg-1), β-

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endosulfan (0.00104 and 0.00885 mg kg-1) and endosulfan sulfate (0.00039 and 0.00427 mg kg-

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1),

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accumulation of α-endosulfan (0.00020 and 0.00077 mg kg-1), β-endosulfan (0.00031 and

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0.00186 mg kg-1) and endosulfan sulfate (0.00014 and 0.00058 mg kg-1) as compared to control

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(un-inoculated soil) of α-endosulfan (0.00051 and 0.00297 mg kg-1), β-endosulfan (0.00085 and

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0.00489 mg kg-1) and endosulfan sulfate (0.00018 and 0.00205 mg kg-1), respectively (Fig. 1f).

respectively (Fig. 1c). Similarly, in shoot at 5 and 50 mg kg-1, PRB101 showed the lowest

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The root and shoot of H. annuus plants grown in bacteria strains IITISM08, PRB77 and

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PRB101 inoculated soil showed minimum uptake of endosulfan as compared to control (un-

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inoculated soil). However, no accumulation of endosulfan was noticed in the root and shoot of H.

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annuus grown in endosulfan unamended soils. These results indicated that the accumulation of

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endosulfan followed the pattern of root > shoot, hence suggested that the maximum uptake of

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endosulfan was mainly taking place via soil-root pathway. The accumulation of endosulfan in

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each plant tissue depends on plant characteristics such as transpiration rate, concentration of

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lipids, plant morphology, leaf surface area (Trapp and McFarlane, 1995; Bakker, 2000; Barber et

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al., 2004) or may be due to the reason of the accumulation of endosulfan from soil through roots

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(Bacci and Gaggi, 1985; Bacci et al., 1992). Several studies have reported the accumulation of

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persistent organic pollutants from contaminated soils (Gao and Zhu, 2004; White et al., 2005;

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Gao et al., 2006; Lin et al., 2007; Kidd et al., 2008), whereas specifically for endosulfan, still

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limited studies were found to determine the accumulation and uptake potential of plants

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(Ramirez-Sandoval et al., 2011; Singh and Singh, 2014b). Sunflower has been successfully used

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for remediation of endosulfan (Mitton et al., 2016). Sun et al. (2004) reported that the

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elimination of aldicarb from the planted soils was occurring through two pathways. One pathway

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was the degradation of aldicarb in soil and another one is the uptake by plants. Aldicarb present

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in the soil can be absorbed by plant roots and translocate throughout the plant parts. This results

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in a reduction of aldicarb level in soil and accumulate in the plant tissues. Plants mediate

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bioremediation by the release of exudates which are useful to the growth and activity of the

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microorganisms. Liste and Alexander (2000) found an increase in pyrene degradation in the

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plant-grown soils. Similarly Fang et al. (2001) found enhanced degradation of atrazine and

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phenanthrene in planted soils.

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Fig. 1. Uptake of endosulfan (mg kg-1) by root (a) 40 days (b) 80 days (c) 120 days after sowing and shoot (d) 40 days (e) 80 days (f) 120 days after sowing of H. annuus. Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan); Control (without inoculum) Values are in Mean ± standard deviation; (n=3); Error bars represent SD. Values with different lower case letters indicate significant difference (p < 0.05) between different treatments by similar inoculation of bacterial strains according to one way ANOVA followed by Tukey’s HSD post hoc test. Values with different upper case letters indicate significant difference (p < 0.05) between different inoculations of bacterial strains within a treatment according to one way ANOVA followed by Tukey’s HSD post hoc test.

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3.2.3. Degradation of endosulfan

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The residual concentrations of endosulfan after 40, 80 and 120 days of seed sowing in the

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soil were estimated to analyze the effect of inoculation of bacterial strains IITISM08, PRB77 and

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PRB101 on remediation of endosulfan contaminated soil (Fig. 2). At 40 days after sowing,

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maximum degradation of endosulfan 59.2, 57.0, 56.5 and 53.3% was found in PRB101

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inoculated plant grown soils as compared to un-inoculated soil (control) 3.8, 3.2, 1.7 and 1.8% in

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5, 10, 25 and 50 mg kg-1 concentrations of endosulfan, respectively (Fig. 2a). Similarly, at 120

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days after sowing, endosulfan degradation was found to be maximum as 92.0, 89.5, 83.5 and

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81.3% in PRB101 inoculated plant grown soils at 5, 10, 25 and 50 mg kg-1 concentration of

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endosulfan as compared to un-inoculated soil (control) of 7.6, 6.9, 5.2 and 4.4%, respectively

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(Fig. 2c). The inoculation of bacterial strains into the soil significantly decreased the

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concentration of endosulfan in comparison to non-inoculated soil, showing the ability of

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bacterial strains to degrade endosulfan. The results also indicated that percentage of endosulfan

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degradation decreases over endosulfan concentration, i.e., 5, 10, 25 and 50 mg kg-1. In agreement

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to this, Dubey and Fulekar (2013) reported lower degradation percentages at higher doses in the

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order of 25 > 50 > 75 >100 mg kg-1 for cypermethrin degradation. The presence of bacterial

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strains protected the plant against endosulfan toxicity, suggesting that plants along with bacterial

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strains play important role in attenuating the endosulfan toxicity. It can be concluded that H.

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annuus plant and bacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus

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sp. PRB101 can be used for the removal of endosulfan present in soil. Similarly, Mitton et al.

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(2016) reported that H. annuus plants were found to be the best candidate for phytoremediation

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as it showed a maximum decrease in endosulfan concentration present in soil facilitated by the

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enhanced biomass production and accumulation in roots, stems and leaves. Kuiper et al. (2004)

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studied degradation of pesticides and suggested that pesticides degrading bacteria protect plants

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against the contamination caused by pesticides.

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304 305 306 307 308 309 310 311 312 313 314 315

Fig. 2. Biodegradation of endosulfan in (a) 40 days (b) 80 days and (c) 120 days after sowing.

316

equation (r2) and half-life values (T1/2) (Table 1). Kinetic analysis showed that the process of

317

degradation of endosulfan observed in soil by inoculation of bacterial strains IITISM08, PRB77

318

and PRB101 were described by the rate constant (k) of 0.0047, 0.0074 0.0198 mg kg-1 day-1,

319

respectively, at 5 mg kg-1 concentration of endosulfan supplemented in soil, while at 50 mg kg-1

320

of endosulfan the rate constant (k) were 0.0035, 0.0056 and 0.0139 mg kg-1 day-1, respectively.

321

Moreover, the rate constant of endosulfan at 5 and 50 mg kg-1 of endosulfan amended in soil

322

(without inoculum) were found as 0.0007 and 0.0004 mg kg-1 day-1. The results indicated that

323

maximum degradation of endosulfan was observed in bacterial strains inoculated soil as

324

compared to un-inoculated soil (control), suggesting that bacterial strains had a positive effect on

325

endosulfan degradation in soils. Abraham and Silambarasan (2014) found that the rate constant

326

(k) of endosulfan degradation by a bacterial consortium was 203.1 mg L-1 d-1 in aqueous medium

327

and 178.4 mg kg-1 d-1 in soil. The half-lives of endosulfan was 3.4 days in aqueous medium and

328

3.8 days in soil.

Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan). Values are in Mean ± standard deviation; (n=3); Error bars represent SD. Values with different lower case letters indicate significant difference (p < 0.05) between different treatments by similar inoculation of bacterial strains according to one way ANOVA followed by Tukey’s HSD post hoc test. Values with different upper case letters indicate significant differences (p < 0.05) between different inoculations of bacterial strains within a treatment according to one way ANOVA followed by Tukey’s HSD post hoc test.

The data of residues of endosulfan were statistically interpreted for computation of the regression

329

The half-lives of endosulfan at 5 and 50 mg kg-1 concentration of endosulfan spiked un-

330

inoculated soil were found as 990.0 and 1732.5 days, respectively, while the half-lives of

331

endosulfan amended in soil inoculated with IITISM08, PRB77 and PRB101 were 147, 93 and 35

332

days at 5 mg kg-1 concentration and 198, 123 and 49 days at 50 mg kg-1 concentration of

333

endosulfan, respectively. The results indicated that bacterial strains IITISM08, PRB77 and 16

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334

PRB101 have the potential to reduce the half-lives of endosulfan in spiked soil at different

335

treatments. Kong et al. (2018) reported the half-lives of α-endosulfan as 43.4, 24.6, and 21.9

336

days and β- endosulfan as 95.7, 38 and 37 days in soil with different treatments as soil with

337

endosulfan (NE), soil with endosulfan and bacterial strain NS (NEN) and soil with endosulfan

338

and bacterial strain JW2 (NEJ), respectively.

339 340

Table. 1 Degradation kinetics of endosulfan in soil by different bacterial strains Strain

Treatment

Regression equation

Half-life (days)

Control

E5 E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50

-0.0007x -0.0006x -0.0005x -0.0004x -0.0047x -0.0045x -0.0042x -0.0035x -0.0074x -0.0067x -0.0063x -0.0056x -0.0198x -0.0178x -0.0149x -0.0139x

990.0 1155.0 1386.0 1732.5 147.4 154.0 165.0 198.0 93.6 103.4 110.0 123.7 35.0 38.9 46.5 49.8

IITISM08

PRB77

PRB101

341 342

Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan); Control (without inoculum)

343

The rate of degradation of endosulfan as showed by the regression equation suggested that

344

persistence of endosulfan increased with increase in concentration of endosulfan amended in

345

soil. (Fig. 3).

17

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346 347 348 349 350 351

Fig. 3. Degradation kinetics of endosulfan in H. annuus plant grown soil inoculated with (a) Control (without inoculum) (b) IITISM08 (c) PRB77 and (d) PRB101 Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan).

352 353

3.2.4. Enumeration of bacteria inoculated in soil

354

The endosulfan resistant bacterial strains were studied for their potential to colonize in

355

the rhizosphere soils at 40, 80 and 120 days after sowing (Table 2). The CFU g−1 of all the three

356

bacterial strains were found to be increased along with the increase in concentrations of

357

endosulfan. The numbers of bacterial strains inoculated in plant-grown soils were maximum at

358

80 days after sowing (second harvesting) and decreased towards the end of the experiment (120

359

days after sowing). Similarly, Sun et al. (2004) reported that after 7 days of inoculation,

360

microbial population in planted soil began to decline. Korade and Fulekar (2009) also reported

361

that the microbial numbers were found to be increasing with the increase in concentration of 18

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362

chlorpyrifos within the incubation of 7 days which further became constant towards the end of

363

the experiment. Ahmad et al. (2012) studied the combined use of ryegrass and Bacillus pumilus

364

C2A1 for remediating soil contaminated with chlorpyrifos and found that the microbial counts in

365

plant grown inoculated soil were maximum after 30 days (i.e., at the time of second harvesting)

366

and reduced after 45 days (i.e., towards the end of the experiment).

367 368

Table 2 Enumeration of bacteria (Log CFU g-1 soil) inoculated in soil. Treatments

E5 E10 E25 E50

Bacterial strain (CFU g-1) IITISM08 40 days 80 days 3.11 4.07 3.76 4.49 4.32 5.23 4.84 5.74

120 days 2.26 2.56 3.00 4.01

PRB77 40 days 3.85 4.23 4.77 5.04

80 days 4.46 4.77 5.27 5.62

120 days 2.47 2.85 3.17 4.27

PRB101 40 days 5.76 6.11 6.46 6.81

80 days 6.00 6.38 6.83 7.39

120 days 4.87 5.07 5.36 5.53

369 370

Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan).

371

3.2.5. Lipid Peroxidation

372

At 120 days after sowing, at 5 and 50 mg kg-1 concentration of endosulfan, roots of H.

373

annuus plants grown in PRB101 inoculated soil, showed a minimum level of MDA contents as

374

0.67 and 1.27 mg kg-1 over control 1.37 and 1.84 mg kg-1, respectively. Moreover, leaves of H.

375

annuus plants grown in PRB101 inoculated soil, showed a minimum level of MDA contents as

376

0.38 and 0.78 mg kg-1 as compared to control of 0.98 and 1.30 mg kg-1, respectively at 5 and 50

377

mg kg-1 concentration of endosulfan (Table 3).

378

In the present study, decrease in MDA contents in the root and leaves of H. annuus

379

grown in endosulfan spiked soils inoculated with bacterial strains IITISM08, PRB77 and

380

PRB101 were observed as compared to un-inoculated soils. Decrease in MDA contents is related

381

with minimum lipid peroxidation and therefore to less damage due to oxidative stress.

382 383 19

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384 385

Table 3 Malondialdehyde (MDA mg kg-1) formation by root and leaves of H. annuus

386

Plant tissue

387

Root

Days

40 days

388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429

80 days

120 days

Leaves

40 days

80 days

120 days

Treatment

E0 E5 E10 E25 E50 E0 E5 E10 E25 E50 E0 E5 E10 E25 E50 E0 E5 E10 E25 E50 E0 E5 E10 E25 E50 E0 E5 E10 E25 E50

Malondialdehyde (MDA mg kg-1) Control

IITISM08

PRB77

PRB101

0.75±0.4Aa 1.91±2.9Aa 2.02±0.9Aa 2.11±1.3Aa 2.34±1.2Aa 0.69±0.2Aa 1.72±0.7Aa 1.86±0.7Aa 2.08±0.9Aa 2.20±1.1Aa 0.55±0.3Aa 1.37±0.7Aa 1.54±0.5Aa 1.67±1.2Aa 1.84±1.1Aa 0.67±0.2Aa 1.37±0.7Aa 1.56±0.5Aa 1.70±0.7Aa 1.94±0.9Aa 0.54±0.2Aa 1.15±0.6Aa 1.28±0.7Aa 1.39±0.9Aa 1.58±1.3Aa 0.44±0.2Aa 0.98±0.3Aa 1.06±0.6Aa 1.10±0.1Aa 1.30±0.6Aa

0.66±0.5Aa 1.80±1.5Aa 1.97±1.0Aa 2.05±1.0Aa 2.26±1.0Aa 0.64±0.5Aa 1.64±1.2Aa 1.77±1.2Aa 1.97±1.0Aa 2.13±1.2Aa 0.54±0.2Aa 1.35±1.0Aa 1.44±0.9Aa 1.63±0.6Aa 1.75±1.0Aa 0.66±0.3Aa 1.32±1.5Aa 1.51±0.7Aa 1.59±1.3Aa 1.89±0.5Aa 0.64±0.3Aa 1.12±1.2Aa 1.20±0.3Aa 1.35±0.6Aa 1.54±0.7Aa 0.44±0.2Aa 0.94±1.0Aa 1.02±0.3Aa 1.08±0.5Aa 1.27±0.4Aa

0.64±0.1Aa 1.75±0.8Aa 1.93±0.9Aa 1.97±0.9Aa 2.16±0.5Aa 0.55±0.3Aa 1.51±0.6Aa 1.69±0.7Aa 1.88±1.0Aa 2.02±0.9Aa 0.54±0.2Aa 1.25±0.8Aa 1.36±0.6Aa 1.55±0.9Aa 1.71±0.8Aa 0.63±0.2Aab 1.17±0.3Aab 1.40±0.4Aab 1.57±0.5Aab 1.78±0.2Aa 0.59±0.3Aab 1.07±0.5Aab 1.14±0.4Aab 1.30±0.6Aab 1.50±0.4Aab 0.44±0.1Ab 0.91±0.4Aab 0.97±0.3Aab 1.07±0.4Aab 1.20±0.3Aab

0.52±0.4Aa 1.10±0.7Aa 1.19±0.8Aa 1.45±0.5Aa 1.53±1.6Aa 0.46±0.3Aa 0.81±0.5Aa 1.07±0.9Aa 1.16±0.7Aa 1.39±0.8Aa 0.39±0.2Aa 0.67±0.4Aa 0.81±0.7Aa 0.97±0.6Aa 1.27±0.9Aa 0.49±0.3Aa 0.75±0.2Aa 0.90±0.6Aa 1.14±0.4Aa 1.18±1.1Aa 0.44±0.3Aa 0.64±0.3Aa 0.60±0.3Aa 0.81±0.7Aa 0.97±0.5Aa 0.31±0.1Aa 0.38±0.2Aa 0.52±0.4Aa 0.66±0.3Aa 0.78±0.2Aa

Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan); Control (without inoculum) Values are in Mean ± standard deviation; (n=3). Values with different lower case letters indicate significant difference (p < 0.05) between different treatments by similar inoculation of bacterial strains in individual plant tissue (root and leaves) according to one way ANOVA followed by Tukey’s HSD post hoc test. Values with different upper case letters indicate significant differences (p < 0.05) between different inoculations of bacterial strains within a treatment in the individual plant tissue (root and leaves) according to one way ANOVA followed by Tukey’s HSD post hoc test.

3.2.6. Plant growth, chlorophyll and protein content under endosulfan stress

430

In the presence of endosulfan, decrease in root length (RL) and shoot length (SL) was

431

noticed. At 120 days after sowing, the minimum percent of growth inhibition at 5 and 50 mg kg-1

20

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432

concentration of endosulfan in RL (10.3% and 38.2%) and SL (4.79% and 27.5%) was observed

433

in PRB101 inoculated soil as compared to un-inoculated soil (42.3% and 72.2% of RL and

434

26.0% and 67.8% of SL) (Table 4). In agreement to this, Ahmad et al. (2012) found that RL and

435

SL of ryegrass increased significantly in B. pumilus inoculated chlorpyrifos (25 and 50 mg kg-1)

436

spiked soil as compared to un-inoculated soil.

437

Decrease in root and shoot weight (dry) was observed with an increase in endosulfan

438

concentration. At 120 days after sowing, the lowest percent of growth inhibition at 5 and 50 mg

439

kg-1 endosulfan concentration in root dry weight (5.31% and 42.9%) was found in PRB101

440

inoculated H. annuus planted soil as compared to un-inoculated soil (36.4% and 84.4%).

441

Similarly, in shoot dry weight, lowest percent of growth inhibition (4.49% and 20.8%) was found

442

in PRB101 inoculated H. annuus planted soil as compared to un-inoculated soil (34.0% and

443

76.7%) (Table 4).

444

At 120 days after sowing, 5 mg kg-1 concentration of endosulfan, the minimum percent of

445

growth inhibition of 10.6% and 5.54% was observed in chlorophyll and protein content of H.

446

annuus grown in PRB101 inoculated soil as compared to control (un-inoculated) of 46.6% and

447

36.5%, respectively, whereas at 50 mg kg-1 concentration, the minimum percent of growth

448

inhibition was found as 61.5% for chlorophyll content and 29.8% for protein content as

449

compared to control 79.0% and 69.6%, respectively (Table 4). Chlorophyll and protein content

450

decreases over increase in concentration of endosulfan.

451 452

Table 4 Plant growth, chlorophyll and protein content under endosulfan stress Plant growth parameter

Treatme nts

Bacterial strains IITISM08 PRB77

Control

PRB101

Days Root length

E5

40 29.4

80 33.9

120 42.3

40 21.7

80 24.8

21

120 28.9

40 17.1

80 19.3

120 20.6

40 6.9

80 8.5

120 10.3

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(%GI) Shoot length (%GI) Root weight (%GI) Shoot weight (%GI) Chlorophyll content (%GI) Protein content (%GI)

E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50 E5 E10 E25 E50

46.7 53.6 67.5 22.0 35.9 49.7 64.6 32.8 41.9 54.8 79.7 25.9 40.8 56.0 69.8 41.9 50.5 63.4 75.2 31.4 38.4 47.3 63.9

49.2 56.9 69.3 24.7 37.9 51.8 66.2 34.2 44.2 59.9 83.3 29.7 43.0 59.0 73.1 44.3 53.9 68.6 77.3 34.3 43.0 52.2 66.4

52.4 62.5 72.2 26.0 40.9 54.5 67.8 36.4 46.3 61.0 84.4 34.0 46.5 62.6 76.7 46.6 57.4 72.9 79.0 36.5 48.2 57.7 69.6

45.6 50.9 56.1 13.2 33.5 42.6 57.6 28.6 38.2 51.5 76.1 20.9 37.3 51.9 64.4 32.7 45.4 61.8 69.0 24.1 37.6 46.2 60.0

46.5 54.0 59.5 18.7 35.3 45.6 60.7 31.9 41.3 54.0 78.8 24.2 39.7 55.8 69.3 38.3 49.3 65.7 73.9 28.3 41.3 49.1 64.7

48.8 57.6 64.9 22.3 37.3 47.3 62.2 33.1 44.2 58.4 81.5 30.9 41.9 59.0 73.8 43.6 53.6 69.0 77.2 32.5 46.9 55.5 67.8

41.1 48.2 54.8 12.3 27.5 39.6 54.3 24.0 35.3 47.3 71.6 23.5 33.1 44.9 61.9 26.5 43.0 58.2 67.0 23.8 30.7 45.8 54.4

44.5 50.1 56.8 16.3 29.5 43.1 56.3 27.5 36.9 50.4 75.6 26.0 35.9 49.4 63.6 31.4 46.6 60.9 71.4 27.7 35.9 48.7 58.7

45.9 54.3 59.6 20.0 34.3 45.2 58.3 31.8 40.0 55.5 78.1 28.8 38.1 51.6 64.4 37.5 50.0 63.9 74.2 29.8 41.8 51.3 62.4

10.6 23.6 32.9 2.82 8.66 19.5 23.5 3.37 6.74 21.3 37.9 3.15 7.20 12.6 15.7 6.2 12.5 30.7 48.8 3.5 13.5 18.0 25.7

13.3 25.6 35.7 3.68 11.2 21.5 25.9 4.02 10.3 26.8 41.9 3.57 9.26 14.9 18.1 9.8 15.4 35.9 56.3 4.1 16.3 22.5 27.5

15.8 29.0 38.2 4.79 13.8 23.6 27.5 5.31 11.8 28.5 42.9 4.49 13.0 17.7 20.8 10.6 19.5 41.4 61.5 5.54 18.8 25.6 29.8

453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

Note: E5 (soil+5 mg kg−1 endosulfan), E10 (soil+10 mg kg−1 endosulfan), E25 (soil+25 mg kg−1 endosulfan), E50 (soil+50 mg kg−1 endosulfan); Control (without inoculum)

484

Bacterial inoculation in H. annuus planted soil significantly increases the root length,

485

shoot length, root dry weight and shoot dry weight, chlorophyll content and protein content as

%GI (Growth inhibition) = 100 * (Pc - Pt) / Pc (where Pc; root or shoot length/root or shoot weight/ chlorophyll or protein content of the control (E0) and Pt is the root or shoot length/root or shoot weight/ chlorophyll or protein content of the treated samples). Plants grown in endosulfan non spiked soil (E0), 100% corresponds to 23.1 cm (Control), 26.7 cm (IITISM08), 32.1 cm (PRB77), and 34.6 cm (PRB101) for root length at 40 days. 27.4 cm (Control), 29.4 cm (IITISM08), 35.7 cm (PRB77), and 37.5 cm (PRB101) for root length at 80 days. 32.8 cm (Control), 34.2 cm (IITISM08), 37.2 cm (PRB77), and 41.5 cm (PRB101) for root length at 120 days. 37.6 cm (Control), 39.9 cm (IITISM08), 42.1 cm (PRB77), and 49.6 cm (PRB101) for shoot length at 40 days. 43.2 cm (Control), 45.3 cm (IITISM08), 47.7 cm (PRB77), and 54.3 cm (PRB101) for shoot length at 80 days. 48.8 cm (Control), 51.9 cm (IITISM08), 53.5 cm (PRB77), and 58.4 cm (PRB101) for shoot length at 120 days. 2.86 g (Control), 2.75 g (IITISM08), 2.60 g (PRB77), and 3.56 g (PRB101) for root weight at 40 days. 3.12 g (Control), 3.00 g (IITISM08), 2.83 g (PRB77), and 3.98 g (PRB101) for root weight at 80 days. 3.54 g (Control), 3.28 g (IITISM08), 3.07 g (PRB77), and 4.14 g (PRB101) for root weight at 120 days. 3.6 g (Control), 3.9 g (IITISM08), 4.0 g (PRB77), and 4.4 g (PRB101) for shoot weight at 40 days. 4.1 g (Control), 4.2 g (IITISM08), 4.6 g (PRB77), and 4.7 g (PRB101) for shoot weight at 80 days. 4.9 g (Control), 4.6 g (IITISM08), 5.0 g (PRB77), and 5.1 g (PRB101) for shoot weight at 120 days. 0.93 mg g-1 (Control), 0.55 mg g-1 (IITISM08), 0.79 mg g-1 (PRB77), and 1.27 mg g-1 (PRB101) for chlorophyll content at 40 days. 1.15 mg g-1 (Control), 0.73 mg g-1 (IITISM08), 1.05 mg g-1 (PRB77), and 1.42 mg g-1 (PRB101) for chlorophyll content at 80 days. 1.48 mg g-1 (Control), 1.10 mg g-1 (IITISM08), 1.36 mg g-1 (PRB77), and 1.69 mg g-1 (PRB101) for chlorophyll content at 120 days. 13.4 mg g-1 (Control), 10.9 mg g-1 (IITISM08), 14.38 mg g-1 (PRB77), and 17.8 mg g-1 (PRB101) for protein content at 40 days. 17.6 mg g-1 (Control), 15.3 mg g-1 (IITISM08), 19.6 mg g-1 (PRB77), and 21.9 mg g-1 (PRB101) for protein content at 80 days. 22.5 mg g-1 (Control), 21.0 mg g-1 (IITISM08), 25.6 mg g-1 (PRB77), and 26.8 mg g-1 (PRB101) for protein content at 120 days.

22

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486

compared to un-inoculated soil. Toxicity of endosulfan causes changes in the function and

487

structure of the chloroplast, this might be due to the fact that organic pollutant can alter the lipid

488

composition by influencing the enzymes in C3 cycle and photosynthetic pigments (Knox and

489

Dodge, 1985; Vazquez et al., 1987; Abhilash and Singh, 2010ab; Dubey et al., 2014). Dubey et

490

al. (2014) found that reduction in protein content of Spinacia oleracea due to the adverse effect

491

of lindane resulted in protein degradation which leads to increased proteolytic activity and

492

oxidative modification in the plant.

493

Previous studies reported that pesticides influence several plant mechanisms that eventually

494

affect plant growth (El-Nahhal et al., 2016; El-Nahhal and Hamaduna, 2017ab). Moreover, Perez

495

et al. (2008) found that endosulfan (0.01-5 µL g-1) interacts with the mitotic spindle interrupting

496

normal chromosome migration resulted in genotoxic to Bidens laevis. The presence of lindane

497

and endosulfan in soils decreases seedling growth in Brassica chinensis (Chouychai, 2012).

498

Similarly, hexachlorocyclohexane (HCH) and its isomers showed their phytotoxic action in rice

499

seedlings by interacting with indole-3-ylacetic acid (IAA)-regulated growth and hindering Ca2+ -

500

ATPase activity (Sharada et al., 1992).

501 502

4. Conclusion

503

The findings of this study concluded that inoculation of endosulfan degrading bacterial

504

strains Paenibacillus sp. IITISM08, Bacillus sp. PRB101 and Bacillus sp. PRB77 in the H.

505

annuus planted soil results in decrease of soil pesticide levels in conjugation with high biomass

506

production and accumulation potential of the plant. The inoculation of bacterial strains in

507

endosulfan soiked soils resulted in reduction in malondialdehyde content in roots and leaves of

508

H. annuus plant. The combined use of microorganisms and plants might be used as an effective,

509

economic and ecological alternative to accelerate the removal of endosulfan present in soil. 23

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510

Acknowledgement

511

The authors would like to thank the Department of Environmental Science and Engineering,

512

Indian Institute of Technology (ISM) for providing research facilities.

513 514

Author’s contribution

515

VK designed experiments. RR performed experiments. RR, VK, PG, ZU and AC analyzed data

516

and wrote the manuscript.

517 518

Declarations of interest: None.

519 520

This research did not receive any specific grant from funding agencies in the public, commercial,

521

or not-for-profit sectors.

522 523

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524

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HIGHLIGHTS 

Inoculation of endosulfan degrading bacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 in Helianthus annuus (sunflower) planted soil enhanced remediation of endosulfan.



Bacterial isolates reduced the accumulation of endosulfan in roots and shoots of Helianthus annuus.



Inoculation of bacterial isolates increased plant biomass production and endosulfan degradation



Lipid peroxidation correlated positively with endosulfan levels in plants.