Role of plant growth promoting bacteria and a leguminous plant in metal sequestration from metal contaminated environments by Brassica juncea
Adediran, Gbotemi Abraham
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The worldwide occurrence of sites contaminated with toxic metals and the associated high costs of remediating them using chemical and mechanical methods have led to calls to develop inexpensive and sustainable approaches based on the use of plants that naturally accumulate large amounts of metals in their tissues. The ability of plants to remediate metals has been rigorously studied and some species have been identified as excellent phytoremediators. However, the growth of phytoremediators is often retarded under high soil metal concentrations, rendering them ineffective. Meanwhile, some plants do not have remediating abilities but are capable of growing in contaminated environments with little or no sign of stress. Despite the volume of research dedicated to the screening and evaluation of phytoremediators, major questions remain about why some plants survive but do not remediate while the growth of phytoremediators is mostly hindered. The growth and metal-remediating efficiency of plants exposed to toxic concentrations of metals can be enhanced by inoculating phytoremediating plants with certain bacteria but the mechanisms behind this process remain unclear. Furthermore, the use of leguminous plants to improve the growth of a target plant under a mixed planting system has long been recognised as an effective yield-enhancing cropping system. However, the possibility of a non-remediating but tolerant leguminous plant conferring metal tolerance to a phytoremediator has not been explored. This thesis reports results from repeated glasshouse and lab-based growth experiments on the phytoremediating plant Brassica juncea exposed to 400 – 600 mg Zn kg-1. The aim was to investigate the abilities of two plant growth promoting bacteria (PGPB) species Pseudomonas brassicacearum and Rhizobium leguminosarum, and a leguminous plant Vicia sativa to promote B. juncea growth and enhance remediation of Zn-contaminated soil. B. juncea plant roots were analysed using synchrotron based micro-focus X-ray Fluorescence (μXRF) imaging and X-ray Absorption Near Edge Structure (μXANES) analysis to probe Zn speciation. P. brassicacearum exhibited the poorest plant growth promoting ability, while R. leguminosarum alone and in combination with P. brassicacearum significantly enhanced B. juncea growth and Zn bioaccumulation. X-ray Absorption Spectroscopy (XAS) analysis showed that reduced plant growth was due to root accumulation of Zn as Zn sulphate, Zn oxalate and Zn polygalacturonic acids. The better growth and increased metal accumulation observed in plants inoculated with R. leguminosarum and its combination with P. brassicacearum was attributed to root storage of Zn in the chelated forms of Zn phytate and Zn cysteine. A subcellular analysis of plant root also showed that the PGPB enhanced tolerance to Zn contamination by enhancing epidermal Zn compartmentalisation depending on the nature of root colonization, and induced changes in Zn speciation to less toxic Zn species in the epidermis and endodermis of plant root. The thesis therefore identifies enhanced Zn compartmentalization at the root epidermis and bacterial mediated changes in Zn toxicity through changes in Zn speciation as key complimentary mechanisms of plant growth promotion and enhanced Zn accumulation in plants by PGPB. Further experiments investigating alternative phytoremediation strategies showed that the use of the leguminous plant V. sativa in a mixed planting system with B. juncea plants completely out performed the effects of bacteria in promoting the growth and remediation potential of B. juncea under Zn contamination. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving maximum phytoremediation efficiency. The novel legume assistedmicrobial phytoremediation method that is reported in this thesis is the first to demonstrate complete plant growth recovery in plants exposed to 400 – 450 mg kg-1 soil Zn contamination for 5 weeks. Survival of V. sativa was attributed to its root storage of Zn in the chelated forms of Zn histidine and cysteine whereas in the roots of stunted B. juncea plants the majority of Zn was present as Zn oxalate and toxic Zn sulphate. Although the use of natural and synthetic chelates has been reported to enhance phytoremediation, this thesis recommends a legume-assisted-microbialphytoremediation system as a more sustainable method for Zn bioremediation.