Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus

Abstract

One hundred Cu-resistant-endophytic bacteria were isolated from Cu-tolerant plants grown on Cu mine wasteland, of which, eight Cu-resistant and 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were obtained based on the ACC deaminase activity of the bacteria and characterized with respect to metal resistance, production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores and mineral phosphate solubilization. Ralstonia sp. J1-22-2, Pantoea agglomerans Jp3-3, and Pseudomonas thivervalensis Y1-3-9 with higher ACC deaminase activity (ranging from 213 to 370 μM α-ketobutyrate mg⁻¹ h⁻¹) were evaluated for promoting plant growth and Cu uptake of rape grown in quartz sand containing 0, 2.5, and 5 mg kg⁻¹ of Cu in pot experiments. The eight bacteria were found to exhibit different multiple heavy metal resistance characteristics, to show different levels of ACC deaminase activity and to produce indole acetic acid. Seven bacteria produced siderophores and solubilized inorganic phosphate. Pot experiments showed that inoculation with the strains (J1-22-2, Jp3-3, and Y1-3-9) was found to increase the biomass of rape. Increases in above-ground tissue Cu contents of rape cultivated in 2.5 and 5 mg kg⁻¹ of Cu-contaminated substrates varied from 9% to 31% and from 3 to 4-fold respectively in inoculated-rape plants compared to the uninoculated control. The maximum Cu uptake of rape was observed after inoculation with P. agglomerans Jp3-3. The results show that metal-resistant and plant growth promoting endophytic bacteria play an important role in plant growth and Cu uptake which may provide a new endophytic bacterial-assisted phytoremediation of Cu-contaminated environment.

Introduction

Due to the fact that heavy metals cannot be biologically degraded to harmless products and hence persist in the environment indefinitely, heavy metal contamination in soils is becoming one of the most severe environmental problems. Although Cu is an essential element in metabolic process in plants or animals, excessive Cu concentrations in the contaminated soils can pose significant risks to human health and ecosystems. Therefore, the development of a remediation strategy for metal contaminated soils is urgent for environmental conservation and human health (Abou-Shanab et al., 2006). Phytoremediation to clean up heavy metal contaminated soil has gained more attention than conventional technology as environmental friendly and cost effective (Haque et al., 2008, Chehregani et al., 2009, Kotrba et al., 2009). The success of the phytoremediation process depends on an adequate plant yield and high heavy metal concentrations in above-ground tissues of plants (Rajkumar et al., 2009). However, most hyperaccumulators identified so far are not suitable for field phytoremediation applications due to their small biomass and slow growth (Rajkumar et al., 2009). This has prompted us to explore the possibilities of enhancing the biomass and metal uptake of metal accumulators using plant growth-promoting bacteria (PGPB) as bioinoculants (Abou-Shanab et al., 2006, Sheng and Xia, 2006, Rajkumar et al., 2009). Plant-associated bacteria play a key role in host adaptation to a changing environment (Sturz and Nowak, 2000). Recently, the benefits of combining heavy metal-resistant-endophytic bacteria with plants for increased remediation of pollutants have been successfully tried for toxic metal removal from metal contaminated soils (Rajkumar et al., 2009). Endophytic bacteria have the capacity to promote plant growth and development under adverse conditions by various mechanisms such as nitrogen fixation, solubilization of mineral phosphate, production of indole acetic acid (IAA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Puente et al., 2009, Rajkumar et al., 2009). ACC deaminase can metabolize ACC (an immediate precursor of ethylene in plants) into α-ketobutyric acid and ammonia. Some ACC deaminase-producing PGPB promote plant growth by lowering the level of ethylene in plants growing in the presence of heavy metals (Glick, 2005, Ma et al., 2009). Although bacterial-assisted phytoremediation has been studied (Zaidi et al., 2006, Dell’Amico et al., 2008, Sheng et al., 2008, Mastretta et al., 2009), there are very little data on the characterization and effects of ACC deaminase-producing endophytic bacteria isolated from Cu-tolerant plants on plant growth and Cu uptake under Cu-contaminated conditions. There is also very little concern on the effective matching of PGPB and plant for the bioremediation of Cu-contaminated soils. Endophytic bacteria may be of particular interest as they have the advantage of being relatively protected from the competitive, high-stress environment of the soil (Sturz et al., 2000). A better understanding of the characteristics of heavy metal-resistant and ACC deaminase-producing endophytic bacteria is a critical prerequisite for the development of effective phytoremediation of heavy metal contaminated soils.

The objectives of this study were to isolate and characterize Cu-resistant and ACC deaminase-producing endophytic bacteria from Cu-tolerant plants grown in Cu mine wasteland, select PGPB and elucidate the effects of inoculating PGPB on the plant growth and Cu uptake of rape for improving the efficiency of phytoextraction of Cu-polluted soils.