Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

Abstract

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Introduction

Plants interact with a great diversity of microorganisms, including enteric bacteria. These interactions, which are lined by the characteristics of both, host plant and bacteria, result in associative, commensal, symbiotic, or parasitic relationships between both partners. Members of the Enterobacteriaceae are distributed in many environments, with some being saprophytes and others being parasites of plants and animals. Several studies have shown that Enterobacteriaceae may have beneficial effects on plant development when they are associated with plants (Lodewyckx et al., 2002, Taghavi et al., 2009). They may improve plant growth via nitrogen fixation, suppression of plant pathogens and production of phytohormones and enzymes involved in the metabolism of growth regulators such as ethylene, 1-aminocyclopropane 1-carboxylic acid (ACC), auxins and indole-3-acetic acid (IAA) (Gyaneshwar et al., 2001, Kämpfer et al., 2005, Taghavi et al., 2009). Organisms such as Enterobacter radicincitans, Enterobacter arachidis, Enterobacter oryzae, and Enterobacter sp. CBMB30, which were isolated from the wheat phyllosphere, groundnut rhizosphere, poplar and rice endosphere, respectively, are known as plant growth-promoting bacteria (Lee et al., 2006, Peng et al., 2009, Taghavi et al., 2009, Madhaiyan et al., 2010).

In previous experiments we repeatedly isolated Enterobacter-related strains from the rhizosphere and endosphere of plants (Italian ryegrass and birdsfoot trefoil) grown in diesel-contaminated soils (Yousaf et al., 2010a). Further characterization revealed that several strains belong to Enterobacter ludwigii. This species is known for its clinical relevance as most isolates have been isolated from clinical specimens (Hoffmann et al., 2005). E. ludwigii belongs to the Enterobacter cloacae complex, which has been frequently isolated from nosocomial infections; however, it is not clear whether E. ludwigii is a true pathogen or has a rather commensal character (Paauw et al., 2008). Generally, few studies on E. ludwigii are available, but it has been reported as a plant-associated bacterium with plant growth-promoting and biocontrol capacities (Shoebitz et al., 2009).

Global industrialization over the past years has resulted in numerous sites with strong contamination of the soil with persistent organic and inorganic contaminants. Aliphatic hydrocarbons (e.g. diesel fuel and engine oils) make up a substantial proportion of substances found at contaminated sites (Stroud et al., 2007). The use of plants and their associated microorganisms for the treatment of hydrocarbon-contaminated soils has attained increasing acceptance as a viable clean-up technology (Lelie et al., 2001). The efficiency of a phytoremediation process depends mainly on the presence and activity of plant-associated microorganisms carrying degradation genes required for the enzymatic break-down of contaminants. The rhizosphere and plant endosphere have been reported to host pollutant-degrading bacteria (Siciliano et al., 2001, Andria et al., 2009) and highly diverse alkane degrading bacteria containing alkane degrading genes have been isolated from the plant environment (Kaimi et al., 2007). Expression analysis of alkane monooxygenase (alkB) and a cytochrome P450 hydroxylase (CYP153 gene) indicated degradation in the rhizosphere as well as in the plant interior (Powell et al., 2006, Andria et al., 2009, Afzal et al., 2011).

In this study we characterized in detail selected alkane degrading Enterobacter strains, which were previously isolated from Italian ryegrass and birdsfoot trefoil (Yousaf et al., 2010a) and identified as E. ludwigii. In plant experiments, we studied in detail the hydrocarbon degradation and plant colonization capacities of these strains.