Bacillus pumilus ES4: Candidate plant growth-promoting bacterium to enhance establishment of plants in mine tailings

https://doi.org/10.1016/j.envexpbot.2010.04.014Get rights and content

Abstract

Three plant growth-promoting bacteria (PGPB; Bacillus pumilus ES4, B. pumilus RIZO1, and Azospirillum brasilense Cd) were tested for their ability to enhance plant growth and development of the native Sonoran Desert shrub quailbush (Atriplex lentiformis) and for their effect on the native bacterial community in moderately acidic, high-metal content tailings (AHMT) and in neutral, low-metal content natural tailings (NLMT) in controlled greenhouse experiments. Inoculation of quailbush with all three PGPB significantly enhanced plant growth parameters, such as germination, root length, dry weight of shoots and roots, and root/shoot ratio in both types of tailings. The effect of inoculation on the indigenous bacterial community by the most successful PGPB B. pumilus ES4 was evaluated by denaturating gradient gel electrophoresis (PCR-DGGE) fingerprinting and root colonization was followed by specific fluorescent in situ hybridization (FISH). Inoculation with this strain significantly changed the bacterial community over a period of 60 days. FISH analysis showed that the preferred site of colonization was the root tips and root elongation area. This study shows that inoculation of native perennial plants with PGPB can be used for developing technologies for phytostabilizing mine tailings.

Introduction

In the deserts of the southwestern United States and northern Mexico, large mounds of mine tailings, the main waste product of mineral ore processing of abandoned and productive mines, are potentially a health hazard to nearby urban populations in the form of wind-blown dust and ground water sources. This happens because tailings, lacking plant cover and soil structure, easily degrade by wind and rain action and serve as a continuous source of metal pollution (Pilon-Smits, 2005). Phytostabilization, using native plants has been proposed as an economic strategy to reduce these hazards (McCutcheon and Schnoor, 2003). This phytoremediation approach involves creation of a plant cover on the tailings that is sufficient to prevent erosion. A major challenge is that some of these tailings cannot serve, in their natural state, as growth substrate for most plant species because of metal toxicity, low pH, lack of essential minerals, lack of clay and organic matter to retain water, lack of soil structure, lack of a seed bank of nearby native plants, or some combination of these environmental parameters (Mendez and Maier, 2008). Thus, tailings can remain devoid of plants for decades or have only a slight plant cover (Gonzalez-Chavez et al., 2009). A partial solution and an upgrade of this inhospitable substrate to a “soil-like” status can be accomplished by adding large quantities of compost, biosolids, and irrigation (Ye et al., 2001, Chiu et al., 2006). The drawback of the compost-water strategy is that applying large quantities of compost is often not economically feasible because of the extensive area of tailings and the remoteness of the sites. Furthermore, water and irrigation facilities are largely absent in deserts, especially at long-abandoned mines.

Inoculation with plant growth-promoting bacteria (PGPB; Bashan and Holguin, 1998) has been proposed to aid in establishment of plants on tailings at reduced compost concentrations (Petrisor et al., 2004, Zhuang et al., 2007, Mendez and Maier, 2008). Successful attempts have been made to isolate and use potential PGPB from tailings (Grandlic et al., 2008). The premise for this approach is that PGPB isolated from tailings may aid in plant survival and growth through several mechanisms including: increasing nutrient availability to plants, increasing plant resistance to metal toxicity, or decreasing toxic metal bioavailability in the rhizosphere (Burd et al., 1998, Burd et al., 2000, Pishchik et al., 2002, Glick, 2003, Belimov et al., 2004, Reed and Glick, 2005, Reed et al., 2005, Vivas et al., 2006, Wu et al., 2006a, Li et al., 2007, Rajkumar and Freitas, 2008a, Rajkumar and Freitas, 2008b).

Alternatively, there are many well-studied PGPB used in other applications that could be useful in helping plant establishment in mine tailings. For example, strains from the genus Bacillus have been shown to enhance the growth of agricultural crops, wild plants, trees, microalgae, and model plants, through different mechanisms of plant growth-promotion (Bashan et al., 2000, Enebak et al., 1998, Hernandez et al., 2009, Kloepper et al., 2004a, Kloepper et al., 2004b, Ryu et al., 2005, Vessey, 2003). Bacillus is also a genus commonly found in various types of mine tailings (Natarajan, 1998, Vijayalakshmi and Raichur, 2003, Wu et al., 2006b, Tsuruta, 2007, Zhang et al., 2007). A second genus, Azospirillum, is also commonly used for its PGP activities. Azospirillum is perhaps the best studied of the PGPB except for rhizobia (Bashan et al., 2004).

The hypothesis of this study is that inoculation with classical agricultural PGPB can support establishment and growth of the common, native Sonoran Desert shrub, quailbush (Atriplex lentiformis) in mine tailings at reduced compost concentrations. The specific objectives were to: (1) Evaluate the growth of quailbush in two types of tailings: an acidic, high-metal content tailing and neutral low-metal content tailing supplemented with a suboptimal level of compost and inoculated with one of three PGPB (Bacillus pumilus ES4, B. pumilus RIZO1, and A. brasilenese Cd), comparing the performance of the three PGPB on plant growth; (2) Monitor root colonization capacity of the most successful PGPB using the technique of fluorescent in situ hybridization (FISH) and confocal laser microscopy; and (3) Assess whether there is an effect in structure on the tailings bacterial community by PGPB inoculation using PCR-DGGE fingerprint analysis.

Section snippets

Organisms, bacterial growth conditions and inoculant preparation

The plant growth-promoting bacteria Azospirillum brasilense Cd (DSM 1843, the type strain of A. brasilense; Braunschweig, Germany), B. pumilus ES4, and B. pumilus RIZO1 (Puente et al., 2004) and the evergreen shrub quailbush (A. lentiformis) native to southwestern North America, served as model organisms. The two B. pumilus strains were identified by sequencing their entire 16S rRNA gene (100% similarity to deposited B. pumilus) and their sequences were deposited in the GenBank (B. pumilus

Mine tailing analysis

The NLMT tailings have the following physiochemical properties: pH 7.0; 0.2% total carbon; 0.02% total organic carbon; <0.01% total nitrogen; 450 mg kg−1 total phosphorus; 1.4 mg kg−1 organic phosphorus; 7400 mg kg−1 total sulfur; 1550 mg kg−1 SO4−2; 2.3 mmhos cm−1 electrical conductivity; 3.89 meq 100 g−1 CEC; texture, 76.8% sand, 16.6% silt, 6.6% clay. Elemental analysis (mg kg−1): Fe (12,600); Ca (9600); Mg (3550); K (3040); Na (783); Cu (423); Mn (190); Zn (39); Ni (11); Cr (8); Pb (3.4); As (1.4); Se

Discussion

Phytostabilization of mine tailings is a type of phytoremediation that uses plants capable of surviving on these stressed substrates, with the objective of eliminating dispersal of contaminant and particulates from the site. In general, this has been done with large quantities of organic matter coupled with irrigation. Alternatively, the use of PGPB in phytostabilization of mine tailings is proposed (Petrisor et al., 2004, Zhuang et al., 2007, Mendez and Maier, 2008). There are two

Acknowledgements

We thank Julia Neilsen, Antje Legatzki, Sadie Iverson, and Fernando Solis-Dominguez from our research group at the University of Arizona for advice and discussion, Anton Hartmann and Michael Schmid of the Hemholtz Zentrum, München, Germany for advice on FISH development, and Christopher Grandlic at Synthetic Genomics, California, for advice on compost application in tailings. This research was supported by the National Institute of Environmental Health Sciences Superfund Basic Research Program,

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