Symbiotic microbes of Saxifraga stellaris ssp. alpigena from the copper creek of Schwarzwand (Austrian Alps) enhance plant tolerance to copper

Highlights

• Saxifraga stellaris is a copper-resistant plant.

• S. stellaris growing in a former mining area harbors beneficial endophytes.

• Endophytic fungus and bacterium influenced the Cu tolerance of S. stellaris.

Abstract

Saxifraga stellaris var. alpigena grows as one of the very few higher plants in the copper rich area of the “blue creek” in Austria. Two endophytes were isolated from it: Mortierella sp. (fungus), and Stenotrophomonas maltophilia (bacterium). Both microbes were practically inseparable due to resistance of the bacteria to the antibiotics tested. On PDA media, the fungus showed higher tolerance to copper than the bacterium, which disappeared from both the media and the surface of the fungus in the presence of 150 μM of Cu. However, at this Cu concentration, the bacteria were still detectable inside the mycelium and reappeared on the outside when transferred to media of lower Cu concentration. Microscopic studies of in vitro cultivated plants showed that the fungus was present in both, the roots and shoots of the plant. The effects of endophytes on plant performance were assessed in rhizoboxes filled with Cu-rich substratum; plants inoculated with both microbes showed better growth, survival and photosynthesis performance than the non-inoculated controls. The results of this study prove the beneficial influence of the isolated endophytes on the Cu tolerance of S. stellaris, and indicate the ecological potential of applying microbial consortia to plants under extreme environmental conditions.

Introduction

Copper (Cu) is an essential micronutrient needed by practically all organisms, but concentrations higher than 60 ppm are considered an environmental risk (Nies, 1992, 1999; Mirlean et al., 2007). Nowadays, contamination by this metal is a widespread problem that results from metal-mining activities, sewage-sludge disposal and the use of agrochemicals such as pesticides (Mirlean et al., 2007; Norgrove, 2007). Although Cu is often used against pathogens, it is becoming widely accepted that some microbes tolerate high concentrations of Cu and that they moreover protect plants against biotic- and abiotic-stress factors and even promote their growth (Zhang et al., 2011; Glick, 2014), especially under extreme conditions. Such microbes, isolated from internal parts of plants that survive in high concentrations of Cu, e.g. in copper mine wastes, could become an important tool in phytoremediation under extreme conditions.

Endophytes are organisms that colonize the living, internal tissues of their host plant but cause no obvious symptoms (at least not for a period of time) (Petrini, 1991; Schulz and Boyle, 2006). Endophyte-plant interactions may result in improved access to nutrients for the endophyte together with protection of the plants from environmental stresses, such as drought (Ravel et al., 1995; Cheplick et al., 2000), heat (Redman et al., 2002), heavy metals (Monnet et al., 2001), low pH (Lewis, 2004), high salinity (Waller et al., 2005), insect pests and herbivores (Siegel and Bush, 1996; Schardl and Phillips, 1997) and competition by other microorganisms (White et al., 2000; Schulz and Boyle, 2005). Various substances are involved in plant growth promotion and protection (Sirrenberg et al., 2007; Dai et al., 2008).

Historic mining sites at Schwarzwand near Salzburg offer the possibility of studying such plant-microbe interactions. Although the mine was closed a long time ago, the rocks of the creek on the steep slope are still covered by bluish precipitations (Fig. S1) containing up to 2% Cu (Saukel, 1980). The blue layer consists of calcium carbonate covered by copper minerals (Günther, 2006). As shown by Adlassnig et al. (2013), it is a biofilm dominated by the filamentous cyanobacterium Phormidium sp. (up to 80%), accompanied by other prokaryotes and mosses, and encrusted by minerals, such as gypsum and sampleite. Water flowing down the creek loses most of its Cu because the blue microbial mat plays an important role as a biofilter and removes metals from the water. It contains up to 20.000 ppm Cu in its dry mass (Adlassnig et al., 2013).

Saxifraga stellaris subsp. alpigena Temesy (syn. S. stellaris var. robusta Engl.), the starry saxifrage or hairy kidney-wort, is one of the few plants that survives under these conditions (Adlassnig et al., 2013). It roots in the Cu-containing blue sludge of the river and river banks, and from there grows rosettes of leaves and a stem carrying 5–10 flowers in a loose panicle. Leaves can be immersed in the copper sludge, at least temporarily, because they are protected by a hydrophobic cuticle, which makes direct metal penetration much more difficult.

Copper is an important micronutrient for plants for growth and development (Yruela, 2005), however, Cu in excess leads to significant changes in growth and metabolism. For example, Sheldon and Menzies (2005) observed inhibition of root growth and severe changes in root morphology. Copper at higher concentrations shows also negative effects on photosynthetic activity (Pádua et al., 2010), lipid content in plasma membrane (Quartacci et al., 2001). Induction of oxidative stress was also reported (Gill et al., 2012).

The main aims of the present research were i) to understand the remarkable and exceptional tolerance of S. stellaris ssp. alpigena to copper, ii) to isolate its endophytic microbes and iii) to evaluate their role in the plant's tolerance to copper.